OSK1 peptide analogs and pharmaceutical compositions

Abstract
Disclosed is a composition of matter comprising an OSK1 peptide analog, and in some embodiments, a pharmaceutically acceptable salt thereof. A pharmaceutical composition comprises the composition and a pharmaceutically acceptable carrier. Also disclosed are DNAs encoding the inventive composition of matter, an expression vector comprising the DNA, and host cells comprising the expression vector. Methods of treating an autoimmune disorder and of preventing or mitigating a relapse of a symptom of multiple sclerosis are also disclosed.
Description
BACKGROUND OF THE INVENTION

1. Field of the Invention


The present invention is related to the biochemical arts, in particular to therapeutic peptides and conjugates.


2. Discussion of the Related Art


Ion channels are a diverse group of molecules that permit the exchange of small inorganic ions across membranes. All cells require ion channels for function, but this is especially so for excitable cells such as those present in the nervous system and the heart. The electrical signals orchestrated by ion channels control the thinking brain, the beating heart and the contracting muscle. Ion channels play a role in regulating cell volume, and they control a wide variety of signaling processes.


The ion channel family includes Na+, K+, and Ca2+ cation and Cl anion channels. Collectively, ion channels are distinguished as either ligand-gated or voltage-gated. Ligand-gated channels include both extracellular and intracellular ligand-gated channels. The extracellular ligand-gated channels include the nicotinic acetylcholine receptor (nAChR), the serotonin (5-hdroxytryptamine, 5-HT) receptors, the glycine and γ-butyric acid receptors (GABA) and the glutamate-activated channels including kanate, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) and N-methyl-D-aspartate receptors (NMDA) receptors. (Harte and Ouzounis (2002), FEBS Lett. 514: 129-34). Intracellular ligand gated channels include those activated by cyclic nucleotides (e.g. cAMP, cGMP), Ca2+ and G-proteins. (Harte and Ouzounis (2002), FEBS Lett. 514: 129-34). The voltage-gated ion channels are categorized by their selectivity for inorganic ion species, including sodium, potassium, calcium and chloride ion channels. (Harte and Ouzounis (2002), FEBS Lett. 514: 129-34).


A unified nomenclature for classification of voltage-gated channels was recently presented. (Catterall et al. (2000), Pharmacol. Rev. 55: 573-4; Gutman et al. (2000), Pharmacol. Rev. 55, 583-6; Catterall et al. (2000) Pharmacol. Rev. 55: 579-81; Catterall et al. (2000), Pharmacol. Rev. 55: 575-8; Hofmann et al. (2000), Pharmacol. Rev. 55: 587-9; Clapham et al. (2000), Pharmacol Rev. 55: 591-6; Chandy (1991), Nature 352: 26; Goldin et al. (2000), Neuron 28: 365-8; Ertel et al. (2000), Neuron 25: 533-5).


The K+ channels constitute the largest and best characterized family of ion channels described to date. Potassium channels are subdivided into three general groups: the 6 transmembrane (6™) K+ channels, the 2™-2™/leak K+ channels and the 2™/K+ inward rectifying channels. (Tang et al. (2004), Ann. Rev. Physiol. 66, 131-159). These three groups are further subdivided into families based on sequence similarity. The voltage-gated K+ channels, including (Kv1-6, Kv8-9), EAG, KQT, and Slo (BKCa), are family members of the 6™ group. The 2™-2™ group comprises TWIK, TREK, TASK, TRAAK, and THIK, whereas the 2™/Kir group consists of Kir1-7. Two additional classes of ion channels include the inward rectifier potassium (IRK) and ATP-gated purinergic (P2X) channels. (Harte and Ouzounis (2002), FEBS Lett. 514: 129-34).


Toxin peptides produced by a variety of organisms have evolved to target ion channels. Snakes, scorpions, spiders, bees, snails and sea anemone are a few examples of organisms that produce venom that can serve as a rich source of small bioactive toxin peptides or “toxins” that potently and selectively target ion channels and receptors. In most cases, these toxin peptides have evolved as potent antagonists or inhibitors of ion channels, by binding to the channel pore and physically blocking the ion conduction pathway. In some other cases, as with some of the tarantula toxin peptides, the peptide is found to antagonize channel function by binding to a region outside the pore (e.g., the voltage sensor domain).


The toxin peptides are usually between about 20 and about 80 amino acids in length, contain 2-5 disulfide linkages and form a very compact structure (see, e.g., FIG. 10). Toxin peptides (e.g., from the venom of scorpions, sea anemones and cone snails) have been isolated and characterized for their impact on ion channels. Such peptides appear to have evolved from a relatively small number of structural frameworks that are particularly well suited to addressing the critical issues of potency and stability. The majority of scorpion and Conus toxin peptides, for example, contain 10-40 amino acids and up to five disulfide bonds, forming extremely compact and constrained structure (microproteins) often resistant to proteolysis. The conotoxin and scorpion toxin peptides can be divided into a number of superfamilies based on their disulfide connections and peptide folds. The solution structure of many of these has been determined by NMR spectroscopy, illustrating their compact structure and verifying conservation of their family fold. (E.g., Tudor et al., Ionisation behaviour and solution properties of the potassium-channel blocker ShK toxin, Eur. J. Biochem. 251(1-2):133-41 (1998); Pennington et al., Role of disulfide bonds in the structure and potassium channel blocking activity of ShK toxin, Biochem. 38(44): 14549-58 (1999); Jaravine et al., Three-dimensional structure of toxin OSK1 from Orthochirus scrobiculosus scorpion venom, Biochem. 36(6):1223-32 (1997); del Rio-Portillo et al.; NMR solution structure of Cn12, a novel peptide from the Mexican scorpion Centruroides noxius with a typical beta-toxin sequence but with alpha-like physiological activity, Eur. J. Biochem. 271(12): 2504-16 (2004); Prochnicka-Chalufour et al., Solution structure of discrepin, a new K+-channel blocking peptide from the alpha-KTx15 subfamily, Biochem. 45(6):1795-1804 (2006)).


Conserved disulfide structures can also reflect the individual pharmacological activity of the toxin family. (Nicke et al. (2004), Eur. J. Biochem. 271: 2305-19, Table 1; Adams (1999), Drug Develop. Res. 46: 219-34). For example, α-conotoxins have well-defined four cysteine/two disulfide loop structures (Loughnan, 2004) and inhibit nicotinic acetylcholine receptors. In contrast, ω-conotoxins have six cysteine/three disulfide loop consensus structures (Nielsen, 2000) and block calcium channels. Structural subsets of toxins have evolved to inhibit either voltage-gated or calcium-activated potassium channels. FIG. 9 shows that a limited number of conserved disulfide architectures shared by a variety of venomous animals from bee to snail and scorpion to snake target ion channels. FIG. 7A-7B shows alignment of alpha-scorpion toxin family and illustrates that a conserved structural framework is used to derive toxins targeting a vast array of potassium channels.


Due to their potent and selective blockade of specific ion channels, toxin peptides have been used for many years as tools to investigate the pharmacology of ion channels. Other than excitable cells and tissues such as those present in heart, muscle and brain, ion channels are also important to non-excitable cells such as immune cells. Accordingly, the potential therapeutic utility of toxin peptides has been considered for treating various immune disorders, in particular by inhibition of potassium channels such as Kv1.3 and IKCa1 since these channels indirectly control calcium signaling pathway in lymphocytes. [e.g., Kem et al., ShK toxin compositions and methods of use, U.S. Pat. No. 6,077,680; Lebrun et al., Neuropeptides originating in scorpion, U.S. Pat. No. 6,689,749; Beeton et al., Targeting effector memory T cells with a selective peptide inhibitor of Kv1.3 channels for therapy of autoimmune diseases, Molec. Pharmacol. 67(4):1369-81 (2005); Mouhat et al., K+ channel types targeted by synthetic OSK1, a toxin from Orthochirus scrobiculosus scorpion venom, Biochem. J. 385:95-104 (2005); Mouhat et al., Pharmacological profiling of Orthochirus scrobiculosus toxin 1 analogs with a trimmed N-terminal domain, Molec. Pharmacol. 69:354-62 (2006); Mouhat et al., OsK1 derivatives, WO 2006/002850 A2; B. S. Jensen et al. The Ca2+-activated K+ Channel of Intermediate Conductance: A Molecular Target for Novel Treatments?, Current Drug Targets 2:401-422 (2001); Rauer et al., Structure-guided Transformation of Charybdotoxin Yields an Analog That Selectively Targets Ca2+-activated over Voltage-gated K+ Channels, J. Biol. Chem. 275: 1201-1208 (2000); Castle et al., Maurotoxin: A Potent Inhibitor of Intermediate Conductance Ca2+-Activated Potassium Channels, Molecular Pharmacol. 63: 409-418 (2003); Chandy et al., K+ channels as targets for specific Immunomodulation, Trends in Pharmacol. Sciences 25: 280-289 (2004); Lewis & Garcia, Therapeutic Potential of Venom Peptides, Nat. Rev. Drug Discov. 2: 790-802 (2003)].


Small molecules inhibitors of Kv1.3 and IKCa1 potassium channels and the major calcium entry channel in T cells, CRAC, have also been developed to treat immune disorders [A. Schmitz et al. (2005) Molecul. Pharmacol. 68, 1254; K. G. Chandy et al. (2004) TIPS 25, 280; H. Wulff et al. (2001) J. Biol. Chem. 276, 32040; C. Zitt et al. (2004) J. Biol. Chem. 279, 12427], but obtaining small molecules with selectivity toward some of these targets has been difficult.


Calcium mobilization in lymphocytes is known to be a critical pathway in activation of inflammatory responses [M. W. Winslow et al. (2003) Current Opinion Immunol. 15, 299]. Compared to other cells, T cells show a unique sensitivity to increased levels of intracellular calcium and ion channels both directly and indirectly control this process. Inositol triphosphate (IP3) is the natural second messenger which activates the calcium signaling pathway. IP3 is produced following ligand-induced activation of the T cell receptor (TCR) and upon binding to its intracellular receptor (a channel) causes unloading of intracellular calcium stores. The endoplasmic reticulum provides one key calcium store. Thapsigargin, an inhibitor of the sarcoplasmic-endoplasmic reticulum calcium ATPase (SERCA), also causes unloading of intracellular stores and activation of the calcium signaling pathway in lymphocytes. Therefore, thapsigargin can be used as a specific stimulus of the calcium signaling pathway in T cells. The unloading of intracellular calcium stores in T cells is known to cause activation of a calcium channel on the cell surface which allows for influx of calcium from outside the cell. This store operated calcium channel (SOCC) on T cells is referred to as “CRAC” (calcium release activated channel) and sustained influx of calcium through this channel is known to be critical for full T cell activation [S. Feske et al. (2005) J. Exp. Med. 202, 651 and N. Venkatesh et al. (2004) PNAS 101, 8969]. For many years it has been appreciated that in order to maintain continued calcium influx into T cells, the cell membrane must remain in a hyperpolarized condition through efflux of potassium ions. In T cells, potassium efflux is accomplished by the voltage-gated potassium channel Kv1.3 and the calcium-activated potassium channel IKCa1 [K. G. Chandy et al. (2004) TIPS 25, 280]. These potassium channels therefore indirectly control the calcium signaling pathway, by allowing for the necessary potassium efflux that allows for a sustained influx of calcium through CRAC.


Sustained increases in intracellular calcium activate a variety of pathways in T cells, including those leading to activation of NFAT, NF-kB and AP-1 [Quintana-A (2005) Pflugers Arch.—Eur. J. Physiol. 450, 1]. These events lead to various T cell responses including alteration of cell size and membrane organization, activation of cell surface effector molecules, cytokine production and proliferation. Several calcium sensing molecules transmit the calcium signal and orchestrate the cellular response. Calmodulin is one molecule that binds calcium, but many others have been identified (M. J. Berridge et al. (2003) Nat. Rev. Mol. Cell. Biol. 4, 517). The calcium-calmodulin dependent phosphatase calcineurin is activated upon sustained increases in intracellular calcium and dephosphorylates cytosolic NFAT. Dephosphorylated NFAT quickly translocates to the nucleus and is widely accepted as a critical transcription factor for T cell activation (F. Macian (2005) Nat. Rev. Immunol. 5, 472 and N. Venkatesh et al. (2004) PNAS 101, 8969). Inhibitors of calcineurin, such as cyclosporin A (Neoral, Sand immune) and FK506 (Tacrolimus) are a main stay for treatment of severe immune disorders such as those resulting in rejection following solid organ transplant (I. M. Gonzalez-Pinto et al. (2005) Transplant. Proc. 37, 1713 and D. R. J. Kuypers (2005) Transplant International 18, 140). Neoral has been approved for the treatment of transplant rejection, severe rheumatoid arthritis (D. E. Yocum et al. (2000) Rheumatol. 39, 156) and severe psoriasis (J. Koo (1998) British J. Dermatol. 139, 88). Preclinical and clinical data has also been provided suggesting calcineurin inhibitors may have utility in treatment of inflammatory bowel disease (IBD; Baumgart D C (2006) Am. J. Gastroenterol. March 30; Epub ahead of print), multiple sclerosis (Ann. Neurol. (1990) 27, 591) and asthma (S. Rohatagi et al. (2000) J. Clin. Pharmacol. 40, 1211). Lupus represents another disorder that may benefit from agents blocking activation of helper T cells. Despite the importance of calcineurin in regulating NFAT in T cells, calcineurin is also expressed in other tissues (e.g. kidney) and cyclosporine A & FK506 have a narrow safety margin due to mechanism based toxicity. Renal toxicity and hypertension are common side effects that have limited the promise of cyclosporine & FK506. Due to concerns regarding toxicity, calcineurin inhibitors are used mostly to treat only severe immune disease (Bissonnette-R et al. (2006) J. Am. Acad. Dermatol. 54, 472). Kv1.3 inhibitors offer a safer alternative to calcineurin inhibitors for the treatment of immune disorders. This is because Kv1.3 also operates to control the calcium signaling pathway in T cells, but does so through a distinct mechanism to that of calcineurin inhibitors, and evidence on Kv1.3 expression and function show that Kv1.3 has a more restricted role in T cell biology relative to calcineurin, which functions also in a variety of non-lymphoid cells and tissues.


Calcium mobilization in immune cells also activates production of the cytokines interleukin 2 (IL-2) and interferon gamma (IFNg) which are critical mediators of inflammation. IL-2 induces a variety of biological responses ranging from expansion and differentiation of CD4+ and CD8+ T cells, to enhancement of proliferation and antibody secretion by B cells, to activation of NK cells [S. L. Gaffen & K. D. Liu (2004) Cytokine 28, 109]. Secretion of IL-2 occurs quickly following T cell activation and T cells represent the predominant source of this cytokine. Shortly following activation, the high affinity IL-2 receptor (IL2-R) is upregulated on T cells endowing them with an ability to proliferate in response to IL-2. T cells, NK cells, B cells and professional antigen presenting cells (APCs) can all secrete IFNg upon activation. T cells represent the principle source of IFNg production in mediating adaptive immune responses, whereas natural killer (NK) cells & APCs are likely an important source during host defense against infection [K. Schroder et al. (2004) J. Leukoc. Biol. 75, 163]. IFNg, originally called macrophage-activating factor, upregulates antigen processing and presentation by monocytes, macrophages and dendritic cells. IFNg mediates a diverse array of biological activities in many cell types [U. Boehm et al. (1997) Annu. Rev. Immunol. 15, 749] including growth & differentiation, enhancement of NK cell activity and regulation of B cell immunoglobulin production and class switching.


CD40L is another cytokine expressed on activated T cells following calcium mobilization and upon binding to its receptor on B cells provides critical help allowing for B cell germinal center formation, B cell differentiation and antibody isotype switching. CD40L-mediated activation of CD40 on B cells can induce profound differentiation and clonal expansion of immunoglobulin (Ig) producing B cells [S. Quezada et al. (2004) Annu. Rev. Immunol. 22, 307]. The CD40 receptor can also be found on dendritic cells and CD40L signaling can mediate dendritic cell activation and differentiation as well. The antigen presenting capacity of B cells and dendritic cells is promoted by CD40L binding, further illustrating the broad role of this cytokine in adaptive immunity. Given the essential role of CD40 signaling to B cell biology, neutralizing antibodies to CD40L have been examined in preclinical and clinical studies for utility in treatment of systemic lupus erythematosis (SLE),—a disorder characterized by deposition of antibody complexes in tissues, inflammation and organ damage [J. Yazdany and J Davis (2004) Lupus 13, 377].


Production of toxin peptides is a complex process in venomous organisms, and is an even more complex process synthetically. Due to their conserved disulfide structures and need for efficient oxidative refolding, toxin peptides present challenges to synthesis. Although toxin peptides have been used for years as highly selective pharmacological inhibitors of ion channels, the high cost of synthesis and refolding of the toxin peptides and their short half-life in vivo have impeded the pursuit of these peptides as a therapeutic modality. Far more effort has been expended to identify small molecule inhibitors as therapeutic antagonists of ion channels, than has been given the toxin peptides themselves. One exception is the recent approval of the small ω-conotoxin MVIIA peptide (Ziconotide™) for treatment of intractable pain. The synthetic and refolding production process for Ziconotide™, however, is costly and only results in a small peptide product with a very short half-life in vivo (about 4 hours).


A cost-effective process for producing therapeutics, such as but not limited to, inhibitors of ion channels, is a desideratum provided by the present invention, which involves toxin peptides fused, or otherwise covalently conjugated to a vehicle.


SUMMARY OF THE INVENTION

The present invention relates to a composition of matter of the formula:





(X1)a—(F1)d—(X2)b—(F2)e—(X3)c  (I)


and multimers thereof, wherein:


F1 and F2 are half-life extending moieties, and d and e are each independently 0 or 1, provided that at least one of d and e is 1;


X1, X2, and X3 are each independently -(L)f-P-(L)g-, and f and g are each independently 0 or 1;


P is a toxin peptide of no more than about 80 amino acid residues in length, comprising at least two intrapeptide disulfide bonds, and at least one P is an OSK1 peptide analog;


L is an optional linker (present when f=1 and/or g=1); and


a, b, and c are each independently 0 or 1, provided that at least one of a, b and c is 1.


The present invention thus concerns molecules having variations on Formula I, such as the formulae:





P-(L)g-F1 (i.e., b, c, and e equal to 0);  (II)





F1-(L)g-P (i.e., a, c, and e equal to 0);  (III)





P-(L)g-F1-(L)f-P or (X1)a—F1—(X2)b (i.e., c and e equal to 0);  (IV)





F1-(L)f-P-(L)g-F2 (i.e., a and c equal to 0);  (V)





F1-(L)f-P-(L)g-F2-(L)f-P (i.e., a equal to 0);  (VI)





F1—F2-(L)f-P (i.e., a and b equal to 0);  (VII)





P-(L)g-F1—F2 (i.e., b and c equal to 0);  (VIII)





P-(L)g-F1—F2-(L)f-P (i.e., b equal to 0);  (IX)


and any multimers of any of these, when stated conventionally with the N-terminus of the peptide(s) on the left. All of such molecules of Formulae II-IX are within the meaning of Structural Formula I. Within the meaning of Formula I, the toxin peptide (P), if more than one is present, can be independently the same or different from the OSK1 peptide analog, or any other toxin peptide(s) also present in the inventive composition, and the linker moiety ((L)f and/or (L)g), if present, can be independently the same or different from any other linker, or linkers, that may be present in the inventive composition. Conjugation of the toxin peptide(s) to the half-life extending moiety, or moieties, can be via the N-terminal and/or C-terminal of the toxin peptide, or can be intercalary as to its primary amino acid sequence, F1 being linked closer to the toxin peptide's N-terminus than is linked F2. Examples of useful half-life extending moieties (F1 or F2) include immunoglobulin Fc domain (e.g., a human immunoglobulin Fc domain, including Fc of IgG1, IgG2, IgG3 or IgG4) or a portion thereof, human serum albumin (HSA), or poly(ethylene glycol) (PEG). These and other half-life extending moieties described herein are useful, either individually or in combination. A monovalent dimeric Fc-toxin peptide fusion (as represented schematically in FIG. 2B), for example, an Fc-OSK1 peptide analog fusion or Fc-ShK peptide analog fusion, is an example of the inventive composition of matter encompassed by Formula VII above.


The present invention also relates to a composition of matter, which includes, conjugated or unconjugated, a toxin peptide analog of ShK, OSK1, ChTx, or Maurotoxin modified from the native sequences at one or more amino acid residues, having greater Kv1.3 or IKCa1 antagonist activity, and/or target selectivity, compared to a ShK, OSK1, or Maurotoxin (MTX) peptides having a native sequence. The toxin peptide analogs comprise an amino acid sequence selected from any of the following:


SEQ ID NOS: 88, 89, 92, 148 through 200, 548 through 561, 884 through 949, or 1295 through 1300 as set forth in Table 2; or


SEQ ID NOS: 980 through 1274, 1303, or 1308 as set forth in Table 7; or any of SEQ ID NOS: 1391 through 4912, 4916, 4920 through 5006, 5009, 5010, and 5012 through 5015 as set forth in Table 7A, Table 7B, Table 7C, Table 7D, Table 7E, Table 7F, Table 7G, Table 7H, Table 7I or Table 7J.


SEQ ID NOS: 330 through 337, 341, 1301, 1302, 1304 through 1307, 1309, 1311, 1312, and 1315 through 1336 as set forth in Table 13; or


SEQ ID NOS: 36, 59, 344-346, or 1369 through 1390 as set forth in Table 14.


The present invention also relates to other toxin peptide analogs that comprise an amino acid sequence selected from, or comprise the amino acid primary sequence of, any of the following:


SEQ ID NOS: 201 through 225 as set forth in Table 3; or


SEQ ID NOS: 242 through 248 or 250 through 260 as set forth in Table 4; or


SEQ ID NOS: 261 through 275 as set forth in Table 5; or


SEQ ID NOS: 276 through 293 as set forth in Table 6; or


SEQ ID NOS: 299 through 315 as set forth in Table 8; or


SEQ ID NOS: 316 through 318 as set forth in Table 9; or


SEQ ID NO: 319 as set forth in Table 10; or


SEQ ID NO: 327 or 328 as set forth in Table 11; or


SEQ ID NOS: 330 through 337, 341, 1301, 1302, 1304 through 1307, 1309, 1311, 1312, or 1315 through 1336 as set forth in Table 13;


SEQ ID NOS: 1369 through 1390 as set forth in Table 14; or


SEQ ID NOS: 348 through 353 as set forth in Table 16; or


SEQ ID NOS: 357 through 362, 364 through 368, 370, 372 through 385, or 387 through 398 as set forth in Table 19; or


SEQ ID NOS: 399 through 408 as set forth in Table 20; or


SEQ ID NOS: 410 through 421 as set forth in Table 22; or


SEQ ID NOS: 422, 424, 426, or 428 as set forth in Table 23; or


SEQ ID NOS: 430 through 437 as set forth in Table 24; or


SEQ ID NOS: 438 through 445 as set forth in Table 25; or


SEQ ID NOS: 447, 449, 451, 453, 455, or 457 as set forth in Table 26; or


SEQ ID NOS: 470 through 482 or 484 through 493 as set forth in Table 28; or


SEQ ID NOS: 495 through 506 as set forth in Table 29; or


SEQ ID NOS: 507 through 518 as set forth in Table 30.


The present invention is also directed to a pharmaceutical composition that includes the inventive composition of matter and a pharmaceutically acceptable carrier.


The compositions of this invention can be prepared by conventional synthetic methods, recombinant DNA techniques, or any other methods of preparing peptides and fusion proteins well known in the art. Compositions of this invention that have non-peptide portions can be synthesized by conventional organic chemistry reactions, in addition to conventional peptide chemistry reactions when applicable. Thus the present invention also relates to DNAs encoding the inventive compositions and expression vectors and host cells for recombinant expression.


The primary use contemplated is as therapeutic and/or prophylactic agents. The inventive compositions incorporating the toxin peptide can have activity and/or ion channel target selectivity comparable to—or even greater than—the unconjugated peptide.


Accordingly, the present invention includes a method of treating an autoimmune disorder, which involves administering to a patient who has been diagnosed with an autoimmune disorder, such as multiple sclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease (IBD, including Crohn's Disease and ulcerative colitis), contact-mediated dermatitis, rheumatoid arthritis, psoriatic arthritis, asthma, allergy, restinosis, systemic sclerosis, fibrosis, scleroderma, glomerulonephritis, Sjogren syndrome, inflammatory bone resorption, transplant rejection, graft-versus-host disease, or lupus, a therapeutically effective amount of the inventive composition of matter (preferably comprising a Kv1.3 antagonist peptide or IKCa1 antagonist peptide), whereby at least one symptom of the disorder is alleviated in the patient. In addition, the present invention also relates to the use of one or more of the inventive compositions of matter in the manufacture of a medicament for the treatment or prevention of an autoimmune disorder, such as, but not limited to, any of the above-listed autoimmune disorders, e.g. multiple sclerosis, type 1 diabetes or IBD.


The present invention is further directed to a method of preventing or mitigating a relapse of a symptom of multiple sclerosis, which method involves administering to a patient, who has previously experienced at least one symptom of multiple sclerosis, a prophylactically effective amount of the inventive composition of matter (preferably comprising a Kv1.3 antagonist peptide or IKCa1 antagonist peptide), such that the at least one symptom of multiple sclerosis is prevented from recurring or is mitigated.


Although mostly contemplated as therapeutic agents, compositions of this invention can also be useful in screening for therapeutic or diagnostic agents. For example, one can use an Fc-peptide in an assay employing anti-Fc coated plates. The half-life extending moiety, such as Fc, can make insoluble peptides soluble and thus useful in a number of assays.


Numerous additional aspects and advantages of the present invention will become apparent upon consideration of the figures and detailed description of the invention.


U.S. Nonprovisional patent application Ser. No. 11/406,454, filed Apr. 17, 2006, is hereby incorporated by reference in its entirety.





BRIEF DESCRIPTION OF THE FIGURES


FIG. 1 shows schematic structures of some exemplary Fc dimers that can be derived from an IgG1 antibody. “Fc” in the figure represents any of the Fc variants within the meaning of “Fc domain” herein. “X1” and “X2” represent peptides or linker-peptide combinations as defined hereinafter. The specific dimers are as follows:



FIG. 1A and FIG. 1D: Single disulfide-bonded dimers;



FIG. 1B and FIG. 1E: Doubly disulfide-bonded dimers;



FIG. 1C and FIG. 1F: Noncovalent dimers.



FIG. 2A-C show schematic structures of some embodiments of the composition of the invention that shows a single unit of the pharmacologically active toxin peptide. FIG. 2A shows a single chain molecule and can also represent the DNA construct for the molecule. FIG. 2B shows a dimer in which the linker-peptide portion is present on only one chain of the dimer (i.e., a “monovalent” dimer). FIG. 2C shows a dimer having the peptide portion on both chains. The dimer of FIG. 2C will form spontaneously in certain host cells upon expression of a DNA construct encoding the single chain shown in FIG. 2A. In other host cells, the cells could be placed in conditions favoring formation of dimers or the dimers can be formed in vitro.



FIG. 3A-3B shows exemplary nucleic acid and amino acid sequences (SEQ ID NOS: 1 and 2, respectively) of human IgG1 Fc that is optimized for mammalian expression and can be used in this invention.



FIG. 4A-4B shows exemplary nucleic acid and amino acid sequences (SEQ ID NOS: 3 and 4, respectively) of human IgG1 Fc that is optimized for bacterial expression and can be used in this invention.



FIG. 5A shows the amino acid sequence of the mature ShK peptide (SEQ ID NO: 5), which can be encoded for by a nucleic acid sequence containing codons optimized for expression in mammalian cell, bacteria or yeast.



FIG. 5B shows the three disulfide bonds (—S—S—) formed by the six cysteines within the ShK peptide (SEQ ID NO: 10).



FIG. 6 shows an alignment of the voltage-gated potassium channel inhibitor Stichodactyla helianthus (ShK) with other closely related members of the sea anemone toxin family. The sequence of the 35 amino acid mature ShK toxin (Accession #P29187) isolated from the venom of Stichodactyla helianthus is shown aligned to other closely related members of the sea anemone family. The consensus sequence and predicted disulfide linkages are shown, with highly conserved residues being shaded. The HmK peptide toxin sequence shown (Swiss-Protein Accession #097436) is of the immature precursor from the Magnificent sea anemone (Radianthus magnifica; Heteractis maqnifica) and the putative signal peptide is underlined. The mature HmK peptide toxin would be predicted to be 35 amino acids in length and span residues 40 through 74. AeK is the mature peptide toxin, isolated from the venom of the sea anemone Actinia equine (Accession #P81897). The sequence of the mature peptide toxin AsKS (Accession #Q9TWG1) and BgK (Accession #P29186) isolated from the venom of the sea anemone Anemonia sulcata and Bunodosoma granulifera, respectively, are also shown. FIG. 6A shows the amino acid alignment (SEQ ID NO: 10) of ShK to other members of the sea anemone family of toxins, HmK (SEQ ID NO: 6 (Mature Peptide), (SEQ ID NO: 542, Signal and Mature Peptide portions)), AeK (SEQ ID NO: 7), AsKs (SEQ ID NO: 8), and BgK (SEQ ID NO: 9). The predicted disulfide linkages are shown and conserved residues are highlighted. (HmK, SEQ ID NO: 543; ShK, SEQ ID NO: 10; AeK, SEQ ID NO: 544; AsKS, SEQ ID NO: 545). FIG. 6B shows a disulfide linkage map for this family having 3 disulfide linkages (C1-C6, C2-C4, C3-C5).



FIG. 7A-7B shows an amino acid alignment of the alpha-scorpion toxin family of potassium channel inhibitors. (BmKK1, SEQ ID NO: 11; BmKK4, SEQ ID NO: 12; PBTx1, SEQ ID NO: 14; Tc32, SEQ ID NO: 13; BmKK6, SEQ ID NO: 15; P01, SEQ ID NO: 16; Pi2, SEQ ID NO: 17; Pi3, SEQ ID NO: 18; Pi4, SEQ ID NO: 19; MTX, SEQ ID NO: 20; Pi1, SEQ ID NO: 21; HsTx1, SEQ ID NO: 61; AgTx2, SEQ ID NO: 23; KTX1, SEQ ID NO: 24; OSK1, SEQ ID NO: 25; BmKTX, SEQ ID NO: 22; HgTX1, SEQ ID NO: 27; MgTx, SEQ ID NO: 28; C11Tx1, SEQ ID NO: 29; NTX, SEQ ID NO: 30; Tc30, SEQ ID NO: 31; TsTX-Ka, SEQ ID NO: 32; PBTx3, SEQ ID NO: 33; Lqh 15-1, SEQ ID NO: 34; MartenTx, SEQ ID NO: 37; ChTx, SEQ ID NO:36; ChTx-Lq2, SEQ ID NO: 42; IbTx, SEQ ID NO: 38; SloTx, SEQ ID NO: 39; BmTx1; SEQ ID NO: 43; BuTx, SEQ ID NO: 41; AmmTx3, SEQ ID NO: 44; AaTX1, SEQ ID NO: 45; BmTX3, SEQ ID NO: 46; Tc1, SEQ ID NO: 48; OSK2, SEQ ID NO: 49; TsK, SEQ ID NO: 54; CoTx1, SEQ ID NO:55; CoTx2, SEQ ID NO: 871; BmPo5, SEQ ID NO: 60; ScyTx, SEQ ID NO: 51; P05, SEQ ID NO: 52; Tamapin, SEQ ID NO: 53; and TmTx, SEQ ID NO: 691. Highly conserved residues are shaded and a consensus sequence is listed. Subfamilies of the α-KTx are listed and are from Rodriguez de la Vega, R. C. et al. (2003) TIPS 24: 222-227. A list of some ion channels reported to antagonized is listed (IK=IKCa, BK=BKCa, SK=SKCa, Kv=voltage-gated K+ channels). Although most family members in this alignment represent the mature peptide product, several represent immature or modified forms of the peptide and these include: BmKK1, BmKK4, BmKK6, BmKTX, MartenTx, ChTx, ChTx-Lq2, BmTx1, AaTx1, BmTX3, TsK, CoTx1, BmP05.



FIG. 8 shows an alignment of toxin peptides converted to peptibodies in this invention (Apamin, SEQ ID NO: 68; HaTx1, SEQ ID NO: 494; ProTx1, SEQ ID NO: 56; PaTx2, SEQ ID NO: 57; ShK[2-35], SEQ ID NO: 92; ShK[1-35], SEQ ID NO: 5; HmK, SEQ ID NO: 6; ChTx (K32E), SEQ ID NO: 59; ChTx, SEQ ID NO: 36; IbTx, SEQ ID NO: 38; OSK1 (E16K, K20D), SEQ ID NO: 296; OSK1, SEQ ID NO: 25; AgTx2, SEQ ID NO: 23; KTX1, SEQ ID NO: 24; MgTx, SEQ ID NO: 28; NTX, SEQ ID NO: 30; MTX, SEQ ID NO: 20; Pi2, SEQ ID NO: 17; HsTx1, SEQ ID NO: 61; Anuroctoxin [AnTx], SEQ ID NO: 62; BeKm1, SEQ ID NO: 63; ScyTx, SEQ ID NO: 51; ωGVIA, SEQ ID NO: 64; ωMVIIa, SEQ ID NO: 65; Ptu1, SEQ ID NO: 66; and CTX, SEQ ID NO: 67). The original sources of the toxins is indicated, as well as, the number of cysteines in each. Key ion channels targeted are listed. The alignment shows clustering of toxin peptides based on their source and ion channel target impact.



FIG. 9 shows disulfide arrangements within the toxin family. The number of disulfides and the disulfide bonding order for each subfamily is indicated. A partial list of toxins that fall within each disulfide linkage category is presented.



FIG. 10 illustrates that solution structures of toxins reveal a compact structure. Solution structures of native toxins from sea anemone (ShK), scorpion (MgTx, MTX, HsTx1), marine cone snail (wGVIA) and tarantula (HaTx1) indicate the 28 to 39 amino acid peptides all form a compact structure. The toxins shown have 3 or 4 disulfide linkages and fall within 4 of the 6 subfamilies shown in FIG. 9. The solution structures of native toxins from sea anemone (ShK), scorpion (MgTx, MTX, HsTx1), marine cone snail (wGVIA) and tarantula (HaTx1) were derived from Protein Data Bank (PDB) accession numbers 1ROO (mmdbld:5247), 1MTX (mmdbld:4064), 1TXM (mmdbld:6201), 1QUZ (mmdbld:36904), 1OMZ (mmdbld:1816) and 1D1H (mmdbld:14344) using the MMDB Entrez 3D-structure database [J. Chen et al. (2003) Nucleic Acids Res. 31, 474] and viewer.



FIG. 11A-C shows the nucleic acid (SEQ ID NO: 69 and SEQ ID NO: 1358) and encoded amino acid (SEQ ID NO:70, SEQ ID NO:1359 and SEQ ID NO: 1360) sequences of residues 5131-6660 of pAMG21ampR-Fc-pep. The sequences of the Fc domain (SEQ ID NOS: 71 and 72) exclude the five C-terminal glycine residues. This vector enables production of peptibodies in which the peptide-linker portion is at the C-terminus of the Fc domain.



FIG. 11D shows a circle diagram of a peptibody bacterial expression vector pAMG21ampR-Fc-pep having a chloramphenicol acetyltransferase gene (cat; “CmR” site) that is replaced with the peptide-linker sequence.



FIG. 12A-C shows the nucleic acid (SEQ ID NO: 73 and SEQ ID NO: 1361) and encoded amino acid (SEQ ID NO:74, SEQ ID NO: 1362 and SEQ ID NO: 1363) sequences of residues 5131-6319 of pAMG21ampR-Pep-Fc. The sequences of the Fc domain (SEQ ID NOS: 75 and 76) exclude the five N-terminal glycine residues. This vector enables production of peptibodies in which the peptide-linker portion is at the N-terminus of the Fc domain.



FIG. 12D shows a circle diagram of a peptibody bacterial expression vector having a zeocin resistance (ble; “ZeoR”) site that is replaced with the peptide-linker sequence.



FIG. 12E-G shows the nucleic acid (SEQ ID NO:1339) and encoded amino acid sequences of pAMG21ampR-Pep-Fc (SEQ ID NO:1340, SEQ ID NO:1341, and SEQ ID NO:1342). The sequences of the Fc domain (SEQ ID NOS: 75 and 76) exclude the five N-terminal glycine residues. This vector enables production of peptibodies in which the peptide-linker portion is at the N-terminus of the Fc domain.



FIG. 13A is a circle diagram of mammalian expression vector pCDNA3.1(+) CMVi.



FIG. 13B is a circle diagram of mammalian expression vector pCDNA3.1 (+) CMVi-Fc-2×G4S-Activin RIIb that contains a Fc region from human IgG1, a 10 amino acid linker and the activin RIIb gene.



FIG. 13C is a circle diagram of the CHO expression vector pDSRa22 containing the Fc-L10-ShK[2-35] coding sequence.



FIG. 14A-14B shows the nucleotide and encoded amino acid sequences (SEQ. ID. NOS: 77 and 78, respectively) of the molecule identified as “Fc-L10-ShK[1-35]” in Example 1 hereinafter. The L10 linker amino acid sequence (SEQ ID NO: 79) is underlined.



FIG. 15A-15B shows the nucleotide and encoded amino acid sequences (SEQ. ID. NOS: 80 and 81, respectively) of the molecule identified as “Fc-L10-ShK[2-35]” in Example 2 hereinafter. The same L10 linker amino acid sequence (SEQ ID NO: 79) as used in Fc-L10-ShK[1-35] (FIG. 14A-14B) is underlined.



FIG. 16A-16B shows the nucleotide and encoded amino acid sequences (SEQ. ID. NOS: 82 and 83, respectively) of the molecule identified as “Fc-L25-ShK[2-35]” in Example 2 hereinafter. The L25 linker amino acid sequence (SEQ ID NO: 84) is underlined.



FIG. 17 shows a scheme for N-terminal PEGylation of ShK peptide (SEQ ID NO: 5 and SEQ ID NO:10) by reductive amination, which is also described in Example 32 hereinafter.



FIG. 18 shows a scheme for N-terminal PEGylation of ShK peptide (SEQ ID NO: 5 and SEQ ID NO:10) via amide formation, which is also described in Example 34 hereinafter.



FIG. 19 shows a scheme for N-terminal PEGylation of ShK peptide (SEQ ID NO: 5 and SEQ ID NO:10) by chemoselective oxime formation, which is also described in Example 33 hereinafter.



FIG. 20A shows a reversed-phase HPLC analysis at 214 nm and FIG. 20B shows electrospray mass analysis of folded ShK[2-35], also described as folded-“Des-Arg1-ShK” (Peptide 2).



FIG. 21 shows reversed phase HPLC analysis at 214 nm of N-terminally PEGylated ShK[2-35], also referred to as N-Terminally PEGylated-“Des-Arg1-ShK”.



FIG. 22A shows a reversed-phase HPLC analysis at 214 nm of folded ShK[1-35], also referred to as “ShK”.



FIG. 22B shows electrospray mass analysis of folded ShK[1-35], also referred to as “ShK”.



FIG. 23 illustrates a scheme for N-terminal PEGylation of ShK[2-35] (SEQ ID NO: 92 and SEQ ID NO: 58, also referred to as “Des-Arg1-ShK” or “ShK d1”) by reductive amination, which is also described in Example 31 hereinafter.



FIG. 24A shows a western blot of conditioned medium from HEK 293 cells transiently transfected with Fc-L10-ShK[1-35]. Lane 1: molecular weight markers; Lane 2: 15 μl Fc-L10-ShK; Lane 3: 10 μl Fc-L10-ShK; Lane 4: 5 μl Fc-L10-ShK; Lane 5; molecular weight markers; Lane 6: blank; Lane 7: 15 μl No DNA control; Lane 8: 10 μl No DNA control; Lane 9: 5 μl No DNA control; Lane 10; molecular weight markers.



FIG. 24B shows a western blot of with 15 μl of conditioned medium from clones of Chinese Hamster Ovary (CHO) cells stably transfected with Fc-L-ShK[1-35]. Lanes 1-15 were loaded as follows: blank, BB6, molecular weight markers, BB5, BB4, BB3, BB2, BB1, blank, BD6, BD5, molecular weight markers, BD4, BD3, BD2.



FIG. 25A shows a western blot of a non-reducing SDS-PAGE gel containing conditioned medium from 293T cells transiently transfected with Fc-L-SmIIIA.



FIG. 25B shows a western blot of a reducing SDS-PAGE gel containing conditioned medium from 293T cells transiently transfected with Fc-L-SmIIIA.



FIG. 26A shows a Spectral scan of 10 μl purified bivalent dimeric Fc-L10-ShK[1-35] product from stably transfected CHO cells diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1-cm path length quartz cuvette.



FIG. 26B shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final bivalent dimeric Fc-L10-ShK[1-35] product. Lanes 1-12 were loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.



FIG. 26C shows size exclusion chromatography on 20 μg of the final bivalent dimeric Fc-L10-ShK[1-35] product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, and pH 6.9 at 1 ml/min observing the absorbance at 280 nm.



FIG. 26D shows a MALDI mass spectral analysis of the final sample of bivalent dimeric Fc-L10-ShK[1-35] analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.



FIG. 26E shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final monovalent dimeric Fc-L10-ShK[1-35] product. Lanes 1-12 were loaded as follows: Novex Mark12 wide range protein standards (10 μL), 0.5 μg product non-reduced (1.3 μL), blank, 2.0 μg product non-reduced (5 μL), blank, 10 μg product non-reduced (25 μL), Novex Mark12 wide range protein standards (10 μL), 0.5 μg product reduced (1.3 μL), blank, 2.0 μg product reduced (5 μL), blank, and 10 μg product reduced (25 μL).



FIG. 26F shows size exclusion chromatography on 20 μg of the final monovalent dimeric Fc-L10-ShK[1-35] product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, and pH 6.9 at 1 ml/min observing the absorbance at 280 nm.



FIG. 27A shows a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final purified bivalent dimeric Fc-L10-ShK[2-35] product from stably transfected CHO cells. Lane 1-12 were loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.



FIG. 27B shows size exclusion chromatography on 50 μg of the purified bivalent dimeric Fc-L10-ShK[2-35] injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, and pH 6.9 at 1 ml/min observing the absorbance at 280 nm.



FIG. 27C shows a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of bivalent dimeric Fc-L5-ShK[2-35] purified from stably transfected CHO cells. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.



FIG. 27D shows a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of bivalent dimeric Fc-L25-ShK[2-35] purified from stably transfected CHO cells. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.



FIG. 27E shows a spectral scan of 10 μl of the bivalent dimeric Fc-L10-ShK[2-35] product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.



FIG. 27F shows a MALDI mass spectral analysis of the final sample of bivalent dimeric Fc-L110-ShK[2-35] analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from about 200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.



FIG. 27G shows a spectral scan of 10 μl of the bivalent dimeric Fc-L5-ShK[2-35] product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.



FIG. 27H shows the size exclusion chromatography on 50 mg of the final bivalent dimeric Fc-L5-ShK[2-35] product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.



FIG. 27I shows a MALDI mass spectral analysis of the final sample of bivalent dimeric Fc-L5-ShK[2-35] analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.



FIG. 27J shows a Spectral scan of 20 μl of the product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.



FIG. 27K shows the size exclusion chromatography on 50 μg of the final bivalent dimeric Fc-L25-ShK[2-35] product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.



FIG. 27L shows a MALDI mass spectral analysis of the final sample of bivalent dimeric Fc-L25-ShK[2-35] analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from about 200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.



FIG. 28A shows a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of Fc-L10-KTX1 purified and refolded from bacterial cells. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.



FIG. 28B shows size exclusion chromatography on 45 μg of purified Fc-L10-KTX1 injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.



FIG. 28C shows a Spectral scan of 20 μl of the Fc-L10-KTX1 product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.



FIG. 28D shows a MALDI mass spectral analysis of the final sample of Fc-L10-KTX1 analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.



FIG. 29A shows a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of Fc-L-AgTx2 purified and refolded from bacterial cells. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.



FIG. 29B shows a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of Fc-L10-HaTx1 purified and refolded from bacterial cells. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced, spectral scan of the purified material.



FIG. 29C shows a Spectral scan of 20 μl of the Fc-L110-AgTx2 product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.



FIG. 29D shows the Size exclusion chromatography on 20 μg of the final Fc-L10-AgTx2 product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.



FIG. 29E shows a MALDI mass spectral analysis of the final sample of Fc-L10-AgTx2 analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from about 200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.



FIG. 29F shows a Spectral scan of 20 μl of the Fc-L10-HaTx1 product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.



FIG. 29G shows the size exclusion chromatography on 20 μg of the final Fc-L10-HaTx1 product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.



FIG. 29H shows a MALDI mass spectral analysis of the final sample of Fc-L10-HaTx1 analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.



FIG. 30A shows Fc-L10-ShK[1-35] purified from CHO cells produces a concentration dependent block of the outward potassium current recorded from HEK293 cell stably expressing the human Kv1.3 channel.



FIG. 30B shows the time course of potassium current block by Fc-L10-ShK[1-35] at various concentrations. The IC50 was estimated to be 15±2 pM (n=4 cells).



FIG. 30C shows synthetic ShK[1-35] (also referred to as “ShK” alone) produces a concentration dependent block of the outward potassium current recorded from HEK293 cell stably expressing human Kv1.3 channel.



FIG. 30D shows the time course of ShK[1-35] block at various concentrations. The IC50 for ShK was estimated to be 12±1 pM (n=4 cells).



FIG. 31A shows synthetic peptide analog ShK[2-35] producing a concentration dependent block of the outward potassium current as recorded from HEK293 cells stably expressing human Kv1.3 channel with an IC50 of 49±5 pM (n=3 cells).



FIG. 31B shows the CHO-derived Fc-L10-ShK[2-35] peptibody producing a concentration dependent block of the outward potassium current as recorded from HEK293 cell stably expressing human Kv1.3 channel with an IC50 of 115±18 pM (n=3 cells).



FIG. 31C shows the Fc-L5-ShK[2-35] peptibody produces a concentration dependent block of the outward potassium current recorded from HEK293 cell stably expressing human Kv1.3 channel with an IC50 of 100 pM (n=3 cells).



FIG. 32A shows Fc-L-KTX1 peptibody purified from bacterial cells producing a concentration dependent block of the outward potassium current as recorded from HEK293 cell stably expressing human Kv1.3 channel.



FIG. 32B shows the time course of potassium current block by Fc-L10-KTX1 at various concentrations.



FIG. 33 shows by immunohistochemistry that CHO-derived Fc-L10-ShK[1-35] peptibody stains HEK 293 cells stably transfected with human Kv1.3 (FIG. 33A), whereas untransfected HEK 293 cells are not stained with the peptibody (FIG. 33B).



FIG. 34 shows results of an enzyme-immunoassay using fixed HEK 293 cells stably transfected with human Kv1.3. FIG. 34A shows the CHO-derived Fc-L10-ShK[1-35] (referred to here simply as “Fc-L10-ShK”) peptibody shows a dose-dependent increase in response, whereas the CHO-Fc control (“Fc control”) does not. FIG. 34B shows the Fc-L10-ShK[1-35] peptibody (referred to here as “Fc-ShK”) does not elicit a response from untransfected HEK 293 cells using similar conditions and also shows other negative controls.



FIG. 35 shows the CHO-derived Fc-L10-ShK[1-35] peptibody shows a dose-dependent inhibition of IL-2 (FIG. 35A) and IFNγ (FIG. 35B) production from PMA and αCD3 antibody stimulated human PBMCs. The peptibody shows a novel pharmacology exhibiting a complete inhibition of the response, whereas the synthetic ShK[1-35] peptide alone shows only a partial inhibition.



FIG. 36 shows the mammalian-derived Fc-L10-ShK[1-35] peptibody inhibits T cell proliferation (3H-thymidine incorporation) in human PBMCs from two normal donors stimulated with antibodies to CD3 and CD28. FIG. 36A shows the response of donor 1 and FIG. 36B the response of donor 2. Pre-incubation with the anti-CD32 (FcgRII) blocking antibody did not alter the sensitivity toward the peptibody.



FIG. 37 shows the purified CHO-derived Fc-L10-ShK[1-35] peptibody causes a dose-dependent inhibition of IL-2 (FIG. 37A) and IFNγ (FIG. 37B) production from αCD3 and αCD28 antibody stimulated human PBMCs.



FIG. 38A shows the PEGylated ShK[2-35] synthetic peptide produces a concentration dependent block of the outward potassium current recorded from HEK293 cell stably expressing human Kv1.3 channel and the time course of potassium current block at various concentrations is shown in FIG. 38B.



FIG. 39A shows a spectral scan of 50 μl of the Fc-L10-ShK(1-35) product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.



FIG. 39B shows a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final Fc-L10-ShK(1-35) product. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.



FIG. 39C shows the Size exclusion chromatography on 50 μg of the final Fc-L10-ShK(1-35) product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.



FIG. 40A shows a Spectral scan of 20 μl of the Fc-L10-ShK(2-35) product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.



FIG. 40B shows a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final Fc-L10-ShK(2-35) product. Lanes 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.



FIG. 40C shows the size exclusion chromatography on 50 μg of the final Fc-L10-ShK(2-35) product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.



FIG. 40D shows a MALDI mass spectral analysis of the final sample of Fc-L10-ShK(2-35) analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.



FIG. 41A shows spectral scan of 50 μl of the Fc-L10-OSK1 product diluted in 700 μl Formulation Buffer using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.



FIG. 41B shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final Fc-L10-OSK1 product. Lanes 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.



FIG. 41C shows size exclusion chromatography on 123 μg of the final Fc-L10-OSK1 product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.



FIG. 41D shows liquid chromatography—mass spectral analysis of approximately 4 μg of the final Fc-L110-OSK1 sample using a Vydac C4 column with part of the effluent directed into a LCQ ion trap mass spectrometer. The mass spectrum was deconvoluted using the Bioworks software provided by the mass spectrometer manufacturer.



FIG. 42A-B shows nucleotide and amino acid sequences (SEQ ID NO: 1040 and SEQ ID NO: 1041, respectively) of Fc-L10-OSK1.



FIG. 43A-B shows nucleotide and amino acid sequences (SEQ ID NO: 1042 and SEQ ID NO: 1043, respectively) of Fc-L10-OSK1[K7S].



FIG. 44A-B shows nucleotide and amino acid sequences (SEQ ID NO: 1044 and SEQ ID NO: 1045, respectively) of Fc-L10-OSK1[E16K,K20D].



FIG. 45A-B shows nucleotide and amino acid sequences (SEQ ID NO: 1046 and SEQ ID NO: 1047, respectively) of Fc-L10-OSK1[K7S,E16K,K20D].



FIG. 46 shows a Western blot (from tris-glycine 4-20% SDS-PAGE) with anti-human Fc antibodies. Lanes 1-6 were loaded as follows: 15 μl of Fc-L10-OSK1[K7S,E16K,K20D]; 15 μl of Fc-L10-OSK1[E16K,K20D]; 15 μl of Fc-L10-OSK1[K7S]; 15 μl of Fc-L10-OSK1; 15 μl of “No DNA” control; molecular weight markers.



FIG. 47 shows a Western blot (from tris-glycine 4-20% SDS-PAGE) with anti-human Fc antibodies. Lanes 1-5 were loaded as follows: 21 of Fc-L10-OSK1; 5 μl of Fc-L10-OSK1; 10 μl of Fc-L10-OSK1; 20 ng Human IgG standard; molecular weight markers.



FIG. 48 shows a Western blot (from tris-glycine 4-20% SDS-PAGE) with anti-human Fc antibodies. Lanes 1-13 were loaded as follows: 20 ng Human IgG standard; D1; C3; C2; B6; B5; B2; B1; A6; A5; A4; A3; A2 (5 μl of clone-conditioned medium loaded in lanes 2-13).



FIG. 49A shows a spectral scan of 50 μl of the Fc-L10-OSK1 product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.



FIG. 49B shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final Fc-L10-OSK1 product. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.



FIG. 49C shows Size exclusion chromatography on 149 μg of the final Fc-L10-OSK1 product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.



FIG. 49D shows MALDI mass spectral analysis of the final sample of Fc-L10-OsK1 analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.



FIG. 50A shows a spectral scan of 50 μl of the Fc-L10-OsK1(K7S) product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.



FIG. 50B shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final Fc-L10-OsK1(K7S) product. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.



FIG. 50C shows size exclusion chromatography on 50 μg of the final Fc-L10-OsK1(K7S) product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.



FIG. 50D shows MALDI mass spectral analysis of a sample of the final product Fc-L10-OsK1 (K7S) analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.



FIG. 51A shows a spectral scan of 50 μl of the Fc-L10-OsK1(E16K, K20D) product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.



FIG. 51B shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final Fc-L10-OsK1(E16K, K20D) product. Lane 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.



FIG. 51C shows size exclusion chromatography on 50 μg of the final Fc-L10-OsK1(E16K, K20D) product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.



FIG. 51D shows MALDI mass spectral analysis of a sample of the final product Fc-L10-OsK1 (E16K, K20D) analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.



FIG. 52A shows a spectral scan of 50 μl of the Fc-L110-OsK1 (K7S, E16K, K20D) product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.



FIG. 52B shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final Fc-L10-OsK1(K7S, E16K, K20D) product. Lanes 1-12 are loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.



FIG. 52C shows size exclusion chromatography on 50 μg of the final Fc-L10-OsK1(K7S, E16K, K20D) product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.



FIG. 52D shows MALDI mass spectral analysis of a sample of the final product Fc-L10-OsK1(K7S, E16K, K20D) analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.



FIG. 53 shows inhibition of the outward potassium current recorded from HEK293 cell stably expressing human Kv1.3 channel by synthetic Osk1, a 38-residue toxin peptide of the Asian scorpion Orthochirus scrobiculosus venom. FIG. 53A shows a concentration dependent block of the outward potassium current recorded from HEK293 cell stably expressing human Kv1.3 channel by the synthetic Osk1 toxin peptide. FIG. 53B shows the time course of the synthetic Osk1 toxin peptide block at various concentrations. The IC50 for the synthetic Osk1 toxin peptide was estimated to be 39±12 pM (n=4 cells).



FIG. 54 shows that modification of the synthetic OSK1 toxin peptide by fusion to the Fc-fragment of an antibody (OSK1-peptibody) retained the inhibitory activity against the human Kv1.3 channel. FIG. 54A shows a concentration dependent block of the outward potassium current recorded from HEK293 cells stably expressing human Kv1.3 channel by OSK1 linked to a human IgG1 Fc-fragment with a linker chain length of 10 amino acid residues (Fc-L10-OSK1). The fusion construct was stably expressed in Chinese Hamster Ovarian (CHO) cells. FIG. 54B shows the time course of the Fc-L10-OSK1 block at various concentrations. The IC50 for Fc-L10-OSK1 was estimated to be 198±35 pM (n=6 cells), approximately 5-fold less potent than the synthetic OSK1 toxin peptide.



FIG. 55 shows that a single amino-acid residue substitution of the OSK1-peptibody retained the inhibitory activity against the human Kv1.3 channel. FIG. 55A shows a concentration dependent block of the outward potassium current recorded from HEK293 cell stably expressing human Kv1.3 channel by OSK1-peptibody with a single amino acid substitution (lysine to serine at the 7th position from N-terminal, [K7S]) and linked to a human IgG1 Fc-fragment with a linker chain length of 10 amino acid residues (Fc-L10-OSK1[K7S]). The fusion construct was stably expressed in Chinese Hamster Ovarian (CHO) cells. FIG. 55B shows the time course of potassium current block by Fc-L10-OSK1[K7S] at various concentrations. The IC50 was estimated to be 372±71 pM (n=4 cells), approximately 10-fold less potent than the synthetic OSK1 toxin peptide.



FIG. 56 shows that a two amino-acid residue substitution of the OSK1-peptibody retained the inhibitory activity against the human Kv1.3 channel. FIG. 56A shows a concentration dependent block of the outward potassium current recorded from HEK293 cell stably expressing human Kv1.3 channel by OSK1-peptibody with two amino acid substitutions (glutamic acid to lysine and lysine to aspartic acid at the 16th and 20th position from N-terminal respectively, [E16KK20D]) and linked to a human IgG1 Fc-fragment with a linker chain length of 10 amino acid residues (Fc-L10-OSK1[E16KK20D]). The fusion construct was stably expressed in Chinese Hamster Ovarian (CHO) cells. FIG. 56B shows the time course of potassium current block by Fc-L10-OSK1[E16KK20D] at various concentrations. The IC50 was estimated to be 248±63 pM (n=3 cells), approximately 6-fold less potent than the synthetic OSK1 toxin peptide.



FIG. 57 shows that a triple amino-acid residue substitution of the OSK1-peptibody retained the inhibitory activity against the human Kv1.3 channel, but the potency of inhibition was significantly reduced when compared to the synthetic OSK1 toxin peptide. FIG. 57A shows a concentration dependent block of the outward potassium current recorded from HEK293 cell stably expressing human Kv1.3 channel by OSK1-peptibody with triple amino acid substitutions (lysine to serine, glutamic acid to lysine and lysine to aspartic acid at the 7th, 16th and 20th position from N-terminal respectively, [K7SE16KK20D]) and linked to a human IgG1 Fc-fragment with a linker chain length of 10 amino acid residues (Fc-L10-OSK1[K7SE16KK20D]). The fusion construct was stably expressed in Chinese Hamster Ovarian (CHO) cells. FIG. 57B shows the time course of potassium current block by Fc-L10-OSK1[K7SE16KK20D] at various concentrations. The IC50 was estimated to be 812±84 pM (n=3 cells), approximately 21-fold less potent than the synthetic OSK1 toxin peptide.



FIG. 58 shows Standard curves for ShK (FIG. 58A) and 20K PEG-ShK[1-35] (FIG. 58B) containing linear regression equations for each Standard at a given percentage of serum.



FIG. 59 shows the pharmacokinetic profile in rats of 20K PEG ShK[1-35] molecule after IV injection.



FIG. 60 shows Kv1.3 inhibitory activity in serum samples (5%) of rats receiving a single equal molar IV injection of Kv1.3 inhibitors ShK versus 20K PEG-ShK[1-35].



FIG. 61 illustrates an Adoptive Transfer EAE model experimental design (n=5 rats per treatment group). Dosing values in microgram per kilogram (mg/kg) are based on peptide content.



FIG. 62 shows that treatment with PEG-ShK ameliorated disease in rats in the adoptive transfer EAE model. Clinical scoring: 0=No signs, 0.5=distal limp tail, 1.0=limp tail, 2.0=mild paraparesis, ataxia, 3.0=moderate paraparesis, 3.5=one hind leg paralysis, 4.0=complete hind leg paralysis, 5.0=complete hind leg paralysis and incontinence, 5.5=tetraplegia, 6.0=moribund state or death. Rats reaching a score of 5.5 to 6 died or were euthanized. Mean±sem values are shown. (n=5 rats per treatment group.)



FIG. 63 shows that treatment with PEG-ShK prevented loss of body weight in the adoptive transfer EAE model. Rats were weighed on days −1, 4, 6, and 8 (for surviving rats). Mean±sem values are shown.



FIG. 64 shows that thapsigargin-induced IL-2 production in human whole blood was suppressed by the Kv1.3 channel inhibitors ShK[1-35] and Fc-L10-ShK[2-35]. The calcineurin inhibitor cyclosporine A also blocked the response. The BKCa channel inhibitor iberiotoxin (IbTx) showed no significant activity. The response of whole blood from two separate donors is shown in FIG. 64A and FIG. 64B.



FIG. 65 shows that thapsigargin-induced IFN-g production in human whole blood was suppressed by the Kv1.3 channel inhibitors ShK[1-35] and Fc-L10-ShK[2-35]. The calcineurin inhibitor cyclosporine A also blocked the response. The BKCa channel inhibitor iberiotoxin (IbTx) showed no significant activity. The response of whole blood from two separate donors is shown in FIG. 65A and FIG. 65B.



FIG. 66 shows that thapsigargin-induced upregulation of CD40L on T cells in human whole blood was suppressed by the Kv1.3 channel inhibitors ShK[1-35] and Fc-L10-ShK[1-35] (Fc-ShK). The calcineurin inhibitor cyclosporine A (CsA) also blocked the response. FIG. 66A shows results of an experiment looking at the response of total CD4+ T cells. FIG. 66B shows results of an experiment that looked at total CD4+ T cells, as well as CD4+CD45+ and CD4+CD45-T cells. In FIG. 66B, the BKCa channel inhibitor iberiotoxin (IbTx) and the Kv1.1 channel inhibitor dendrotoxin-K (DTX-K) showed no significant activity.



FIG. 67 shows that thapsigargin-induced upregulation of the IL-2R on T cells in human whole blood was suppressed by the Kv1.3 channel inhibitors ShK[1-35] and Fc-L10-ShK[1-35] (Fc-ShK). The calcineurin inhibitor cyclosporine A (CsA) also blocked the response. FIG. 67A shows results of an experiment looking at the response of total CD4+ T cells. FIG. 67B shows results of an experiment that looked at total CD4+ T cells, as well as CD4+CD45+ and CD4+CD45-T cells. In FIG. 67B, the BKCa channel inhibitor iberiotoxin (IbTx) and the Kv1.1 channel inhibitor dendrotoxin-K (DTX-K) showed no significant activity.



FIG. 68 shows cation exchange chromatograms of PEG-peptide purification on SP Sepharose HP columns for PEG-Shk purification (FIG. 68A) and PEG-OSK-1 purification (FIG. 68B).



FIG. 69 shows RP-HPLC chromatograms on final PEG-peptide pools to demonstrate purity of PEG-Shk purity >99% (FIG. 69A) and PEG-Osk1 purity >97% (FIG. 69B).



FIG. 70 shows the amino acid sequence (SEQ ID NO: 976) of an exemplary FcLoop-L2-OsK1-L2 having three linked domains: Fc N-terminal domain (amino acid residues 1-139); OsK1 (underlined amino acid residues 142-179); and Fc C-terminal domain (amino acid residues 182-270).



FIG. 71 shows the amino acid sequence (SEQ ID NO: 977) of an exemplary FcLoop-L2-ShK-L2 having three linked domains: Fc N-terminal domain (amino acid residues 1-139); ShK (underlined amino acid residues 142-176); and Fc C-terminal domain (amino acid residues 179-267).



FIG. 72 shows the amino acid sequence (SEQ ID NO: 978) of an exemplary FcLoop-L2-ShK-L4 having three linked domains: Fc N-terminal domain (amino acid residues 1-139); ShK (underlined amino acid residues 142-176); and Fc C-terminal domain (amino acid residues 181-269).



FIG. 73 shows the amino acid sequence (SEQ ID NO: 979) of an exemplary FcLoop-L4-OsK1-L2 having three linked domains: Fc N-terminal domain (amino acid residues 1-139); OsK1(underlined amino acid residues 144-181); and Fc C-terminal domain (amino acid residues 184-272).



FIG. 74 shows that the 20K PEGylated ShK[1-35] provided potent blockade of human Kv1.3 as determined by whole cell patch clamp electrophysiology on HEK293/Kv1.3 cells. The data represents blockade of peak current.



FIG. 75 shows schematic structures of some other exemplary embodiments of the composition of matter of the invention. “X2” and “X3” represent toxin peptides or linker-toxin peptide combinations (i.e., -(L)f-P-(L)g-) as defined herein. As described herein but not shown in FIG. 75, an additional X1 domain and one or more additional PEG moieties are also encompassed in other embodiments. The specific embodiments shown here are as follows:



FIG. 75C, FIG. 75D, FIG. 75G and FIG. 75H: show a single chain molecule and can also represent the DNA construct for the molecule.



FIG. 75A, FIG. 75B, FIG. 75E and FIG. 75F: show doubly disulfide-bonded Fc dimers (in position F2); FIG. 75A and FIG. 75B show a dimer having the toxin peptide portion on both chains in position X3; FIG. 75E and FIG. 75F show a dimer having the toxin peptide portion on both chains In position X2.



FIG. 76A shows a spectral scan of 50 μl of the ShK[2-35]-Fc product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.



FIG. 76B shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final ShK[2-35]-Fc product. Lanes 1-12 were loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.



FIG. 76C shows size exclusion chromatography on 70 μg of the final ShK[2-35]-Fc product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.



FIG. 76D shows LC-MS analysis of the final ShK[2-35]-Fc sample using an Agilent 1100 HPCL running reverse phase chromatography, with the column effluent directly coupled to an electrospray source of a Thermo Finnigan LCQ ion trap mass spectrometer. Relevant spectra were summed and deconvoluted to mass data with the Bioworks software package.



FIG. 77A shows a spectral scan of 20 μl of the met-ShK[1-35]-Fc product diluted in 700 μl PBS (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.



FIG. 77B shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final met-ShK[1-35]-Fc product. Lanes 1-12 were loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, Novex Mark12 wide range protein standards, 0.5 μg product reduced, blank, 2.0 μg product reduced, blank, and 10 μg product reduced.



FIG. 77C shows size exclusion chromatography on 93 μg of the final met-ShK[1-35]-Fc product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.



FIG. 77D shows MALDI mass spectral analysis of the final met-ShK[1-35]-Fc sample analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses.



FIG. 78 shows a spectral scan of 10 μl of the CH2-OSK1 fusion protein product diluted in 150 μl water (blanking buffer) using a Hewlett Packard 8453 spectrophotometer and a 1 cm path length quartz cuvette.



FIG. 79 shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final CH2-OSK1 fusion protein product. Lane 1-7 were loaded as follows: Novex Mark12 wide range protein standards, 0.5 μg product non-reduced, blank, 2.0 μg product non-reduced, blank, 10 μg product non-reduced, and Novex Mark12 wide range protein standards.



FIG. 80 shows size exclusion chromatography on 50 μg of the final CH2-OSK1 fusion protein product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm.



FIG. 81 shows liquid chromatography—mass spectral analysis of the CH2-OSK1 fusion protein sample using a Vydac C4 column with part of the effluent directed into a LCQ ion trap mass spectrometer. The mass spectrum was deconvoluted using the Bioworks software provided by the mass spectrometer manufacturer.



FIG. 82 shows cation exchange chromatogram of PEG-CH2-OSK1 reaction mixture. Vertical lines delineate fractions pooled to obtain mono-PEGylated CH2-OSK1.



FIG. 83 shows Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE of the final PEGylated CH2-OSK1 pool. Lane 1-2 were loaded as follows: Novex Mark12 wide range protein standards, 2.0 μg product non-reduced.



FIG. 84 shows whole cell patch clamp (WCPC) and PatchXpress (PX) electrophysiology comparing the activity of OSK1[Ala-12] (SEQ ID No:1410) on human Kv1.3 and human Kv1.1 heterologously overexpressed on CHO and HEK293 cells, respectively. The table summarizes the calculated IC50 values and the plots show the individual traces of the impact of various concentrations of analog on the relative Kv1.3 or Kv1.1 current (percent of control, POC).



FIG. 85 shows whole cell patch clamp electrophysiology comparing the activity of OSK1[Ala-29] (SEQ ID No:1424) on human Kv1.3 and human Kv1.1 heterologously overexpressed on CHO and HEK293 cells, respectively. Concentration response curves of OSK1[Ala-29] on CHO/Kv1.3 (circle, square and diamond, IC50=0.033 nM, n=3) and on HEK/Kv1.1 (filled triangle, IC50=2.7 nM, n=1).



FIG. 86 shows a dose-response curve for OSK1[Ala-29] (SEQ ID No:1424) against human Kv1.3 (CHO) (panel A) and human Kv1.1 (HEK293) (panel B) as determined by high-throughput 384-well planar patch clamp electrophysiology using the IonWorks Quattro system.



FIG. 87A-B show Western blots of Tris-glycine 4-20% SDS-PAGE (FIG. 87A with longer exposure time and FIG. 87B with shorter exposure time) of a monovalent dimeric Fc-L-ShK(2-35) molecule product expressed by and released into the conditioned media from mammalian cells transiently transfected with pTT5-Fc-Fc-L10-Shk(2-35), which was sampled after the indicated number of days. Lanes 3-8 were loaded with 20 μL of conditioned medium per lane. The immunoblot was probed with anti-human IgG-Fc-HRP (Pierce). The lanes were loaded as follows: 1) MW Markers; 2) purified Fc-L10-ShK(2-35), 10 ng; 3) 293-6E-HD (5-day); 4) 293-6E-HD (6-day); 5) 293-6E-PEI (5-day); 6) 293-6E-PEI (6-day); 7) CHO-S (5-day); 8) CHO-S (6-day). Four bands were expected in the reduced gel: Linker-Fc-Shk(2-35) (one cut at 3′ furin site; predicted MW: 33.4 kDa); Fc-ShK(2-35) (both furin sites cut; predicted MW: 30.4 kDa); Fc-linker (one cut at 5′ furin site; predicted MW: 29.1 kDa); Fc (both furin sites cut; predicted MW: 25.8 kDa). Further mass spec or amino acid sequence analysis of the individual bands is needed to identify these bands and their relative ratios.



FIG. 88 shows a western blot of serum samples from a pharmacokinetic study on monovalent dimeric Fc-ShK(1-35) in SD rats. Various times (0.083-168 hours) after a single 1 mg/kg intravenous injection of monovalent dimeric Fc-L10-ShK(1-35) (see, Example 2), blood was drawn, and serum was collected. A Costar EIA/RIA 96 well plate was coated with 2 μg/ml polyclonal goat anti-human Fc antibody overnight at 4° C. Capture antibody was removed and the plate was washed with PBST and then blocked with Blotto. After the plate was washed, serum samples diluted in PBST/0.1% BSA were added. Binding was allowed to occur at room temperature for several hours, and then the plate was again washed. Samples were eluted from the plate with reducing Laemmle buffer, heated, then run on SDS-Page gels. Run in an adjacent lane (“5 ng Control”) of the gel as a standard was 5 ng of the purified monovalent dimeric Fc-L10-ShK(1-35) fusion protein used in the pharmacokinetic study. Proteins were transferred to PVDF membranes by western blot. Membranes were blocked with Blotto followed by incubation with goat anti-Human Fc-HRP conjugated antibody. After the membranes were washed, signal was detected via chemiluminescence using a CCD camera.



FIG. 89 shows the NMR solution structure of OSK1 and sites identified by analoging to be important for Kv1.3 activity and selectivity. Space filling structures are shown in FIGS. 89A, 89B and 89D. The color rendering in FIG. 89A depicts amino acid charge. In FIG. 89B, several key OSK1 amino acid residues found to be important for Kv1.3 activity (Tables 37-40) are lightly shaded and labeled Phe25, Gly26, Lys27, Met29 and Asn30. In FIG. 89D residues Ser11, Met29 and His34 are labeled. Some analogues of these residues were found to result in improved Kv1.3 selectivity over Kv1.1 (Tables 41). FIG. 89C shows the three beta strands and single alpha helix of OSK1. The amino acid sequence of native OSK1 (SEQ ID No: 25) is shown in FIG. 89E, with residues forming the molecules beta strands (β1, β2, β3) and alpha helix (al) underlined. The OSK1 structures shown were derived from PDB:1SCO, and were rendered using Cn3D vers4.1.



FIG. 90A-D illustrates that toxin peptide inhibitors of Kv1.3 provide potent blockade of the whole blood inflammatory response. The activity of the calcineurin inhibitor cyclosporin A (FIG. 90A) and Kv1.3 peptide inhibitors ShK-Ala22 (FIG. 90B; SEQ ID No: 123), OSK1-Ala29 (FIG. 90C; SEQ ID No: 1424) and OSK1-Ala12 (FIG. 90D; SEQ ID No: 1410) were compared in the whole blood assay of inflammation (Example 46) using the same donor blood sample. The potency (IC50) of each molecule is shown, where for each panel the left curve is the impact on IL-2 production and the right curve is the impact on IFNγ production.



FIG. 91A-B shows an immunoblot analysis of expression of monovalent dimeric IgG1-Fc-L-ShK(2-35) from non-reduced SDS-PAGE. FIG. 91A shows detection of human Fc expression with goat anti-human IgG (H+L)-HRP. FIG. 91B shows detection of ShK(2-35) expression with a goat anti-mouse IgG (H+L)-HRP that cross reacts with human IgG. Lane 1: purified Fc-L10-Shk(2-35); Lane 2: conditioned medium from 293EBNA cells transiently transfected with pTT5-huIgG1+pTT5-hKappa+pCMVi-Fc-L10-ShK(2-35); Lane 3: conditioned media from 293EBNA cells transiently transfected with pTT5-huIgG2+pTT5-hKappa+pCMVi-Fc-L10-Shk(2-35); Lane 4: conditioned media from 293EBNA cells transiently transfected with pTT14 vector alone. The two arrows point to the full length huIgG (mol. wt.˜150 kDa) and monovalent dimeric huIgG-FcShK(2-35) (mol. wt.˜100 kDa); the abundant 60-kDa band is the bivalent dimeric Fc-ShK(2-35).



FIG. 92A-C shows schematic representations of an embodiment of a monovalent “hemibody”-toxin peptide fusion protein construct; the single toxin peptide is represented by an oval. FIG. 92A, which can also represent the DNA construct for the fusion protein, represents an immunoglobulin light chain (LC, open rectangle), an immunoglobulin heavy chain (HC, longer cross-hatched rectangle), and an immunoglobulin Fc domain (Fc, shorter cross-hatched rectangle), each separated by an intervening peptidyl linker sequence (thick lines) comprising at least one protease cleavage site (arrows), e.g., a furin cleavage site. FIG. 92 illustrates the association of the recombinantly expressed LC, HC, and Fc-toxin peptide components connected by the peptidyl linker sequences (thick lines) and, in FIG. 92C, the final monovalent chimeric immunoglobulin (LC+HC)-Fc (i.e., “hemibody”)-toxin peptide fusion protein after cleavage (intracellularly or extracellularly) at the protease cleavage sites, to release the linkers, and formation of disulfide bridges between the light and heavy chains and between the heavy chain and the Fc components (shown as thin horizontal lines between the LC, HC, and Fc components in FIG. 92C).





DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION
Definition of Terms

The terms used throughout this specification are defined as follows, unless otherwise limited in specific instances. As used in the specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.


“Polypeptide” and “protein” are used interchangeably herein and include a molecular chain of two or more amino acids linked through peptide bonds. The terms do not refer to a specific length of the product. Thus, “peptides,” and “oligopeptides,” are included within the definition of polypeptide. The terms include post-translational modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. In addition, protein fragments, analogs, mutated or variant proteins, fusion proteins and the like are included within the meaning of polypeptide. The terms also include molecules in which one or more amino acid analogs or non-canonical or unnatural amino acids are included as can be synthesized, or expressed recombinantly using known protein engineering techniques. In addition, inventive fusion proteins can be derivatized as described herein by well-known organic chemistry techniques.


The term “fusion protein” indicates that the protein includes polypeptide components derived from more than one parental protein or polypeptide. Typically, a fusion protein is expressed from a fusion gene in which a nucleotide sequence encoding a polypeptide sequence from one protein is appended in frame with, and optionally separated by a linker from, a nucleotide sequence encoding a polypeptide sequence from a different protein. The fusion gene can then be expressed by a recombinant host cell as a single protein.


A “domain” of a protein is any portion of the entire protein, up to and including the complete protein, but typically comprising less than the complete protein. A domain can, but need not, fold independently of the rest of the protein chain and/or be correlated with a particular biological, biochemical, or structural function or location (e.g., a ligand binding domain, or a cytosolic, transmembrane or extracellular domain).


A “secreted” protein refers to those proteins capable of being directed to the ER, secretory vesicles, or the extracellular space as a result of a secretory signal peptide sequence, as well as those proteins released into the extracellular space without necessarily containing a signal sequence. If the secreted protein is released into the extracellular space, the secreted protein can undergo extracellular processing to produce a “mature” protein. Release into the extracellular space can occur by many mechanisms, including exocytosis and proteolytic cleavage.


The term “signal peptide” refers to a relatively short (3-60 amino acid residues long) peptide chain that directs the post-translational transport of a protein, e.g., its export to the extracellular space. Thus, secretory signal peptides are encompassed by “signal peptide”. Signal peptides may also be called targeting signals, signal sequences, transit peptides, or localization signals.


The term “recombinant” indicates that the material (e.g., a nucleic acid or a polypeptide) has been artificially or synthetically (i.e., non-naturally) altered by human intervention. The alteration can be performed on the material within, or removed from, its natural environment or state. For example, a “recombinant nucleic acid” is one that is made by recombining nucleic acids, e.g., during cloning, DNA shuffling or other well known molecular biological procedures. A “recombinant DNA molecule,” is comprised of segments of DNA joined together by means of such molecular biological techniques. The term “recombinant protein” or “recombinant polypeptide” as used herein refers to a protein molecule which is expressed using a recombinant DNA molecule. A “recombinant host cell” is a cell that contains and/or expresses a recombinant nucleic acid.


A “polynucleotide sequence” or “nucleotide sequence” or “nucleic acid sequence,” as used interchangeably herein, is a polymer of nucleotides, including an oligonucleotide, a DNA, and RNA, a nucleic acid, or a character string representing a nucleotide polymer, depending on context. From any specified polynucleotide sequence, either the given nucleic acid or the complementary polynucleotide sequence can be determined. Included are DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense or antisense strand.


As used herein, the terms “nucleic acid molecule encoding,” “DNA sequence encoding,” and “DNA encoding” refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of ribonucleotides along the mRNA chain, and also determines the order of amino acids along the polypeptide (protein) chain. The DNA sequence thus codes for the RNA sequence and for the amino acid sequence.


“Expression of a gene” or “expression of a nucleic acid” means transcription of DNA into RNA (optionally including modification of the RNA, e.g., splicing), translation of RNA into a polypeptide (possibly including subsequent post-translational modification of the polypeptide), or both transcription and translation, as indicated by the context.


The term “gene” is used broadly to refer to any nucleic acid associated with a biological function. Genes typically include coding sequences and/or the regulatory sequences required for expression of such coding sequences. The term “gene” applies to a specific genomic or recombinant sequence, as well as to a cDNA or mRNA encoded by that sequence. A “fusion gene” contains a coding region that encodes a fusion protein. Genes also include non-expressed nucleic acid segments that, for example, form recognition sequences for other proteins. Non-expressed regulatory sequences including transcriptional control elements to which regulatory proteins, such as transcription factors, bind, resulting in transcription of adjacent or nearby sequences.


As used herein the term “coding region” when used in reference to a structural gene refers to the nucleotide sequences which encode the amino acids found in the nascent polypeptide as a result of translation of an mRNA molecule. The coding region is bounded, in eukaryotes, on the 5′ side by the nucleotide triplet “ATG” which encodes the initiator methionine and on the 3′ side by one of the three triplets which specify stop codons (i.e., TAA, TAG, TGA).


Transcriptional control signals in eukaryotes comprise “promoter” and “enhancer” elements. Promoters and enhancers consist of short arrays of DNA sequences that interact specifically with cellular proteins involved in transcription (Maniatis, et al., Science 236:1237 (1987)). Promoter and enhancer elements have been isolated from a variety of eukaryotic sources including genes in yeast, insect and mammalian cells and viruses (analogous control elements, i.e., promoters, are also found in prokaryotes). The selection of a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range while others are functional in a limited subset of cell types (for review see Voss, et al., Trends Biochem. Sci., 11:287 (1986) and Maniatis, et al., Science 236:1237 (1987)).


The term “expression vector” as used herein refers to a recombinant DNA molecule containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably linked coding sequence in a particular host cell. Nucleic acid sequences necessary for expression in prokaryotes include a promoter, optionally an operator sequence, a ribosome binding site and possibly other sequences. Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals. A secretory signal peptide sequence can also, optionally, be encoded by the expression vector, operably linked to the coding sequence for the inventive recombinant fusion protein, so that the expressed fusion protein can be secreted by the recombinant host cell, for more facile isolation of the fusion protein from the cell, if desired. Such techniques are well known in the art. (E.g., Goodey, Andrew R.; et al., Peptide and DNA sequences, U.S. Pat. No. 5,302,697; Weiner et al., Compositions and methods for protein secretion, U.S. Pat. No. 6,022,952 and U.S. Pat. No. 6,335,178; Uemura et al., Protein expression vector and utilization thereof, U.S. Pat. No. 7,029,909; Ruben et al., 27 human secreted proteins, US 2003/0104400 A1).


The terms “in operable combination”, “in operable order” and “operably linked” as used herein refer to the linkage of nucleic acid sequences in such a manner or orientation that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced and/or transported.


Recombinant DNA- and/or RNA-mediated protein expression techniques, or any other methods of preparing peptides or, are applicable to the making of the inventive recombinant fusion proteins. For example, the peptides can be made in transformed host cells. Briefly, a recombinant DNA molecule, or construct, coding for the peptide is prepared. Methods of preparing such DNA molecules are well known in the art. For instance, sequences encoding the peptides can be excised from DNA using suitable restriction enzymes. Any of a large number of available and well-known host cells may be used in the practice of this invention. The selection of a particular host is dependent upon a number of factors recognized by the art. These include, for example, compatibility with the chosen expression vector, toxicity of the peptides encoded by the DNA molecule, rate of transformation, ease of recovery of the peptides, expression characteristics, bio-safety and costs. A balance of these factors must be struck with the understanding that not all hosts may be equally effective for the expression of a particular DNA sequence. Within these general guidelines, useful microbial host cells in culture include bacteria (such as Escherichia coli sp.), yeast (such as Saccharomyces sp.) and other fungal cells, insect cells, plant cells, mammalian (including human) cells, e.g., CHO cells and HEK293 cells. Modifications can be made at the DNA level, as well. The peptide-encoding DNA sequence may be changed to codons more compatible with the chosen host cell. For E. coli, optimized codons are known in the art. Codons can be substituted to eliminate restriction sites or to include silent restriction sites, which may aid in processing of the DNA in the selected host cell. Next, the transformed host is cultured and purified. Host cells may be cultured under conventional fermentation conditions so that the desired compounds are expressed. Such fermentation conditions are well known in the art.


The term “half-life extending moiety” (i.e., F1 or F2 in Formula I) refers to a pharmaceutically acceptable moiety, domain, or “vehicle” covalently linked (“conjugated”) to the toxin peptide directly or via a linker, that prevents or mitigates in vivo proteolytic degradation or other activity-diminishing chemical modification of the toxin peptide, increases half-life or other pharmacokinetic properties such as but not limited to increasing the rate of absorption, reduces toxicity, improves solubility, increases biological activity and/or target selectivity of the toxin peptide with respect to a target ion channel of interest, increases manufacturability, and/or reduces immunogenicity of the toxin peptide, compared to an unconjugated form of the toxin peptide.


By “PEGylated peptide” is meant a peptide or protein having a polyethylene glycol (PEG) moiety covalently bound to an amino acid residue of the peptide itself or to a peptidyl or non-peptidyl linker (including but not limited to aromatic or aryl linkers) that is covalently bound to a residue of the peptide.


By “polyethylene glycol” or “PEG” is meant a polyalkylene glycol compound or a derivative thereof, with or without coupling agents or derivatization with coupling or activating moieties (e.g., with aldehyde, hydroxysuccinimidyl, hydrazide, thiol, triflate, tresylate, azirdine, oxirane, orthopyridyl disulphide, vinylsulfone, iodoacetamide or a maleimide moiety). In accordance with the present invention, useful PEG includes substantially linear, straight chain PEG, branched PEG, or dendritic PEG. (See, e.g., Merrill, U.S. Pat. No. 5,171,264; Harris et al., Multiarmed, monofunctional, polymer for coupling to molecules and surfaces, U.S. Pat. No. 5,932,462; Shen, N-maleimidyl polymer derivatives, U.S. Pat. No. 6,602,498).


The term “peptibody” refers to molecules of Formula I in which F1 and/or F2 is an immunoglobulin Fc domain or a portion thereof, such as a CH2 domain of an Fc, or in which the toxin peptide is inserted into a human IgG1 Fc domain loop, such that F1 and F2 are each a portion of an Fc domain with a toxin peptide inserted between them (See, e.g., FIGS. 70-73 and Example 49 herein). Peptibodies of the present invention can also be PEGylated as described further herein, at either an Fc domain or portion thereof, or at the toxin peptide(s) portion of the inventive composition, or both.


The term “native Fc” refers to molecule or sequence comprising the sequence of a non-antigen-binding fragment resulting from digestion of whole antibody, whether in monomeric or multimeric form. The original immunoglobulin source of the native Fc is preferably of human origin and can be any of the immunoglobulins, although IgG1 or IgG2 are preferred. Native Fc's are made up of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent association. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4 depending on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgGA2). One example of a native Fc is a disulfide-bonded dimer resulting from papain digestion of an IgG (see Ellison et al. (1982), Nucleic Acids Res. 10: 4071-9). The term “native Fc” as used herein is generic to the monomeric, dimeric, and multimeric forms.


The term “Fc variant” refers to a molecule or sequence that is modified from a native Fc but still comprises a binding site for the salvage receptor, FcRn. Several published patent documents describe exemplary Fc variants, as well as interaction with the salvage receptor. See International Applications WO 97/34 631 (published 25 Sep. 1997; WO 96/32 478, corresponding to U.S. Pat. No. 6,096,891, issued Aug. 1, 2000, hereby incorporated by reference in its entirety; and WO 04/110 472. Thus, the term “Fc variant” includes a molecule or sequence that is humanized from a non-human native Fc. Furthermore, a native Fc comprises sites that can be removed because they provide structural features or biological activity that are not required for the fusion molecules of the present invention. Thus, the term “Fc variant” includes a molecule or sequence that lacks one or more native Fc sites or residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC). Fc variants are described in further detail hereinafter.


The term “Fc domain” encompasses native Fc and Fc variant molecules and sequences as defined above. As with Fc variants and native Fc's, the term “Fc domain” includes molecules in monomeric or multimeric form, whether digested from whole antibody or produced by other means.


The term “multimer” as applied to Fc domains or molecules comprising Fc domains refers to molecules having two or more polypeptide chains associated covalently, noncovalently, or by both covalent and non-covalent interactions. IgG molecules typically form dimers; IgM, pentamers; IgD, dimers; and IgA, monomers, dimers, trimers, or tetramers. One skilled in the art can form multimers by exploiting the sequence and resulting activity of the native Ig source of the Fc or by derivatizing (as defined below) such a native Fc.


The term “dimer” as applied to Fc domains or molecules comprising Fc domains refers to molecules having two polypeptide chains associated covalently or non-covalently. Thus, exemplary dimers within the scope of this invention are as shown in FIG. 2. A “monovalent dimeric” Fc-toxin peptide fusion, or “monovalent dimer”, is a Fc-toxin peptide fusion that includes a toxin peptide conjugated with only one of the dimerized Fc domains (e.g., as represented schematically in FIG. 2B). A “bivalent dimeric” Fc-toxin peptide fusion, or “bivalent dimer”, is a Fc-toxin peptide fusion having both of the dimerized Fc domains each conjugated separately with a toxin peptide (e.g., as represented schematically in FIG. 2C).


The terms “derivatizing” and “derivative” or “derivatized” comprise processes and resulting compounds respectively in which (1) the compound has a cyclic portion; for example, cross-linking between cysteinyl residues within the compound; (2) the compound is cross-linked or has a cross-linking site; for example, the compound has a cysteinyl residue and thus forms cross-linked dimers in culture or in vivo; (3) one or more peptidyl linkage is replaced by a non-peptidyl linkage; (4) the N-terminus is replaced by —NRR1, NRC(O)R1, —NRC(O)OR1, —NRS(O)2R1, —NHC(O)NHR, a succinimide group, or substituted or unsubstituted benzyloxycarbonyl-NH—, wherein R and R1 and the ring substituents are as defined hereinafter; (5) the C-terminus is replaced by —C(O)R2 or —NR3R4 wherein R2, R3 and R4 are as defined hereinafter; and (6) compounds in which individual amino acid moieties are modified through treatment with agents capable of reacting with selected side chains or terminal residues. Derivatives are further described hereinafter.


The term “peptide” refers to molecules of 2 to about 80 amino acid residues, with molecules of about 10 to about 60 amino acid residues preferred and those of about 30 to about 50 amino acid residues most preferred. Exemplary peptides can be randomly generated by any known method, carried in a peptide library (e.g., a phage display library), or derived by digestion of proteins. In any peptide portion of the inventive compositions, for example a toxin peptide or a peptide linker moiety described herein, additional amino acids can be included on either or both of the N- or C-termini of the given sequence. Of course, these additional amino acid residues should not significantly interfere with the functional activity of the composition.


“Toxin peptides” include peptides having the same amino acid sequence of a naturally occurring pharmacologically active peptide that can be isolated from a venom, and also include modified peptide analogs (spelling used interchangeably with “analogues”) of such naturally occurring molecules.


The term “peptide analog” refers to a peptide having a sequence that differs from a peptide sequence existing in nature by at least one amino acid residue substitution, internal addition, or internal deletion of at least one amino acid, and/or amino- or carboxy-terminal end truncations, or additions). An “internal deletion” refers to absence of an amino acid from a sequence existing in nature at a position other than the N- or C-terminus. Likewise, an “internal addition” refers to presence of an amino acid in a sequence existing in nature at a position other than the N- or C-terminus. “Toxin peptide analogs”, such as, but not limited to, an OSK1 peptide analog, ShK peptide analog, or ChTx peptide analog, contain modifications of a native toxin peptide sequence of interest (e.g., amino acid residue substitutions, internal additions or insertions, internal deletions, and/or amino- or carboxy-terminal end truncations, or additions as previously described above) relative to a native toxin peptide sequence of interest, which is in the case of OSK1: GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK (SEQ ID NO:25).


Examples of toxin peptides useful in practicing the present invention are listed in Tables 1-32. The toxin peptide (“P”, or equivalently shown as “P1” in FIG. 2) comprises at least two intrapeptide disulfide bonds, as shown, for example, in FIG. 9. Accordingly, this invention concerns molecules comprising:

    • a) C1-C3 and C2-C4 disulfide bonding in which C1, C2, C3, and C4 represent the order in which cysteine residues appear in the primary sequence of the toxin peptide stated conventionally with the N-terminus of the peptide on the left, with the first and third cysteines in the amino acid sequence forming a disulfide bond, and the second and fourth cysteines forming a disulfide bond. Examples of toxin peptides with such a C1-C3, C2-C4 disulfide bonding pattern include, but are not limited to, apamin peptides, α-conopeptides, PnIA peptides, PnIB peptides, and MII peptides, and analogs of any of the foregoing.
    • b) C1-C6, C2-C4 and C3-C5 disulfide bonding in which, as described above, C1, C2, C3, C4, C5 and C6 represent the order of cysteine residues appearing in the primary sequence of the toxin peptide stated conventionally with the N-terminus of the peptide(s) on the left, with the first and sixth cysteines in the amino acid sequence forming a disulfide bond, the second and fourth cysteines forming a disulfide bond, and the third and fifth cysteines forming a disulfide bond. Examples of toxin peptides with such a C1-C6, C2-C4, C3-C5 disulfide bonding pattern include, but are not limited to, ShK, BgK, HmK, AeKS, AsK, and DTX1, and analogs of any of the foregoing.
    • c) C1-C4, C2-C5 and C3-C6 disulfide bonding in which, as described above, C1, C2, C3, C4, C5 and C6 represent the order of cysteine residues appearing in the primary sequence of the toxin peptide stated conventionally with the N-terminus of the peptide(s) on the left, with the first and fourth cysteines in the amino acid sequence forming a disulfide bond, the second and fifth cysteines forming a disulfide bond, and the third and sixth cysteines forming a disulfide bond. Examples of toxin peptides with such a C1-C4, C2-C5, C3-C6 disulfide bonding pattern include, but are not limited to, ChTx, MgTx, OSK1, KTX1, AgTx2, Pi2, Pi3, NTX, HgTx1, BeKM1, BmKTX, P01, BmKK6, Tc32, Tc1, BmTx1, BmTX3, IbTx, P05, ScyTx, TsK, HaTx1, ProTX1, PaTX2, Ptu1, ωGVIA, ωMVIIA, and SmIIIa, and analogs of any of the foregoing.
    • d) C1-C5, C2-C6, C3-C7, and C4-C8 disulfide bonding in which C1, C2, C3, C4, C5, C6, C7 and C8 represent the order of cysteine residues appearing in the primary sequence of the toxin peptide stated conventionally with the N-terminus of the peptide(s) on the left, with the first and fifth cysteines in the amino acid sequence forming a disulfide bond, the second and sixth cysteines forming a disulfide bond, the third and seventh cysteines forming a disulfide bond, and the fourth and eighth cysteines forming a disulfide bond. Examples of toxin peptides with such a C1-C5, C2-C6, C3-C7, C4-C8 disulfide bonding pattern include, but are not limited to, Anuoroctoxin (AnTx), Pi1, HsTx1, MTX (P12A, P20A), and Pi4 peptides, and analogs of any of the foregoing.
    • e) C1-C4, C2-C6, C3-C7, and C5-C8 disulfide bonding in which C7, C2, C3, C4, C5, C6, C7 and C8 represent the order of cysteine residues appearing in the primary sequence of the toxin peptide stated conventionally with the N-terminus of the peptide(s) on the left, with the first and fourth cysteines in the amino acid sequence forming a disulfide bond, the second and sixth cysteines forming a disulfide bond, the third and seventh cysteines forming a disulfide bond, and the fifth and eighth cysteines forming a disulfide bond. Examples of toxin peptides with such a C1-C4, C2-C6, C3-C7, C5-C8 disulfide bonding pattern include, but are not limited to, Chlorotoxin, Bm-12b, and, and analogs of either.
    • f) C1-C5, C2-C6, C3-C4, and C7-C8 disulfide bonding in which C1, C2, C3, C4, C5, C6, C7 and C8 represent the order of cysteine residues appearing in the primary sequence of the toxin peptide stated conventionally with the N-terminus of the peptide(s) on the left, with the first and fifth cysteines in the amino acid sequence forming a disulfide bond, the second and sixth cysteines forming a disulfide bond, the third and fourth cysteines forming a disulfide bond, and the seventh and eighth cysteines forming a disulfide bond. Examples of toxin peptides with such a C1-C5, C2-C6, C3-C4, C7-C8 disulfide bonding pattern include, but are not limited to, Maurotoxin peptides and analogs thereof.


The term “randomized” as used to refer to peptide sequences refers to fully random sequences (e.g., selected by phage display methods) and sequences in which one or more residues of a naturally occurring molecule is replaced by an amino acid residue not appearing in that position in the naturally occurring molecule. Exemplary methods for identifying peptide sequences include phage display, E. coli display, ribosome display, yeast-based screening, RNA-peptide screening, chemical screening, rational design, protein structural analysis, and the like.


The term “pharmacologically active” means that a substance so described is determined to have activity that affects a medical parameter (e.g., blood pressure, blood cell count, cholesterol level) or disease state (e.g., cancer, autoimmune disorders). Thus, pharmacologically active peptides comprise agonistic or mimetic and antagonistic peptides as defined below.


The terms “-mimetic peptide” and “-agonist peptide” refer to a peptide having biological activity comparable to a naturally occurring toxin peptide molecule, e.g., naturally occurring ShK toxin peptide. These terms further include peptides that indirectly mimic the activity of a naturally occurring toxin peptide molecule, such as by potentiating the effects of the naturally occurring molecule.


The term “antagonist peptide” or “inhibitor peptide” refers to a peptide that blocks or in some way interferes with the biological activity of a receptor of interest, or has biological activity comparable to a known antagonist or inhibitor of a receptor of interest (such as, but not limited to, an ion channel).


The term “acidic residue” refers to amino acid residues in D- or L-form having sidechains comprising acidic groups. Exemplary acidic residues include D and E.


The term “amide residue” refers to amino acids in D- or L-form having sidechains comprising amide derivatives of acidic groups. Exemplary residues include N and Q.


The term “aromatic residue” refers to amino acid residues in D- or L-form having sidechains comprising aromatic groups. Exemplary aromatic residues include F, Y, and W.


The term “basic residue” refers to amino acid residues in D- or L-form having sidechains comprising basic groups. Exemplary basic residues include H, K, R, N-methyl-arginine, ω-aminoarginine, ω-methyl-arginine, 1-methyl-histidine, 3-methyl-histidine, and homoarginine (hR) residues.


The term “hydrophilic residue” refers to amino acid residues in D- or L-form having sidechains comprising polar groups. Exemplary hydrophilic residues include C, S, T, N, Q, D, E, K, and citrulline (Cit) residues.


The term “nonfunctional residue” refers to amino acid residues in D- or L-form having sidechains that lack acidic, basic, or aromatic groups. Exemplary nonfunctional amino acid residues include M, G, A, V, I, L and norleucine (Nle).


The term “neutral polar residue” refers to amino acid residues in D- or L-form having sidechains that lack basic, acidic, or polar groups. Exemplary neutral polar amino acid residues include A, V, L, I, P, W, M, and F.


The term “polar hydrophobic residue” refers to amino acid residues in D- or L-form having sidechains comprising polar groups. Exemplary polar hydrophobic amino acid residues include T, G, S, Y, C, Q, and N.


The term “hydrophobic residue” refers to amino acid residues in D- or L-form having sidechains that lack basic or acidic groups. Exemplary hydrophobic amino acid residues include A, V, L, I, P, W, M, F, T, G, S, Y, C, Q, and N.


In some useful embodiments of the compositions of the invention, the amino acid sequence of the toxin peptide is modified in one or more ways relative to a native toxin peptide sequence of interest, such as, but not limited to, a native ShK or OSK1 sequence, their peptide analogs, or any other toxin peptides having amino acid sequences as set for in any of Tables 1-32. The one or more useful modifications can include amino acid additions or insertions, amino acid deletions, peptide truncations, amino acid substitutions, and/or chemical derivatization of amino acid residues, accomplished by known chemical techniques. Such modifications can be, for example, for the purpose of enhanced potency, selectivity, and/or proteolytic stability, or the like. Those skilled in the art are aware of techniques for designing peptide analogs with such enhanced properties, such as alanine scanning, rational design based on alignment mediated mutagenesis using known toxin peptide sequences and/or molecular modeling. For example, ShK analogs can be designed to remove protease cleavage sites (e.g., trypsin cleavage sites at K or R residues and/or chymotrypsin cleavage sites at F, Y, or W residues) in a ShK peptide- or ShK analog-containing composition of the invention, based partially on alignment mediated mutagenesis using HmK (see, e.g., FIG. 6) and molecular modeling. (See, e.g., Kalman et al., ShK-Dap22, a potent Kv1.3-specific immunosuppressive polypeptide, J. Biol. Chem. 273(49):32697-707 (1998); Kem et al., U.S. Pat. No. 6,077,680; Mouhat et al., OsK1 derivatives, WO 2006/002850 A2)).


The term “protease” is synonymous with “peptidase”. Proteases comprise two groups of enzymes: the endopeptidases which cleave peptide bonds at points within the protein, and the exopeptidases, which remove one or more amino acids from either N- or C-terminus respectively. The term “proteinase” is also used as a synonym for endopeptidase. The four mechanistic classes of proteinases are: serine proteinases, cysteine proteinases, aspartic proteinases, and metallo-proteinases. In addition to these four mechanistic classes, there is a section of the enzyme nomenclature which is allocated for proteases of unidentified catalytic mechanism. This indicates that the catalytic mechanism has not been identified.


Cleavage subsite nomenclature is commonly adopted from a scheme created by Schechter and Berger (Schechter I. & Berger A., On the size of the active site in proteases. I. Papain, Biochemical and Biophysical Research Communication, 27:157 (1967); Schechter I. & Berger A., On the active site of proteases. 3. Mapping the active site of papain; specific inhibitor peptides of papain, Biochemical and Biophysical Research Communication, 32:898 (1968)). According to this model, amino acid residues in a substrate undergoing cleavage are designated P1, P2, P3, P4 etc. in the N-terminal direction from the cleaved bond. Likewise, the residues in the C-terminal direction are designated P1′, P2′, P3′, P4′. etc.


The skilled artisan is aware of a variety of tools for identifying protease binding or protease cleavage sites of interest. For example, the PeptideCutter software tool is available through the ExPASy (Expert Protein Analysis System) proteomics server of the Swiss Institute of Bioinformatics (SIB; www.expasy.org/tools/peptidecutter). PeptideCutter searches a protein sequence from the SWISS-PROT and/or TrEMBL databases or a user-entered protein sequence for protease cleavage sites. Single proteases and chemicals, a selection or the whole list of proteases and chemicals can be used. Different forms of output of the results are available: tables of cleavage sites either grouped alphabetically according to enzyme names or sequentially according to the amino acid number. A third option for output is a map of cleavage sites. The sequence and the cleavage sites mapped onto it are grouped in blocks, the size of which can be chosen by the user. Other tools are also known for determining protease cleavage sites. (E.g., Turk, B. et al., Determination of protease cleavage site motifs using mixture-based oriented peptide libraries, Nature Biotechnology, 19:661-667 (2001); Barrett A. et al., Handbook of proteolytic enzymes, Academic Press (1998)).


The serine proteinases include the chymotrypsin family, which includes mammalian protease enzymes such as chymotrypsin, trypsin or elastase or kallikrein. The serine proteinases exhibit different substrate specificities, which are related to amino acid substitutions in the various enzyme subsites interacting with the substrate residues. Some enzymes have an extended interaction site with the substrate whereas others have a specificity restricted to the P1 substrate residue.


Trypsin preferentially cleaves at R or K in position P1. A statistical study carried out by Keil (1992) described the negative influences of residues surrounding the Arg- and Lys-bonds (i.e. the positions P2 and P1′, respectively) during trypsin cleavage. (Keil, B., Specificity of proteolysis, Springer-Verlag Berlin-Heidelberg-New York, 335 (1992)). A proline residue in position P1′ normally exerts a strong negative influence on trypsin cleavage. Similarly, the positioning of R and K in P1′ results in an inhibition, as well as negatively charged residues in positions P2 and P1′.


Chymotrypsin preferentially cleaves at a W, Y or F in position P1 (high specificity) and to a lesser extent at L, M or H residue in position P1. (Keil, 1992). Exceptions to these rules are the following: When W is found in position P1, the cleavage is blocked when M or P are found in position P1′ at the same time. Furthermore, a proline residue in position P1′ nearly fully blocks the cleavage independent of the amino acids found in position P1. When an M residue is found in position P1, the cleavage is blocked by the presence of a Y residue in position P1′. Finally, when H is located in position P1, the presence of a D, M or W residue also blocks the cleavage.


Membrane metallo-endopeptidase (NEP; neutral endopeptidase, kidney-brush-border neutral proteinase, enkephalinase, EC 3.4.24.11) cleaves peptides at the amino side of hydrophobic amino acid residues. (Connelly, J C et al., Neutral Endopeptidase 24.11 in Human Neutrophils: Cleavage of Chemotactic Peptide, PNAS, 82(24):8737-8741 (1985)).


Thrombin preferentially cleaves at an R residue in position P1. (Keil, 1992). The natural substrate of thrombin is fibrinogen. Optimum cleavage sites are when an R residue is in position P1 and Gly is in position P2 and position P1′. Likewise, when hydrophobic amino acid residues are found in position P4 and position P3, a proline residue in position P2, an R residue in position P1, and non-acidic amino acid residues in position P1′ and position P2′. A very important residue for its natural substrate fibrinogen is a D residue in P10.


Caspases are a family of cysteine proteases bearing an active site with a conserved amino acid sequence and which cleave peptides specifically following D residues. (Eamshaw W C et al., Mammalian caspases: Structure, activation, substrates, and functions during apoptosis, Annual Review of Biochemistry, 68:383-424 (1999)).


The Arg-C proteinase preferentially cleaves at an R residue in position P1. The cleavage behavior seems to be only moderately affected by residues in position P1′. (Keil, 1992). The Asp-N endopeptidase cleaves specifically bonds with a D residue in position P1′. (Keil, 1992).


Furin is a ubiquitous subtilisin-like proprotein convertase. It is the major processing enzyme of the secretory pathway and intracellularly is localized in the trans-golgi network (van den Ouweland, A. M. W. et al. (1990) Nucl. Acids Res., 18, 664; Steiner, D. F. (1998) Curr. Opin. Chem. Biol., 2, 31-39). The minimal furin cleavage site is Arg-X-X-Arg′. However, the enzyme prefers the site Arg-X-(Lys/Arg)-Arg′. An additional arginine at the P6 position appears to enhance cleavage (Krysan, D. J. et al. (1999) J. Biol. Chem., 274, 23229-23234).


The foregoing is merely exemplary and by no means an exhaustive treatment of knowledge available to the skilled artisan concerning protease binding and/or cleavage sites that the skilled artisan may be interested in eliminating in practicing the invention.


Additional useful embodiments of the toxin peptide, e.g., the OSK1 peptide analog, can result from conservative modifications of the amino acid sequences of the peptides disclosed herein. Conservative modifications will produce peptides having functional, physical, and chemical characteristics similar to those of the parent peptide from which such modifications are made. Such conservatively modified forms of the peptides disclosed herein are also contemplated as being an embodiment of the present invention.


In contrast, substantial modifications in the functional and/or chemical characteristics of the toxin peptides may be accomplished by selecting substitutions in the amino acid sequence that differ significantly in their effect on maintaining (a) the structure of the molecular backbone in the region of the substitution, for example, as an α-helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the molecule.


For example, a “conservative amino acid substitution” may involve a substitution of a native amino acid residue with a normative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position. Furthermore, any native residue in the polypeptide may also be substituted with alanine, as has been previously described for “alanine scanning mutagenesis” (see, for example, MacLennan et al., Acta Physiol. Scand. Suppl., 643:55-67 (1998); Sasaki et al., 1998, Adv. Biophys. 35:1-24 (1998), which discuss alanine scanning mutagenesis).


Desired amino acid substitutions (whether conservative or non-conservative) can be determined by those skilled in the art at the time such substitutions are desired. For example, amino acid substitutions can be used to identify important residues of the peptide sequence, or to increase or decrease the affinity of the peptide or vehicle-conjugated peptide molecules described herein.


Naturally occurring residues may be divided into classes based on common side chain properties:


1) hydrophobic: norleucine (Nor), Met, Ala, Val, Leu, lie;


2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;


3) acidic: Asp, Glu;


4) basic: His, Lys, Arg;


5) residues that influence chain orientation: Gly, Pro; and


6) aromatic: Trp, Tyr, Phe.


Conservative amino acid substitutions may involve exchange of a member of one of these classes with another member of the same class. Conservative amino acid substitutions may encompass non-naturally occurring amino acid residues, which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics and other reversed or inverted forms of amino acid moieties.


Non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class. Such substituted residues may be introduced into regions of the human antibody that are homologous with non-human antibodies, or into the non-homologous regions of the molecule.


In making such changes, according to certain embodiments, the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine (−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine (−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine (−4.5).


The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (see, for example, Kyte et al., 1982, J. MOL Biol. 157:105-131). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, in certain embodiments, the substitution of amino acids whose hydropathic indices are within ±2 is included. In certain embodiments, those that are within ±1 are included, and in certain embodiments, those within ±0.5 are included.


It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functional protein or peptide thereby created is intended for use in immunological embodiments, as disclosed herein. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.


The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5); histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5) and tryptophan (−3.4). In making changes based upon similar hydrophilicity values, in certain embodiments, the substitution of amino acids whose hydrophilicity values are within ±2 is included, in certain embodiments, those that are within ±1 are included, and in certain embodiments, those within ±0.5 are included. One may also identify epitopes from primary amino acid sequences on the basis of hydrophilicity. These regions are also referred to as “epitopic core regions.”


Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine norleucine, alanine, or methionine for another, the substitution of one polar (hydrophilic) amino acid residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic amino acid residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another. The phrase “conservative amino acid substitution” also includes the use of a chemically derivatized residue in place of a non-derivatized residue, provided that such polypeptide displays the requisite biological activity. Other exemplary amino acid substitutions that can be useful in accordance with the present invention are set forth in Table 1A.









TABLE 1A







Some Useful Amino Acid Substitutions










Original
Exemplary



Residues
Substitutions







Ala
Val, Leu, Ile



Arg
Lys, Gln, Asn



Asn
Gln



Asp
Glu



Cys
Ser, Ala



Gln
Asn



Glu
Asp



Gly
Pro, Ala



His
Asn, Gln, Lys, Arg



Ile
Leu, Val, Met, Ala, Phe,




Norleucine



Leu
Norleucine, Ile, Val,




Met, Ala, Phe



Lys
Arg, 1,4-Diamino-




butyric Acid, Gln, Asn



Met
Leu, Phe, Ile



Phe
Leu, Val, Ile, Ala, Tyr



Pro
Ala



Ser
Thr, Ala, Cys



Thr
Ser



Trp
Tyr, Phe



Tyr
Trp, Phe, Thr, Ser



Val
Ile, Met, Leu, Phe, Ala,




Norleucine










In other examples, a toxin peptide amino acid sequence, e.g., an OSK1 peptide analog sequence, modified from a naturally occurring toxin peptide amino acid sequence includes at least one amino acid residue inserted or substituted therein, relative to the amino acid sequence of the native toxin peptide sequence of interest, in which the inserted or substituted amino acid residue has a side chain comprising a nucleophilic or electrophilic reactive functional group by which the peptide is conjugated to a linker or half-life extending moiety. In accordance with the invention, useful examples of such a nucleophilic or electrophilic reactive functional group include, but are not limited to, a thiol, a primary amine, a seleno, a hydrazide, an aldehyde, a carboxylic acid, a ketone, an aminooxy, a masked (protected) aldehyde, or a masked (protected) keto functional group. Examples of amino acid residues having a side chain comprising a nucleophilic reactive functional group include, but are not limited to, a lysine residue, an α,β-diaminopropionic acid residue, an α,γ-diaminobutyric acid residue, an ornithine residue, a cysteine, a homocysteine, a glutamic acid residue, an aspartic acid residue, or a selenocysteine residue. In some embodiments, the toxin peptide amino acid sequence (or “primary sequence”) is modified at one, two, three, four, five or more amino acid residue positions, by having a residue substituted therein different from the native primary sequence (e.g., OSK1 SEQ ID NO:25) or omitted (e.g., an OSK1 peptide analog optionally lacking a residue at positions 36, 37, 36-38, 37-38, or 38).


In further describing toxin peptides herein, a one-letter abbreviation system is frequently applied to designate the identities of the twenty “canonical” amino acid residues generally incorporated into naturally occurring peptides and proteins (Table 1B). Such one-letter abbreviations are entirely interchangeable in meaning with three-letter abbreviations, or non-abbreviated amino acid names. Within the one-letter abbreviation system used herein, an uppercase letter indicates a L-amino acid, and a lower case letter indicates a D-amino acid, unless otherwise noted herein. For example, the abbreviation “R” designates L-arginine and the abbreviation “r” designates D-arginine.









TABLE 1B





One-letter abbreviations for the canonical amino acids


Three-letter abbreviations are in parentheses


















Alanine (Ala)
A



Glutamine (Gln)
Q



Leucine (Leu)
L



Serine (Ser)
S



Arginine (Arg)
R



Glutamic Acid (Glu)
E



Lysine (Lys)
K



Threonine (Thr)
T



Asparagine (Asn)
N



Glycine (Gly)
G



Methionine (Met)
M



Tryptophan (Trp)
W



Aspartic Acid (Asp)
D



Histidine (His)
H



Phenylalanine (Phe)
F



Tyrosine (Tyr)
Y



Cysteine (Cys)
C



Isoleucine (Ile)
I



Proline (Pro)
P



Valine (Val)
V










An amino acid substitution in an amino acid sequence is typically designated herein with a one-letter abbreviation for the amino acid residue in a particular position, followed by the numerical amino acid position relative to the native toxin peptide sequence of interest, which is then followed by the one-letter symbol for the amino acid residue substituted in. For example, “T30D” symbolizes a substitution of a threonine residue by an aspartate residue at amino acid position 30, relative to a hypothetical native toxin peptide sequence. By way of further example, “R18hR” or “R18Cit” indicates a substitution of an arginine residue by a homoarginine or a citrulline residue, respectively, at amino acid position 18, relative to the hypothetical native toxin peptide. An amino acid position within the amino acid sequence of any particular toxin peptide (or peptide analog) described herein may differ from its position relative to the native sequence, i.e., as determined in an alignment of the N-terminal or C-terminal end of the peptide's amino acid sequence with the N-terminal or C-terminal end, as appropriate, of the native toxin peptide sequence. For example, amino acid position 1 of the sequence SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC ShK(2-35); SEQ ID NO:92), a N-terminal truncation of the native ShK sequence, thus aligned with the C-terminal of native ShK(1-35) (SEQ ID NO:5), corresponds to amino acid position 2 relative to the native sequence, and amino acid position 34 of SEQ ID NO:92 corresponds to amino acid position 35 relative to the native sequence (SEQ ID NO:5).


In certain embodiments of the present invention, amino acid substitutions encompass, non-canonical amino acid residues, which include naturally rare (in peptides or proteins) amino acid residues or unnatural amino acid residues. Non-canonical amino acid residues can be incorporated into the peptide by chemical peptide synthesis rather than by synthesis in biological systems, such as recombinantly expressing cells, or alternatively the skilled artisan can employ known techniques of protein engineering that use recombinantly expressing cells. (See, e.g., Link et al., Non-canonical amino acids in protein engineering, Current Opinion in Biotechnology, 14(6):603-609 (2003)). The term “non-canonical amino acid residue” refers to amino acid residues in D- or L-form that are not among the 20 canonical amino acids generally incorporated into naturally occurring proteins, for example, β-amino acids, homoamino acids, cyclic amino acids and amino acids with derivatized side chains. Examples include (in the L-form or D-form; abbreviated as in parentheses): citrulline (Cit), homocitrulline (hCit), Nα-methylcitrulline (NMeCit), Nα-methylhomocitrulline (Nα-MeHoCit), ornithine (Orn), Nα-Methylornithine (Nα-MeOrn or NMeOrn), sarcosine (Sar), homolysine (hLys or hK), homoarginine (hArg or hR), homoglutamine (hQ), Nα-methylarginine (NMeR), Nα-methylleucine (Nα-MeL or NMeL), N-methylhomolysine (NMeHoK), Nα-methylglutamine (NMeQ), norleucine (Nle), norvaline (Nva), 1,2,3,4-tetrahydroisoquinoline (Tic), Octahydroindole-2-carboxylic acid (Oic), 3-(1-naphthyl)alanine (1-Nal), 3-(2-naphthyl)alanine (2-Nal), 1,2,3,4-tetrahydroisoquinoline (Tic), 2-indanylglycine (Igl), para-iodophenylalanine (pI-Phe), para-aminophenylalanine (4AmP or 4-Amino-Phe), 4-guanidino phenylalanine (Guf), glycyllysine (abbreviated herein “K(Nε-glycyl)” or “K(glycyl)” or “K(gly)”), nitrophenylalanine (nitrophe), aminophenylalanine (aminophe or Amino-Phe), benzylphenylalanine (benzylphe), γ-carboxyglutamic acid (γ-carboxyglu), hydroxyproline (hydroxypro), p-carboxyl-phenylalanine (Cpa), α-aminoadipic acid (Aad), Nα-methyl valine (NMeVal), N-α-methyl leucine (NMeLeu), Nα-methylnorleucine (NMeNle), cyclopentylglycine (Cpg), cyclohexylglycine (Chg), acetylarginine (acetylarg), α,β-diaminopropionoic acid (Dpr), α,γ-diaminobutyric acid (Dab), diaminopropionic acid (Dap), cyclohexylalanine (Cha), 4-methyl-phenylalanine (MePhe), β,β-diphenyl-alanine (BiPhA), aminobutyric acid (Abu), 4-phenyl-phenylalanine (or biphenylalanine; 4Bip), α-amino-isobutyric acid (Aib), beta-alanine, beta-aminopropionic acid, piperidinic acid, aminocaproic acid, aminoheptanoic acid, aminopimelic acid, desmosine, diaminopimelic acid, N-ethylglycine, N-ethylaspargine, hydroxylysine, allo-hydroxylysine, isodesmosine, allo-isoleucine, N-methylglycine, N-methylisoleucine, N-methylvaline, 4-hydroxyproline (Hyp), γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, ω-methylarginine, 4-Amino-O-Phthalic Acid (4APA), and other similar amino acids, and derivatized forms of any of these as described herein. Table 1B contains some exemplary non-canonical amino acid residues that are useful in accordance with the present invention and associated abbreviations as typically used herein, although the skilled practitioner will understand that different abbreviations and nomenclatures may be applicable to the same substance and my appear interchangeably herein.


Table 1B. Useful non-canonical amino acids for amino acid addition, insertion, or substitution into toxin peptide sequences, including OSK1 peptide analog sequences, in accordance with the present invention. In the event an abbreviation listed in Table 1B differs from another abbreviation for the same substance disclosed elsewhere herein, both abbreviations are understood to be applicable.
















Abbreviation
Amino Acid









Sar
Sarcosine



Nle
norleucine



Ile
isoleucine



1-Nal
3-(1-naphthyl)alanine



2-Nal
3-(2-naphthyl)alanine



Bip
4,4′-biphenyl alanine



Dip
3,3-diphenylalanine



Nvl
norvaline



NMe-Val
Nα-methyl valine



NMe-Leu
Nα-methyl leucine



NMe-Nle
Nα-methyl norleucine



Cpg
cyclopentyl glycine



Chg
cyclohexyl glycine



Hyp
hydroxy proline



Oic
Octahydroindole-2-Carboxylic Acid



Igl
Indanyl glycine



Aib
aminoisobutyric acid



Aic
2-aminoindane-2-carboxylic acid



Pip
pipecolic acid



BhTic
β-homo Tic



BhPro
β-homo proline



Sar
Sarcosine



Cpg
cyclopentyl glycine



Tiq
1,2,3,4-L-Tetrahydroisoquinoline-1-Carboxylic




acid



Nip
Nipecotic Acid



Thz
Thiazolidine-4-carboxylic acid



Thi
3-thienyl alanine



4GuaPr
4-guanidino proline



4Pip
4-Amino-1-piperidine-4-carboxylic acid



Idc
indoline-2-carboxylic acid



Hydroxyl-Tic
1,2,3,4-Tetrahydroisoquinoline-7-hydroxy-3-




carboxylic acid



Bip
4,4′-biphenyl alanine



Ome-Tyr
O-methyl tyrosine



I-Tyr
Iodotyrosine



Tic
1,2,3,4-L-Tetrahydroisoquinoline-3-Carboxylic




acid



Igl
Indanyl glycine



BhTic
β-homo Tic



BhPhe
β-homo phenylalanine



AMeF
α-methyl Phenyalanine



BPhe
β-phenylalanine



Phg
phenylglycine



Anc
3-amino-2-naphthoic acid



Atc
2-aminotetraline-2-carboxylic acid



NMe-Phe
Nα-methyl phenylalanine



NMe-Lys
Nα-methyl lysine



Tpi
1,2,3,4-Tetrahydronorharman-3-Carboxylic acid



Cpg
cyclopentyl glycine



Dip
3,3-diphenylalanine



4Pal
4-pyridinylalanine



3Pal
3-pyridinylalanine



2Pal
2-pyridinylalanine



4Pip
4-Amino-1-piperidine-4-carboxylic acid



4AmP
4-amino-phenylalanine



Idc
indoline-2-carboxylic acid



Chg
cyclohexyl glycine



hPhe
homophenylalanine



BhTrp
β-homotryptophan



pI-Phe
4-iodophenylalanine



Aic
2-aminoindane-2-carboxylic acid



NMe-Lys
Nα-methyl lysine



Orn
ornithine



Dpr
2,3-Diaminopropionic acid



Dbu
2,4-Diaminobutyric acid



homoLys
homolysine



N-eMe-K
Nε-methyl-lysine



N-eEt-K
Nε-ethyl-lysine



N-eIPr-K
Nε-isopropyl-lysine



bhomoK
β-homolysine



rLys
Lys ψ(CH2NH)-reduced amide bond



rOrn
Orn ψ(CH2NH)-reduced amide bond



Acm
acetamidomethyl



Ahx
6-aminohexanoic acid



εAhx
6-aminohexanoic acid



K(NPeg11)
Nε-(O-(aminoethyl)-O′-(2-propanoyl)-




undecaethyleneglycol)-Lysine



K(NPeg27)
Nε-(O-(aminoethyl)-O′-(2-propanoyl)-




(ethyleneglycol)27-Lysine



Cit
Citrulline



hArg
homoarginine



hCit
homocitrulline



NMe-Arg
Nα-methyl arginine (NMeR)



Guf
4-guanidinyl phenylalanine



bhArg
β-homoarginine



3G-Dpr
2-amino-3-guanidinopropanoic acid



4AmP
4-amino-phenylalanine



4AmPhe
4-amidino-phenylalanine



4AmPig
2-amino-2-(1-carbamimidoylpiperidin-4-




yl)acetic acid



4GuaPr
4-guanidino proline



N-Arg
Nα-[(CH2)3NHCH(NH)NH2] substituted glycine



rArg
Arg ψ(CH2NH)-reduced amide bond



4PipA
4-Piperidinyl alanine



NMe-Arg
Nα-methyl arginine (or NMeR)



NMe-Thr
Nα-methyl threonine (or NMeThr)










Nomenclature and Symbolism for Amino Acids and Peptides by the UPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN) have been published in the following documents: Biochem. J., 1984, 219, 345-373; Eur. J. Biochem., 1984, 138, 9-37; 1985, 152, 1; 1993, 213, 2; Internat. J. Pept. Prot. Res., 1984, 24, following p 84; J. Biol. Chem., 1985, 260, 14-42; Pure Appl. Chem., 1984, 56, 595-624; Amino Acids and Peptides, 1985, 16, 387-410; Biochemical Nomenclature and Related Documents, 2nd edition, Portland Press, 1992, pages 39-69].


As stated herein, in accordance with the present invention, peptide portions of the inventive compositions, such as the toxin peptide or a peptide linker, can also be chemically derivatized at one or more amino acid residues. Peptides that contain derivatized amino acid residues can be synthesized by known organic chemistry techniques. “Chemical derivative” or “chemically derivatized” in the context of a peptide refers to a subject peptide having one or more residues chemically derivatized by reaction of a functional side group. Such derivatized molecules include, for example, those molecules in which free amino groups have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine. Also included as chemical derivatives are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty canonical amino acids, whether in L- or D-form. For example, 4-hydroxyproline may be substituted for proline; 5-hydroxylysine maybe substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.


Useful derivatizations include, in some embodiments, those in which the amino terminal of the toxin peptide, such as but not limited to the OSK1 peptide analog, is chemically blocked so that conjugation with the vehicle will be prevented from taking place at an N-terminal free amino group. There may also be other beneficial effects of such a modification, for example a reduction in the toxin peptide's susceptibility to enzymatic proteolysis. The N-terminus of the toxin peptide, e.g., the OSK1 peptide analog, can be acylated or modified to a substituted amine, or derivatized with another functional group, such as an aromatic or aryl moiety (e.g., an indole acid, benzyl (Bzl or Bn), dibenzyl (DiBzl or Bn2), benzoyl, or benzyloxycarbonyl (Cbz or Z)), N,N-dimethylglycine or creatine. For example, in some embodiments, an acyl moiety, such as, but not limited to, a formyl, acetyl (Ac), propanoyl, butanyl, heptanyl, hexanoyl, octanoyl, or nonanoyl, can be covalently linked to the N-terminal end of the peptide, e.g., the OSK1 peptide analog, which can prevent undesired side reactions during conjugation of the vehicle to the peptide. Alternatively, a fatty acid (e.g. butyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic or the like) or polyethylene glycol moiety can be covalently linked to the N-terminal end of the peptide, e.g., the OSK1 peptide analog. Other exemplary N-terminal derivative groups include —NRR1 (other than —NH2), —NRC(O)R1, —NRC(O)OR1, —NRS(O)2R1, —NHC(O)NHR1, succinimide, or benzyloxycarbonyl-NH-(Cbz-NH—), wherein R and R1 are each independently hydrogen or lower alkyl and wherein the phenyl ring may be substituted with 1 to 3 substituents selected from C1-C4 alkyl, C1-C4 alkoxy, chloro, and bromo.


In some embodiments of the present invention, basic residues (e.g., lysine) of the toxin peptide of interest can be replaced with other residues (nonfunctional residues preferred). Such molecules will be less basic than the molecules from which they are derived and otherwise retain the activity of the molecules from which they are derived, which can result in advantages in stability and immunogenicity; the present invention should not, however, be limited by this theory.


Additionally, physiologically acceptable salts of the inventive compositions are also encompassed, including when the inventive compositions are referred to herein as “molecules” or “compounds.”. By “physiologically acceptable salts” is meant any salts that are known or later discovered to be pharmaceutically acceptable. Some non-limiting examples of pharmaceutically acceptable salts are: acetate; trifluoroacetate; hydrohalides, such as hydrochloride and hydrobromide; sulfate; citrate; maleate; tartrate; glycolate; gluconate; succinate; mesylate; besylate; salts of gallic acid esters (gallic acid is also known as 3, 4, 5 trihydroxybenzoic acid) such as PentaGalloylGlucose (PGG) and epigallocatechin gallate (EGCG), salts of cholesteryl sulfate, pamoate, tannate and oxalate salts.


Structure of Compounds:


In general. Recombinant proteins have been developed as therapeutic agents through, among other means, covalent attachment to half-life extending moieties. Such moieties include the “Fc” domain of an antibody, as is used in Enbrel® (etanercept), as well as biologically suitable polymers (e.g., polyethylene glycol, or “PEG”), as is used in Neulasta® (pegfilgrastim). Feige et al. described the use of such half-life extenders with peptides in U.S. Pat. No. 6,660,843, issued Dec. 9, 2003 (hereby incorporated by reference in its entirety).


The present inventors have determined that molecules of this invention-peptides of about 80 amino acids or less with at least two intrapeptide disulfide bonds-possess therapeutic advantages when covalently attached to half-life extending moieties. Molecules of the present invention can further comprise an additional pharmacologically active, covalently bound peptide, which can be bound to the half-life extending moiety (F1 and/or F2) or to the peptide portion (P). Embodiments of the inventive compositions containing more than one half-life extending moiety (F1 and F2) include those in which F1 and F2 are the same or different half-life extending moieties. Examples (with or without a linker between each domain) include structures as illustrated in FIG. 75 as well as the following embodiments (and others described herein and in the working Examples):


20KPEG—toxin peptide—Fc domain, consistent with the formula [(F1)1—(X2)1—(F2)1];


20KPEG—toxin peptide—Fc CH2 domain, consistent with the formula [(F1)1—(X2)1—(F2)1];


20KPEG—toxin peptide—HSA, consistent with the formula [(F1)1(X2)1—(F2)1];


20KPEG—Fc domain—toxin peptide, consistent with the formula [(F1)1—(F2)1—(X3)1];


20KPEG—Fc CH2 domain—toxin peptide, consistent with the formula [(F1)1—(F2)1—(X3)1]; and


20KPEG—HSA—toxin peptide, consistent with the formula [(F1)1—(F2)1—(X3)1].


Toxin peptides. Any number of toxin peptides (i.e., “P”, or equivalently shown as “P1” in FIG. 2) can be used in conjunction with the present invention. Of particular interest are the toxin peptides ShK, HmK, MgTx, AgTx2, Agatoxins, and HsTx1, as well as modified analogs of these, in particular OsK1 (also referred to as “OSK1”) peptide analogs of the present invention, and other peptides that mimic the activity of such toxin peptides. As stated herein above, if more than one toxin peptide “P” is present in the inventive composition, “P” can be independently the same or different from any other toxin peptide(s) also present in the inventive composition. For example, in a composition having the formula P-(L)g-F1-(L)f-P, both of the toxin peptides, “P”, can be the same peptide analog of ShK, different peptide analogs of ShK, or one can be a peptide analog of ShK and the other a peptide analog of OSK1. In a preferred embodiment, at least one P is a an OSK1 peptide analog as further described herein.


In some embodiments of the invention, other peptides of interest are especially useful in molecules having additional features over the molecules of structural Formula I. In such molecules, the molecule of Formula I further comprises an additional pharmacologically active, covalently bound peptide, which is an agonistic peptide, an antagonistic peptide, or a targeting peptide; this peptide can be conjugated to F1 or F2 or P. Such agonistic peptides have activity agonistic to the toxin peptide but are not required to exert such activity by the same mechanism as the toxin peptide. Peptide antagonists are also useful in embodiments of the invention, with a preference for those with activity that can be complementary to the activity of the toxin peptide. Targeting peptides are also of interest, such as peptides that direct the molecule to particular cell types, organs, and the like. These classes of peptides can be discovered by methods described in the references cited in this specification and other references. Phage display, in particular, is useful in generating toxin peptides for use in the present invention. Affinity selection from libraries of random peptides can be used to identify peptide ligands for any site of any gene product. Dedman et al. (1993), J. Biol. Chem. 268: 23025-30. Phage display is particularly well suited for identifying peptides that bind to such proteins of interest as cell surface receptors or any proteins having linear epitopes. Wilson et al. (1998), Can. J. Microbiol. 44: 313-29; Kay et al. (1998), Drug Disc. Today 3: 370-8. Such proteins are extensively reviewed in Herz et al. (1997), J. Receptor and Signal Transduction Res. 17(5): 671-776, which is hereby incorporated by reference in its entirety. Such proteins of interest are preferred for use in this invention.


Particularly preferred peptides appear in the following tables. These peptides can be prepared by methods disclosed in the art or as described hereinafter. Single letter amino acid abbreviations are used. Unless otherwise specified, each X is independently a nonfunctional residue.









TABLE 1







Kv1.3 inhibitor peptide sequences











Short-hand




Sequence/structure
designation
SEQ ID NO:













LVKCRGTSDCGRPCQQQTGCPNSKCINRMCKCYGC
Pi1
21






TISCTNPKQCYPHCKKETGYPNAKCMNRKCKCFGR
Pi2
17





TISCTNEKQCYPHCKKETGYPNAKCMNRKCKCFGR
Pi3
18





IEAIRCGGSRDCYRPCQKRTGCPNAKCINKTCKCYGCS
Pi4
19





ASCRTPKDCADPCRKETGCPYGKCMNRKCKCNRC
HsTx1
61





GVPINVSCTGSPQCIKPCKDAGMRFGKCMNRKCHCTPK
AgTx2
23





GVPINVKCTGSPQCLKPCKDAGMRFGKCINGKCHCTPK
AgTx1
85





GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK
OSK1
25





ZKECTGPQHCTNFCRKNKCTHGKCMNRKCKCFNCK
Anuroctoxin
62





TIINVKCTSPKQCSKPCKELYGSSAGAKCMNGKCKCYNN
NTX
30





TVIDVKCTSPKQCLPPCKAQFGIRAGAKCMNGKCKCYPH
HgTx1
27





QFTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS
ChTx
36





VFINAKCRGSPECLPKCKEAIGKAAGKCMNGKCKCYP
Titystoxin-Ka
86





VCRDWFKETACRHAKSLGNCRTSQKYRANCAKTCELC
BgK
9





VGINVKCKHSGQCLKPCKDAGMRFGKCINGKCDCTPKG
BmKTX
26





QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGKCRCYS
BmTx1
40





VFINVKCRGSKECLPACKAAVGKAAGKCMNGKCKCYP
Tc30
87





TGPQTTCQAAMCEAGCKGLGKSMESCQGDTCKCKA
Tc32
13
















TABLE 2







ShK peptide and ShK peptide analog sequences











Short-hand
SEQ



Sequence/structure
designation
ID NO:













RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK
5






RSCIDTIPKSRCTAFQSKHSMKYRLSFCRKTSGTC
ShK-S17/S32
88





RSSIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTS
ShK-S3/S35
89





SSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-S1
90





(N-acetylarg)
ShK-N-acetylarg1
91


SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC





SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-d1
92





CIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-d2
93





ASCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-A1
94





RSCADTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-A4
95





RSCADTIPKSRCTAAQCKHSMKYRLSFCRKTCGTC
ShK-A4/A15
96





RSCADTIPKSRCTAAQCKHSMKYRASFCRKTCGTC
ShK-A4/A15/A25
97





RSCIDAIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-A6
98





RSCIDTAPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-A7
99





RSCIDTIAKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-A8
100





RSCIDTIPASRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-A9
101





RSCIDTIPESRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-E9
102





RSCIDTIPQSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-Q9
103





RSCIDTIPKARCTAFQCKHSMKYRLSFCRKTCGTC
ShK-A10
104





RSCIDTIPKSACTAFQCKHSMKYRLSFCRKTCGTC
ShK-A11
105





RSCIDTIPKSECTAFQCKHSMKYRLSFCRKTCGTC
ShK-E11
106





RSCIDTIPKSQCTAFQCKHSMKYRLSFCRKTCGTC
ShK-Q11
107





RSCIDTIPKSRCAAFQCKHSMKYRLSFCRKTCGTC
ShK-A13
108





RSCIDTIPKSRCTAAQCKHSMKYRLSFCRKTCGTC
ShK-A15
109





RSCIDTIPKSRCTAWQCKHSMKYRLSFCRKTCGTC
ShK-W15
110





RSCIDTIPKSRCTAXs15QCKHSMKYRLSFCRKTCGTC
ShK-X15
111





RSCIDTIPKSRCTAAQCKHSMKYRASFCRKTCGTC
ShK-A15/A25
112





RSCIDTIPKSRCTAFACKHSMKYRLSFCRKTCGTC
ShK-A16
113





RSCIDTIPKSRCTAFECKHSMKYRLSFCRKTCGTC
ShK-E16
114





RSCIDTIPKSRCTAFQCAHSMKYRLSFCRKTCGTC
ShK-A18
115





RSCIDTIPKSRCTAFQCEHSMKYRLSFCRKTCGTC
ShK-E18
116





RSCIDTIPKSRCTAFQCKASMKYRLSFCRKTCGTC
ShK-A19
117





RSCIDTIPKSRCTAFQCKKSMKYRLSFCRKTCGTC
ShK-K19
118





RSCIDTIPKSRCTAFQCKHAMKYRLSFCRKTCGTC
ShK-A20
119





RSCIDTIPKSRCTAFQCKHSAKYRLSFCRKTCGTC
ShK-A21
120





RSCIDTIPKSRCTAFQCKHSXs21KYRLSFCRKTCGTC
ShK-X21
121





RSCIDTIPKSRCTAFQCKHS(norleu)KYRLSFCRKTCGTC
ShK-Nle21
122





RSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC
ShK-A22
123





RSCIDTIPKSRCTAFQCKHSMEYRLSFCRKTCGTC
ShK-E22
124





RSCIDTIPKSRCTAFQCKHSMRYRLSFCRKTCGTC
ShK-R22
125





RSCIDTIPKSRCTAFQCKHSMXs22YRLSFCRKTCGTC
ShK-X22
126





RSCIDTIPKSRCTAFQCKHSM(norleu)YRLSFCRKTCGTC
ShK-Nle22
127





RSCIDTIPKSRCTAFQCKHSM(orn)YRLSFCRKTCGTC
ShK-Orn22
128





RSCIDTIPKSRCTAFQCKHSM(homocit)YRLSFCRKTCGTC
ShK-Homocit22
129





RSCIDTIPKSRCTAFQCKHSM(diaminopropionic)YRLS
ShK-Diamino-
130


FCRKTCGTC
propionic22





RSCIDTIPKSRCTAFQCKHSMKARLSFCRKTCGTC
ShK-A23
131





RSCIDTIPKSRCTAFQCKHSMKSRLSFCRKTCGTC
ShK-S23
132





RSCIDTIPKSRCTAFQCKHSMKFRLSFCRKTCGTC
ShK-F23
133





RSCIDTIPKSRCTAFQCKHSMKXs23RLSFCRKTCGTC
ShK-X23
134





RSCIDTIPKSRCTAFQCKHSMK(nitrophe)RLSECRKTCGTC
ShK-Nitrophe23
135





RSCIDTIPKSRCTAFQCKHSMK(aminophe)RLSFCRKTCGTC
ShK-Aminophe23
136





RSCIDTIPKSRCTAFQCKHSMK(benzylphe)RLSFCRKTCG
ShK-Benzylphe23
137


TC





RSCIDTIPKSRCTAFQCKHSMKYALSFCRKTCGTC
ShK-A24
138





RSCIDTIPKSRCTAFQCKHSMKYELSFCRKTCGTC
ShK-E24
139





RSCIDTIPKSRCTAFQCKHSMKYRASFCRKTCGTC
ShK-A25
140





RSCIDTIPKSRCTAFQCKHSMKYRLAFCRKTCGTC
ShK-A26
141





RSCIDTIPKSRCTAFQCKHSMKYRLSACRKTCGTC
ShK-A27
142





RSCIDTIPKSRCTAFQCKHSMKYRLSXs27CRKTCGTC
ShK-X27
143





RSCIDTIPKSRCTAFQCKHSMKYRLSFCAKTCGTC
ShK-A29
144





RSCIDTIPKSRCTAFQCKHSMKYRLSFCRATCGTC
ShK-A30
145





RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKACGTC
ShK-A31
146





RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGAC
ShK-A34
147





SCADTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-A4d1
148





SCADTIPKSRCTAAQCKHSMKYRLSFCRKTCGTC
ShK-A4/A15d1
149





SCADTIPKSRCTAAQCKHSMKYRASFCRKTCGTC
ShK-A4/A15/A25
150



d1





SCIDAIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-A6 d1
151





SCIDTAPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-A7 d1
152





SCIDTIAKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-A8 d1
153





SCIDTIPASRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-A9 d1
154





SCIDTIPESRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-E9 d1
155





SCIDTIPQSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-Q9 d1
156





SCIDTIPKARCTAFQCKHSMKYRLSFCRKTCGTC
ShK-A10 d1
157





SCIDTIPKSACTAFQCKHSMKYRLSFCRKTCGTC
ShK-A11 d1
158





SCIDTIPKSECTAFQCKHSMKYRLSFCRKTCGTC
ShK-E11 d1
159





SCIDTIPKSQCTAFQCKHSMKYRLSFCRKTCGTC
ShK-Q11 d1
160





SCIDTIPKSRCAAFQCKHSMKYRLSFCRKTCGTC
ShK-A13 d1
161





SCIDTIPKSRCTAAQCKHSMKYRLSFCRKTCGTC
ShK-A15 d1
162





SCIDTIPKSRCTAWQCKHSMKYRLSFCRKTCGTC
ShK-W15 d1
163





SCIDTIPKSRCTAXs15QCKHSMKYRLSFCRKTCGTC
ShK-X15 d1
164





SCIDTIPKSRCTAAQCKHSMKYRASFCRKTCGTC
ShK-A15/A25 d1
165





SCIDTIPKSRCTAFACKHSMKYRLSFCRKTCGTC
ShK-A16 d1
166





SCIDTIPKSRCTAFECKHSMKYRLSFCRKTCGTC
ShK-E16 d1
167





SCIDTIPKSRCTAFQCAHSMKYRLSFCRKTCGTC
ShK-A18 d1
168





SCIDTIPKSRCTAFQCEHSMKYRLSFCRKTCGTC
ShK-E18 d1
169





SCIDTIPKSRCTAFQCKASMKYRLSFCRKTCGTC
ShK-A19 d1
170





SCIDTIPKSRCTAFQCKKSMKYRLSFCRKTCGTC
ShK-K19 d1
171





SCIDTIPKSRCTAFQCKHAMKYRLSFCRKTCGTC
ShK-A20 d1
172





SCIDTIPKSRCTAFQCKHSAKYRLSFCRKTCGTC
ShK-A21 d1
173





SCIDTIPKSRCTAFQCKHSXs21KYRLSFCRKTCGTC
ShK-X21 d1
174





SCIDTIPKSRCTAFQCKHS(norleu)KYRLSFCRKTCGTC
ShK-Nle21 d1
175





SCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC
ShK-A22 d1
176





SCIDTIPKSRCTAFQCKHSMEYRLSFCRKTCGTC
ShK-E22 d1
177





SCIDTIPKSRCTAFQCKHSMRYRLSFCRKTCGTC
ShK-R22 d1
178





SCIDTIPKSRCTAFQCKHSMXs22YRLSFCRKTCGTC
ShK-X22 d1
179





SCIDTIPKSRCTAFQCKHSM(norleu)YRLSFCRKTCGTC
ShK-Nle22 d1
180





SCIDTIPKSRCTAFQCKHSM(orn)YRLSFCRKTCGTC
ShK-Orn22 d1
181





SCIDTIPKSRCTAFQCKHSM(homocit)YRLSFCRKTCGTC
ShK-Homocit22
182



d1





SCIDTIPKSRCTAFQCKHSM(diaminopropionic)YRLSF
ShK-Diamino-
183


CRKTCGTC
propionic22 d1





SCIDTIPKSRCTAFQCKHSMKARLSFCRKTCGTC
ShK-A23 d1
184





SCIDTIPKSRCTAFQCKHSMKSRLSFCRKTCGTC
ShK-S23 d1
185





SCIDTIPKSRCTAFQCKHSMKFRLSFCRKTCGTC
ShK-F23 d1
186





SCIDTIPKSRCTAFQCKHSMKXs23RLSFCRKTCGTC
ShK-X23 d1
187





SCIDTIPKSRCTAFQCKHSMK(nitrophe)RLSFCRKTCGTC
ShK-Nitrophe23
188



d1





SCIDTIPKSRCTAFQCKHSMK(aminophe)RLSFCRKTCGTC
ShK-Aminophe23
189



d1





SCIDTIPKSRCTAFQCKHSMK(benzylphe)RLSFCRKTCGTC
ShK-Benzylphe23
190



d1





SCIDTIPKSRCTAFQCKHSMKYALSFCRKTCGTC
ShK-A24 d1
191





SCIDTIPKSRCTAFQCKHSMKYELSFCRKTCGTC
ShK-E24 d1
192





SCIDTIPKSRCTAFQCKHSMKYRASFCRKTCGTC
ShK-A25 d1
193





SCIDTIPKSRCTAFQCKHSMKYRLAFCRKTCGTC
ShK-A26 d1
194





SCIDTIPKSRCTAFQCKHSMKYRLSACRKTCGTC
ShK-A27 d1
195





SCIDTIPKSRCTAFQCKHSMKYRLSXs27CRKTCGTC
ShK-X27 d1
196





SCIDTIPKSRCTAFQCKHSMKYRLSFCAKTCGTC
ShK-A29 d1
197





SCIDTIPKSRCTAFQCKHSMKYRLSFCRATCGTC
ShK-A30 d1
198





SCIDTIPKSRCTAFQCKHSMKYRLSFCRKACGTC
ShK-A31 d1
199





SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGAC
ShK-A34 d1
200





YSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-Y1
548





KSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-K1
549





HSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-H1
550





QSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-Q1
551





PPRSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
PP-ShK
552





MRSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
M-ShK
553





GRSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
G-ShK
554





YSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC
ShK-Y1/A22
555





KSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC
ShK-K1/A22
556





HSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC
ShK-H1/A22
557





QSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC
ShK-Q1/A22
558





PPRSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC
PP-ShK-A22
559





MRSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC
M-ShK-A22
560





GRSCIDTIPKSRCTAFQCKHSMAYRLSFCRKTCGTC
G-ShK-A22
561





RSCIDTIPASRCTAFQCKHSMAYRLSFCRKTCGTC
ShK-A9/A22
884





SCIDTIPASRCTAFQCKHSMAYRLSFCRKTCGTC
ShK-A9/A22 d1
885





RSCIDTIPVSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-V9
886





RSCIDTIPVSRCTAFQCKHSMAYRLSFCRKTCGTC
ShK-V9/A22
887





SCIDTIPVSRCTAFQCKHSMKYRLSFCRKTCGTC
ShK-V9 d1
888





SCIDTIPVSRCTAFQCKHSMAYRLSFCRKTCGTC
ShK-V9/A22 d1
889





RSCIDTIPESRCTAFQCKHSMAYRLSFCRKTCGTC
ShK-E9/A22
890





SCIDTIPESRCTAFQCKHSMAYRLSFCRKTCGTC
ShK-E9/A22 d1
891





RSCIDTIPKSACTAFQCKHSMAYRLSFCRKTCGTC
ShK-A11/A22
892





SCIDTIPKSACTAFQCKHSMAYRLSFCRKTCGTC
ShK-A11/22 d1
893





RSCIDTIPKSECTAFQCKHSMAYRLSFCRKTCGTC
ShK-E11/A22
894





SCIDTIPKSECTAFQCKHSMAYRLSFCRKTCGTC
ShK-E11/A22 d1
895





RSCIDTIPKSRCTDFQCKHSMKYRLSFCRKTCGTC
ShK-D14
896





RSCIDTIPKSRCTDFQCKHSMAYRLSFCRKTCGTC
ShK-D14/A22
897





SCIDTIPKSRCTDFQCKHSMKYRLSFCRKTCGTC
ShK-D14 d1
898





SCIDTIPKSRCTDFQCKHSMAYRLSFCRKTCGTC
ShK-D14/A22 d1
899





RSCIDTIPKSRCTAAQCKHSMAYRLSFCRKTCGTC
ShK-A15A/22
900





SCIDTIPKSRCTAAQCKHSMAYRLSFCRKTCGTC
ShK-A15/A22 d1
901





RSCIDTIPKSRCTAIQCKHSMKYRLSFCRKTCGTC
ShK-I15
902





RSCIDTIPKSRCTAIQCKHSMAYRLSFCRKTCGTC
ShK-I15/A22
903





SCIDTIPKSRCTAIQCKHSMKYRLSFCRKTCGTC
ShK-I15 d1
904





SCIDTIPKSRCTAIQCKHSMAYRLSFCRKTCGTC
ShK-I15/A22 d1
905





RSCIDTIPKSRCTAVQCKHSMKYRLSFCRKTCGTC
ShK-V15
906





RSCIDTIPKSRCTAVQCKHSMAYRLSFCRKTCGTC
ShK-V15/A22
907





SCIDTIPKSRCTAVQCKHSMKYRLSFCRKTCGTC
ShK-V15 d1
908





SCIDTIPKSRCTAVQCKHSMAYRLSFCRKTCGTC
ShK-V15/A22 d1
909





RSCIDTIPKSRCTAFRCKHSMKYRLSFCRKTCGTC
ShK-R16
910





RSCIDTIPKSRCTAFRCKHSMAYRLSFCRKTCGTC
ShK-R16/A22
911





SCIDTIPKSRCTAFRCKHSMKYRLSFCRKTCGTC
ShK-R16 d1
912





SCIDTIPKSRCTAFRCKHSMAYRLSFCRKTCGTC
ShK-R16/A22 d1
913





RSCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC
ShK-K16
914





RSCIDTIPKSRCTAFKCKHSMAYRLSFCRKTCGTC
ShK-K16/A22
915





SCIDTIPKSRCTAFKCKHSMKYRLSFCRKTCGTC
ShK-K16 d1
916





SCIDTIPKSRCTAFKCKHSMAYRLSFCRKTCGTC
ShK-K16/A22 d1
917





RSCIDTIPASECTAFQCKHSMKYRLSFCRKTCGTC
ShK-A9/E11
918





RSCIDTIPASECTAFQCKHSMAYRLSFCRKTCGTC
ShK-A9/E11/A22
919





SCIDTIPASECTAFQCKHSMKYRLSFCRKTCGTC
ShK-A9/E11 d1
920





SCIDTIPASECTAFQCKHSMAYRLSFCRKTCGTC
ShK-A9/E11/A22
921



d1





RSCIDTIPVSECTAFQCKHSMKYRLSFCRKTCGTC
ShK-V9/E11
922





RSCIDTIPVSECTAFQCKHSMAYRLSFCRKTCGTC
ShK-V9/E11/A22
923





SCIDTIPVSECTAFQCKHSMKYRLSFCRKTCGTC
ShK-V9/E11 d1
924





SCIDTIPVSECTAFQCKHSMAYRLSFCRKTCGTC
ShK-V9/E11/A22
925



d1





RSCIDTIPVSACTAFQCKHSMKYRLSFCRKTCGTC
ShK-V9/A11
926





RSCIDTIPVSACTAFQCKHSMAYRLSFCRKTCGTC
ShK-V9/A11/A22
927





SCIDTIPVSACTAFQCKHSMKYRLSFCRKTCGTC
ShK-V9/A11 d1
928





SCIDTIPVSACTAFQCKHSMAYRLSFCRKTCGTC
ShK-V9/A11/A22
929



d1





RSCIDTIPASACTAFQCKHSMKYRLSFCRKTCGTC
ShK-A9/A11
930





RSCIDTIPASACTAFQCKHSMAYRLSFCRKTCGTC
ShK-A9/A11/A22
931





SCIDTIPASACTAFQCKHSMKYRLSFCRKTCGTC
ShK-A9/A11 d1
932





SCIDTIPASACTAFQCKHSMAYRLSFCRKTCGTC
ShK-A9/A11/A22
933



d1





RSCIDTIPKSECTDIRCKHSMKYRLSFCRKTCGTC
ShK-
934



E11/D14/I15/R16





RSCIDTIPKSECTDIRCKHSMAYRLSFCRKTCGTC
ShK-
935



E11/D14/I15/R16/



A22





SCIDTIPKSECTDIRCKHSMKYRLSFCRKTCGTC
ShK-
936



E11/D14/I15/R16



d1





SCIDTIPKSECTDIRCKHSMAYRLSFCRKTCGTC
ShK-
937



E11/D14/I15//R16



A22 d1





RSCIDTIPVSECTDIRCKHSMKYRLSFCRKTCGTC
ShK-
938



V9/E11/D14/I15/



R16





RSCIDTIPVSECTDIRCKHSMAYRLSFCRKTCGTC
ShK-
939



V9/E11/D14/I15/



R16/A22





SCIDTIPVSECTDIRCKHSMKYRLSFCRKTCGTC
ShK-
940



V9/E11/D14/I15/



R16 d1





SCIDTIPVSECTDIRCKHSMAYRLSFCRKTCGTC
ShK-
941



V9/E11/D14/I15/



R16/A22 d1





RSCIDTIPVSECTDIQCKHSMKYRLSFCRKTCGTC
ShK-
942



V9/E11/D14/I15





RSCIDTIPVSECTDIQCKHSMAYRLSFCRKTCGTC
ShK-
943



V9/E11/D14/I15/A22





SCIDTIPVSECTDIQCKHSMKYRLSFCRKTCGTC
ShK-
944



V9/E11/D14/I15



d1





SCIDTIPVSECTDIQCKHSMAYRLSFCRKTCGTC
ShK-
945



V9/E11/D14/I15/A



22 d1





RTCKDLIPVSECTDIRCKHSMKYRLSFCRKTCGTC
ShK-
946



T2/K4/L6/V9/E11/



D14/I15/R16





RTCKDLIPVSECTDIRCKHSMAYRLSFCRKTCGTC
ShK-
947



T2/K4/L6/V9/E11/



D14/I15/R16/A22





TCKDLIPVSECTDIRCKHSMKYRLSFCRKTCGTC
ShK-
948



T2/K4/L6/V9/E11/



D14/I15/R16 d1





TCKDLIPVSECTDIRCKHSMAYRLSFCRKTCGTC
ShK-
949



T2/K4/L6/V9/E11/



D14/I15/R16/A22



d1





(L-Phosphotyrosine)-
ShK(L5)
950


AEEARSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC





QSCADTIPKSRCTAAQCKHSMKYRLSFCRKTCGTC
ShK Q1/A4/A15
1295





QSCADTIPKSRCTAAQCKHSMAYRLSFCRKTCGTC
ShK
1296



Q1/A4/A15/A22





QSCADTIPKSRCTAAQCKHSM(Dap)YRLSFCRKTCGTC
ShK
1297



Q1/A4/A15/Dap22





QSCADTIPKSRCTAAQCKHSMKYRASFCRKTCGTC
ShK
1298



Q1/A4/A15/A25





QSCADTIPKSRCTAAQCKHSMAYRASFCRKTCGTC
ShK
1299



Q1/A4/A15/A22/A25





QSCADTIPKSRCTAAQCKHSM(Dap)YRASFCRKTCGTC
ShK
1300



Q1/A4/A15/Dap22/



A25









Many peptides as described in Table 2 can be prepared as described in U.S. Pat. No. 6,077,680 issued Jun. 20, 2000 to Kem et al., which is hereby incorporated by reference in its entirety. Other peptides of Table 2 can be prepared by techniques known in the art. For example, ShK(L5) (SEQ ID NO: 950) can be prepared as described in Beeton et al., Targeting effector memory T cells with a selective peptide inhibitor of Kv1.3 channels for therapy of autoimmune diseases, Molec. Pharmacol. 67(4): 1369-81 (2005), which is hereby incorporated by reference in its entirety. In Table 2 and throughout the specification, Xs15, Xs21, Xs22, Xs23 and Xs27 each independently refer to nonfunctional amino acid residues.









TABLE 3







HmK, BgK, AeK and AsKS peptide and peptide analog sequences











Short-hand
SEQ



Sequence/structure
designation
ID NO:













RTCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC
HmK
6






ATCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC
HmK-A1
201





STCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC
HmK-S1
202





TCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC
HmK-d1
203





SCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC
HmK-d1/S2
204





TCIDLIPVSECTDIRCRTSMKYRLNLCRKTCGSC
HmK-d1/I4
205





TCKDTIPVSECTDIRCRTSMKYRLNLCRKTCGSC
HmK-d1/T6
206





TCKDLIPKSECTDIRCRTSMKYRLNLCRKTCGSC
HmK-d1/K9
207





TCKDLIPVSRCTDIRCRTSMKYRLNLCRKTCGSC
HmK-
208



d1/R11





TCKDLIPVSECTAIRCRTSMKYRLNLCRKTCGSC
HmK-
209



d1/A14





TCKDLIPVSECTDFRCRTSMKYRLNLCRKTCGSC
HmK-
210



d1/F15





TCKDLIPVSECTDIQCRTSMKYRLNLCRKTCGSC
HmK-
211



d1/Q16





TCKDLIPVSECTDIRCKTSMKYRLNLCRKTCGSC
HmK-
212



d1/K18





TCKDLIPVSECTDIRCRHSMKYRLNLCRKTCGSC
HmK-
213



d1/H19





TCKDLIPVSECTDIRCRTSMKYRLSLCRKTCGSC
HmK-
214



d1/S26





TCKDLIPVSECTDIRCRTSMKYRLNFCRKTCGSC
HmK-
215



d1/F27





TCKDLIPVSECTDIRCRTSMKYRLNLCRKTCGTC
HmK-
216



d1/T34





TCKDLIPVSRCTDIRCRTSMKYRLNFCRKTCGSC
HmK-
217



d1/R11/F27





ATCKDLIPVSRCTDIRCRTSMKYRLNFCRKTCGSC
HmK-
218



A1/R11/F27





TCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGSC
HmK-d1/Z1
219





TCIDTIPKSRCTAFQCRTSMKYRLNFCRKTCGSC
HmK-d1/Z2
220





TCADLIPASRCTAIACRTSMKYRLNFCRKTCGSC
HmK-d1/Z3
221





TCADLIPASRCTAIACKHSMKYRLNFCRKTCGSC
HmK-d1/Z4
222





TCADLIPASRCTAIACAHSMKYRLNFCRKTCGSC
HmK-d1/Z5
223





RTCKDLIPVSECTDIRCRTSMXh22YRLNLCRKTCGSC
HmK-X22
224





ATCKDLXh6PVSRCTDIRCRTSMKXh22RLNXh26CRKTCGSC
HmK-X6,
225



22, 26





VCRDWFKETACRHAKSLGNCRTSQKYRANCAKTCELC
BgK
9





ACRDWFKETACRHAKSLGNCRTSQKYRANCAKTCELC
BgK-A1
226





VCADWFKETACRHAKSLGNCRTSQKYRANCAKTCELC
BgK-A3
227





VCRDAFKETACRHAKSLGNCRTSQKYRANCAKTCELC
BgK-A5
228





VCRDWFKATACRHAKSLGNCRTSQKYRANCAKTCELC
BgK-A8
229





VCRDWFKEAACRHAKSLGNCRTSQKYRANCAKTCELC
BgK-A9
230





VCRDWFKETACAHAKSLGNCRTSQKYRANCAKTCELC
BgK-A12
231





VCRDWFKETACRHAASLGNCRTSQKYRANCAKTCELC
BgK-A15
232





VCRDWFKETACRHAKALGNCRTSQKYRANCAKTCELC
BgK-A16
233





VCRDWFKETACRHAKSAGNCRTSQKYRANCAKTCELC
BgK-A17
234





VCRDWFKETACRHAKSLGNCATSQKYRANCAKTCELC
BgK-A21
235





VCRDWFKETACRHAKSLGNCRASQKYRANCAKTCELC
BgK-A22
236





VCRDWFKETACRHAKSLGNCRTSQKYAANCAKTCELC
BgK-A27
237





VCRDWFKETACRHAKSLGNCRTSQKYRANCAATCELC
BgK-A32
238





VCRDWFKETACRHAKSLGNCRTSQKYRANCAKACELC
BgK-A33
239





VCRDWFKETACRHAKSLGNCRTSQKYRANCAKTCALC
BgK-A35
240





VCRDWFKETACRHAKSLGNCRTSQKYRANCAKTCEAC
BgK-A37
241





GCKDNFSANTCKHVKANNNCGSQKYATNCAKTCGKC
AeK
7





ACKDNFAAATCKHVKENKNCGSQKYATNCAKTCGKC
AsKS
8





In Table 3 and throughout the specification, Xh6, Xh22, Xh26 are each independently nonfunctional residues.













TABLE 4







MgTx peptide and MgTx peptide analog sequences











Short-hand
SEQ



Sequence/structure
designation
ID NO:













TIINVKCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH
MgTx
28






TIINVACTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH
MgTx-A6
242





TIINVSCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH
MgTx-S6
243





TIINVKCTSPAQCLPPCKAQFGQSAGAKCMNGKCKCYPH
MgTx-A11
244





TIINVKCTSPKQCLPPCAAQFGQSAGAKCMNGKCKCYPH
MgTx-A18
245





TIINVKCTSPKQCLPPCKAQFGQSAGAACMNGKCKCYPH
MgTx-A28
246





TIINVKCTSPKQCLPPCKAQFGQSAGAKCMNGACKCYPH
MgTx-A33
247





TIINVKCTSPKQCLPPCKAQFGQSAGAKCMNGKCACYPH
MgTx-A35
248





TIINVKCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPN
MgTx-H39N
249





TIINVACTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPN
MgTx-
250



A6/H39N





TIINVSCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYS
MgTx-
251



S6/38/d39





TIITISCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH
MgTx-T4/I5/S6
252





TISCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH
MgTx-
253



d3/T4/I5/S6





TISCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCFGR
MgTx-Pi2
254





NVACTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH
MgTx-d3/A6
255





QFTNVSCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYS
MgTx-ChTx
256





QFTDVDCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYQ
MgTx-IbTx
257





IINVSCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH
MgTx-Z1
258





IITISCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH
MgTx-Z2
259





GVIINVSCTSPKQCLPPCKAQFGQSAGAKCMNGKCKCYPH
MgTx-Z3
260









Many peptides as described in Table 4 can be prepared as described in WO 95/03065, published Feb. 2, 1995, for which the applicant is Merck & Co., Inc. That application corresponds to U.S. Ser. No. 07/096,942, filed 22 Jul. 1993, which is hereby incorporated by reference in its entirety.









TABLE 5







AgTx2 peptide and AgTx2 peptide analog sequences











Short-hand




Sequence/structure
designation
SEQ ID NO:













GVPINVSCTGSPQCIKPCKDAGMRFGKCMNRKCHCTPK
AgTx2
23






GVPIAVSCTGSPQCIKPCKDAGMRFGKCMNRKCHCTPK
AgTx2-A5
261





GVPINVSCTGSPQCIAPCKDAGMRFGKCMNRKCHCTPK
AgTx2-A16
262





GVPINVSCTGSPQCIKPCADAGMRFGKCMNRKCHCTPK
AgTx2-A19
263





GVPINVSCTGSPQCIKPCKDAGMAFGKCMNRKCHCTPK
AgTx2-A24
264





GVPINVSCTGSPQCIKPCKDAGMRFGACMNRKCHCTPK
AgTx2-A27
265





GVPINVSCTGSPQCIKPCKDAGMRFGKCMNAKCHCTPK
AgTx2-A31
266





GVPINVSCTGSPQCIKPCKDAGMRFGKCMNRACHCTPK
AgTx2-A32
267





GVPINVSCTGSPQCIKPCKDAGMRFGKCMNRKCHCTPA
AgTx2-A38
268





GVPIAVSCTGSPQCIKPCKDAGMRFGKCMNRKCHCTPA
Agx2-A5/A38
269





GVPINVSCTGSPQCTKPCKDAGMRFGKCMNGKCHCTPK
AgTx2-G31
270





GVPIIVSCKGSRQCIKPCKDAGMRFGKCMNGKCHCTPK
AgTx2-
271



OSK_z1





GVPIIVSCKISRQCIKPCKDAGMRFGKCMNGKCHCTPK
AgTx2-
272



OSK_z2





GVPIIVKCKGSRQCIKPCKDAGMRFGKCMNGKCHCTPK
AgTx2-
273



OSK_z3





GVPIIVKCKISRQCIKPCKDAGMRFGKCMNGKCHCTPK
AgTx2-
274



OSK_z4





GVPIIVKCKISRQCIKPCKDAGMRFGKCMNGKCHCTPK
AgTx2-
275



OSK_z5
















TABLE 6







Heteromitrus spinnifer (HsTx1) peptide


and HsTx1 peptide analog sequences











SEQ



Short-hand
ID


Sequence/structure
designation
NO:












ASCRTPKDCADPCRKETGCPYGKCMNRKCKCNRC
HsTx1
61





ASCXTPKDCADPCRKETGCPYGKCMNRKCKCNRC
HsTx1-X4
276





ASCATPKDCADPCRKETGCPYGKCMNRKCKCNRC
HsTx1-A4
277





ASCRTPXDCADPCRKETGCPYGKCMNRKCKCNRC
HsTx1-X7
278





ASCRTPADCADPCRKETGCPYGKCMNRKCKCNRC
HsTx1-A7
279





ASCRTPKDCADPCXKETGCPYGKCMNRKCKCNRC
HsTx1-X14
280





ASCRTPKDCADPCAKETGCPYGKCMNRKCKCNRC
HsTx1-A14
281





ASCRTPKDCADPCRXETGCPYGKCMNRKCKCNRC
HsTx1-X15
282





ASCRTPKDCADPCRAETGCPYGKCMNRKCKCNRC
HsTx1-A15
283





ASCRTPKDCADPCRKETGCPYGXCMNRKCKCNRC
HsTx1-X23
284





ASCRTPKDCADPCRKETGCPYGACMNRKCKCNRC
HsTx1-A23
285





ASCRTPKDCADPCRKETGCPYGKCMNXKCKCNRC
HsTx1-X27
286





ASCRTPKDCADPCRKETGCPYGKCMNAKCKCNRC
HsTx1-A27
287





ASCRTPKDCADPCRKETGCPYGKCMNRXCKCNRC
HsTx1-X28
288





ASCRTPKDCADPCRKETGCPYGKCMNRACKCNRC
HsTx1-A28
289





ASCRTPKDCADPCRKETGCPYGKCMNRKCXCNRC
HsTx1-X30
290





ASCRTPKDCADPCRKETGCPYGKCMNRKCACNRC
HsTx1-A30
291





ASCRTPKDCADPCRKETGCPYGKCMNRKCKCNXC
HsTx1-X33
292





ASCRTPKDCADPCRKETGCPYGKCMNRKCKCNAC
HsTx1-A33
293









Peptides as described in Table 5 can be prepared as described in U.S. Pat. No. 6,689,749, issued Feb. 10, 2004 to Lebrun et al., which is hereby incorporated by reference in its entirety.










TABLE 7







Orthochirus scrobiculosus (OSK1) peptide



and OSK1 peptide analog sequences












SEQ




Short-hand
ID


Sequence/structure
designation
NO:













GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK
OSK1
25






GVIINVSCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK
OSK1-S7
1303





GVIINVKCKISRQCLKPCKKAGMRFGKCMNGKCHCTPK
OSK1-K16
294





GVIINVKCKISRQCLEPCKDAGMRFGKCMNGKCHCTPK
OSK1-D20
295





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
OSK1-K16, D20
296





GVIINVSCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
OSK1-S7, K16, D20
1308





GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK
OSK1-P12, K16, D20
297





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYPK
OSK1-K16, D20, Y36
298





Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK
Ac-OSK1-P12,
562



K16, D20





GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
OSK1-P12, K16,
563



D20-NH2





Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
Ac-OSK1-P12,
564



K16, D20-NH2





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYPK-NH2
OSK1-K16, D20,
565



Y36-NH2





Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYPK
Ac-OSK1-K16,
566



D20, Y36





Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYPK-NH2
Ac-OSK1-K16, D20,
567



Y36-NH2





GVIINVKCKISRQCLKPCKKAGMRFGKCMNGKCHCTPK-NH2
OSK1-K16-NH2
568





Ac-GVIINVKCKISRQCLKPCKKAGMRFGKCMNGKCHCTPK
Ac-OSK1-K16
569





Ac-GVIINVKCKISRQCLKPCKKAGMRFGKCMNGKCHCTPK-NH2
Ac-OSK1-K16-NH2
570





Ac-GVIINVKCKISRQCLEPCKDAGMRFGKCMNGKCHCTPK
Ac-OSK1-D20
571





GVIINVKCKISRQCLEPCKDAGMRFGKCMNGKCHCTPK-NH2
OSK1-D20-NH2
572





Ac-GVIINVKCKISRQCLEPCKDAGMRFGKCMNGKCHCTPK-NH2
Ac-QSK1-D20-NH2
573





GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH2
OSK1-NH2
574





Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK
Ac-OSK1
575





Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH2
Ac-OSK1-NH2
576





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
OSK1-K16, D20-NH2
577





Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
Ac-OSK1-K16,
578



D20





Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
Ac-OSK1-K16, D20-
579



NH2





VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK
Δ1-OSK1
580





Ac-VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK
Ac-Δ1-OSK1
581





VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH2
Δ1-OSK1-NH2
582





Ac-VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH2
Ac-Δ1-OSK1-NH2
583





GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK
OSK1-A34
584





Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK
Ac-OSK1-A34
585





GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK-NH2
OSK1-A34-NH2
586





Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK-NH2
Ac-OSK1-A34-NH2
587





VIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
Δ1-OSK1-K16, D20
588





Ac-VIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
Ac-Δ1-OSK1-K16,
589



D20





VIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
Δ1-OSK1-K16, D20-
590



NH2





Ac-VIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
Ac-Δ1-OSK1-K16,
591



D20-NH2





NVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
(Δ1-4)-OSK1-K16,
592



D20





Ac-NVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
Ac-(Δ1-4)-OSK1-
593



K16, D20





NVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
(Δ1-4)-OSK1-K16,
594



D20-NH2





Ac-NVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
Ac-(Δ1-4)-OSK1-
595



K16, D20-NH2





KCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
(Δ1-6)-OSK1-K16,
596



D20





Ac-KCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
Ac-(Δ1-6)-OSK1-
597



K16, D20





KCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
(Δ1-6)-OSK1-K16,
598



D20-NH2





Ac-KCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
Ac-(Δ1-6)-OSK1-
599



K16, D20-NH2





CKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
(Δ1-7)-OSK1-K16,
600



D20





Ac-CKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
Ac-(Δ1-7)-OSK1-
601



K16, D20





CKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
(Δ1-7)-OSK1-K16,
602



D20-NH2





Ac-CKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
Ac-(Δ1-7)-OSK1-
603



K16, D20-NH2





GVIINVKCKISRQCLKPCKDAGMRNGKCMNGKCHCTPK
OSK1-K16, D20,
604



N25





GVIINVKCKISRQCLKPCKDAGMRNGKCMNGKCHCTPK-NH2
OSK1-K16, D20,
605



N25-NH2





Ac-GVIINVKCKISRQCLKPCKDAGMRNGKCMNGKCHCTPK
Ac-OSK1-K16,
606



D20, N25





Ac-GVIINVKCKISRQCLKPCKDAGMRNGKCMNGKCHCTPK-NH2
Ac-OSK1-K16, D20,
607



N25-NH2





GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK
OSK1-K16, D20,
608



R31





GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-NH2
OSK1-K16, D20,
609



R31-NH2





Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK
Ac-OSK1-K16,
610



D20, R31





Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-NH2
Ac-OSK1-K16, D20,
611



R31-NH2





GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK
OSK1-K12, K16,
612



R19, D20





Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK
Ac-OSK1-K12, K16,
613



R19, D20





GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK-NH2
OSK1-K12, K16,
614



R19, D20-NH2





Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK-NH2
Ac-OSK1-K12, K16,
615



R19, D20-NH2





TIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
Δ1-OSK1-T2, K16,
616



D20





Ac-TIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
Ac-Δ1-OSK1-T2,
617



K16, D20





TIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
Δ1-OSK1-T2, K16,
618



D20-NH2





Ac-TIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
Ac-Δ1-OSK1-T2,
619



K16, D20-NH2





GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK
OSK1-K3
620





Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK
Ac-OSK1-K3
621





GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH2
OSK1-K3-NH2
622





Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-NH2
AC-OSK1-K3-NH2
623





GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK
OSK1-K3, A34
624





GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
OSK1-K3, K16, D20
625





GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCACTPK
OSK1-K3, K16, D20,
626



A34





Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK
Ac-OSK1-K3, A34
627





GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK-NH2
OSK1-K3, A34-NH2
628





Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK-NH2
Ac-OSK1-K3, A34-
629



NH2





Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCACTPK
Ac-OSK1-K3, K16,
630



D20, A34





GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCACTPK-NH2
OSK1-K3, K16, D20,
631



A34-NH2





Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCACTPK-NH2
Ac-OSK1-K3, K16,
632



D20, A34-NH2





Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK
Ac-OSK1-K3, K16,
633



D20





GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
OSK1-K3, K16, D20-
634



NH2





Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK-NH2
Ac-OSK1-K3, K16,
635



D20-NH2





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCT
Δ36-38-OSK1-K16,
636



D20





GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCTPK
OSK1-O16, D20
980





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCTPK
OSK1-hLys
981



16, D20





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCTPK
OSK1-hArg
982



16, D20





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCTPK
OSK1-Cit 16, D20
983





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK
OSK1-hCit
984



16, D20





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCTPK
OSK1-Dpr 16, D20
985





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCHCTPK
OSK1-Dab 16, D20
986





GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCYPK
OSK1-O16, D20, Y36
987





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCYPK
OSK1-hLys
988



16, D20, Y36





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCYPK
OSK1-hArg
989



16, D20, Y36





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCYPK
OSK1-Cit
990



16, D20, Y36





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCYPK
OSK1-hCit
991



16, D20, Y36





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCYPK
OSK1-Dpr
992



16, D20, Y36





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCHCYPK
OSK1-Dab
993



16, D20, Y36





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYPK
OSK1-
994



K16, D20, A34, Y36





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCGCYPK
OSK1-
995



K16, D20, G34, Y36





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACFPK
OSK1-
996



K16, D20, A34, F36





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACWPK
OSK1-
997



K16, D20, A34, W36





GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACYPK
OSK1-
998



K16, E20, A34, Y36





GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACTPK
OSK1-O16, D20, A34
999





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACTPK
OSK1-hLys
1000



16, D20, A34





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACTPK
OSK1-hArg
1001



16, D20, A34





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACTPK
OSK1-Cit
1002



16, D20, A34





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK
OSK1-hCit
1003



16, D20, A34





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACTPK
OSK1-Dpr
1004



16, D20, A34





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACTPK
OSK1-Dab
1005



16, D20, A34





GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHC
Δ36-38, OSK1-
1006



O16, D20,





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHC
Δ36-38, OSK1-
1007



hLys 16, D20





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHC
Δ36-38, OSK1-
1008



hArg 16, D20





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHC
Δ36-38, OSK1-Cit
1009



16, D20





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHC
Δ36-38, OSK1-
1010



hCit 16, D20





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHC
Δ36-38, OSK1-Dpr
1011



16, D20





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCHC
Δ36-38, OSK1-
1012



Dab16, D20





GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCAC
Δ36-38, OSK1-
1013



O16, D20, A34





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCAC
Δ36-38, OSK1-
1014



hLys 16, D20, A34





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCAC
Δ36-38, OSK1-
1015



hArg 16, D20, A34





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCAC
Δ36-38, OSK1-cit
1016



16, D20, A34





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHC
Δ36-38, OSK1-
1017



hCit 16, D20, A34





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCAC
Δ36-38, OSK1-Dpr
1018



16, D20, A34





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCAC
Δ36-38, OSK1-Dab
1019



16, D20, A34





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCGCYGG
OSK1-
1020



K16, D20, G34, Y36, G37,



G38





GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCYGG
OSK1-
1021



O16, D20, Y36, G37, G38





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCYGG
OSK1-hLys
1022



16, D20, Y36, G37, G38





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCYGG
OSK1-hArg
1023



16, D20, Y36, G37, G38





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCYGG
OSK1-Cit
1024



16, D20, Y36, G37, G38





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCYGG
OSK1-hCit
1025



16, D20, Y36, G37, G38





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCYGG
OSK1-Dpr
1026



16, D20, Y36, G37, G38





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYGG
OSK1-
1027



K16, D20, A34, Y36, G37,



G38





GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACYGG
OSK1-
1028



O16, D20, A34, Y36, G37,



G38





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACYGG
OSK1-hLys
1029



16, D20, A34, Y36, G37,



G38





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACYGG
OSK1-hArg
1030



16, D20, A34, Y36, G37,



G38





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACYGG
OSK1-Cit
1031



16, D20, A34, Y36, G37,



G38





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCYGG
OSK1-hCit
1032



16, D20, A34, Y3, G37,



G38





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACYGG
OSK1-Dpr
1033



16, D20, A34, Y36, G37,



G38





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACYGG
OSK1-Dab
1034



16, D20, A34, Y36, G37,



G38





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYG
Δ38, OSK1-
1035



K16, D20, A34, Y36, G37





GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCGGG
OSK1-
1036



O16, D20, G36-38





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCGGG
OSK1-hLys
1037



16, D20, G36-38





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCGGG
OSK1-hArg
1038



16, D20, G36-38





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCGGG
OSK1-Cit
1039



16, D20, G36-38





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCGGG
OSK1-hCit
1040



16, D20, G36-38





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCGGG
OSK1-Dpr
1041



16, D20, G36-38





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACFGG
OSK1-
1042



K16, D20, A34, F36, G37,



G38





GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACGGG
OSK1-
1043



O16, D20, A34, G36-



38





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACGGG
OSK1-hLys
1044



16, D20, A34, G36-38





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACGGG
OSK1-hArg
1045



16, D20, A34, G36-38





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACGGG
OSK1-Cit
1046



16, D20, A34, G36-38





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCACGGG
OSK1-hCit
1047



16, D20, A34, G36-38





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACGGG
OSK1-Dpr
1048



16, D20, A34, G36-38





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACGGG
OSK1-Dab
1049



16, D20, A34, G36-38





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACGG
Δ38, OSK1-
1050



K16, D20, A34, G36-



37





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYG
Δ38, OSK1-
1051



K16, D20, A35, Y36, G37





GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACGG
Δ38, OSK1-
1052



O16, D20, A35, Y36,



G37





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCTPK
OSK1-hLys
1053



16, E20





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCTPK
OSK1-hArg
1054



16, E20





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCTPK
OSK1-Cit 16, E20
1055





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCTPK
OSK1-hCit
1056



16, E20





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCTPK
OSK1-Dpr 16, E20
1057





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCTPK
OSK1-Dab 16, E20
1058





GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCYPK
OSK1-O16, E20, Y36
1059





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCYPK
OSK1-hLys
1060



16, E20, Y36





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCYPK
OSK1-hArg
1061



16, E20, Y36





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCYPK
OSK1-Cit
1062



16, E20, Y36





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYPK
OSK1-hCit
1063



16, E20, Y36





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCYPK
OSK1-Dpr
1064



16, E20, Y36





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCYPK
OSK1-Dab
1065



16, E20, Y36





GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACTPK
OSK1-O16, E20, A34
1066





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACTPK
OSK1-hLys
1067



16, E20, A34





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACTPK
OSK1-hArg
1068



16, E20, A34





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACTPK
OSK1-Cit
1069



16, E20, A34





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCTPK
OSK1-hCit
1070



16, E20, A34





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACTPK
OSK1-Dpr
1071



16, E20, A34





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACTPK
OSK1-Dab
1072



16, E20, A34





GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHC
Δ36-38, OSK1-
1073



O16, E20,





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHC
Δ36-38, OSK1-
1074



hLys 16, E20





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHC
Δ36-38, OSK1-
1075



hArg 16, E20





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHC
Δ36-38, OSK1-Cit
1076



16, E20





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHC
Δ36-38, OSK1-
1077



hCit16, E20





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHC
Δ36-38, OSK1-Dpr
1078



16, E20





GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCAC
Δ36-38, OSK1-
1079



O16, E20, A34





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCAC
Δ36-38, OSK1-
1080



hLys 16, E20, A34





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCAC
Δ36-38, OSK1-
1081



hArg 16, E20, A34





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCAC
Δ36-38, OSK1-Cit
1082



16, E20, A34





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHC
Δ36-38, OSK1-
1083



hCit 16, E20, A34





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCAC
Δ36-38, OSK1-Dpr
1084



16, E20, A34





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCAC
Δ36-38, OSK1-Dab
1085



16, E20, A34





GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCHCYGG
OSK1-
1086



K16, E20, Y36, G37, G38





GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCYGG
OSK1-
1087



O16, E20, Y36, G37, G38





GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCHCYG
Δ38 OSK1-
1088



K16, E20, Y36, G37





GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACYG
Δ38 OSK1-
1089



K16, E20, A34,



Y36, G37





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCYGG
OSK1-hLys
1090



16, E20, Y36, G37, G38





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCYGG
OSK1-hArg
1091



16, E20, Y36, G37, G38





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCYGG
OSK1-Cit
1092



16, E20, Y36, G37, G38





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG
Δ37-38, OSK1-
1093



hCit



16, E20, Y36, G37, G38





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCYGG
OSK1-Dpr
1094



16, E20, Y36, G37, G38





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCYGG
OSK1-Dab
1095



16, E20, Y36, G37, G38





GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACYG
Δ38, OSK1-
1096



K16, E20, A34, Y36, G37





GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACYGG
OSK1-
1097



O16, E20, A34, Y36, G37,



G38





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACYGG
OSK1-hLys
1098



16, E20, A34, Y36, G37,



G38





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACYGG
OSK1-hArg
1099



16, E20, A34, Y36, G37,



G38





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACYGG
OSK1-Cit
1100



16, E20, A34, Y36, G37,



G38





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG
OSK1-hCit
1101



16, E20, A34, Y3, G37,



G38





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACYGG
OSK1-Dpr
1102



16, E20, A34, Y36, G37,



G38





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACYGG
OSK1-Dab
1103



16, E20, A34, Y36, G37,



G38





GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACFGG
OSK1-
1104



K16, D20, A34, F36, G37,



G38





GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCGGG
OSK1-
1105



O16, E20, G36-38





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCGGG
OSK1-hLys
1106



16, E20, G36-38





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCGGG
OSK1-hArg
1107



16, E20, G36-38





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCGGG
OSK1-Cit
1108



16, E20, G36-38





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCGGG
OSK1-hCit
1109



16, E20, G36-38





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCGGG
OSK1-Dpr
1110



16, E20, G36-38





GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACGGG
OSK1-
1111



O16, E20, A34, G36-



38





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACGGG
OSK1-hLys
1112



16, E20, A34, G36-38





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACGGG
OSK1-hArg
1113



16, E20, A34, G36-38





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACGGG
OSK1-Cit
1114



16, E20, A34, G36-38





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCACTP
OSK1-hCit
1115



16, E20, A34, G36-38





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACTP
OSK1-Dpr
1116



16, E20, A34, G36-38





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACTP
OSK1-Dab
1117



16, E20, A34, G36-38





GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCTPK-NH2
OSK1-O16, D20-
1118



amide





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCTPK-
OSK1-hLys
1119


NH2
16, D20-amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCTPK-
OSK1-hArg
1120


NH2
16, D20-amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCTPK-NH2
OSK1-Cit
1121



16, D20-amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK-
OSK1-hCit
1122


NH2
16, D20-amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCTPK-NH2
OSK1-Dpr
1123



16, D20-amide





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCHCTPK-NH2
OSK1-Dab 16, D20
1124





GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCYPK-NH2
OSK1-
1125



O16, D20, Y36-



amide





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCYPK-
OSK1-hLys
1126


NH2
16, D20, Y36-



amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCYPK-
OSK1-hArg
1127


NH2
16, D20, Y36-



amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCYPK-NH2
OSK1-Cit
1128



16, D20, Y36-



amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCYPK-
OSK1-hCit
1129


NH2
16, D20, Y36-



amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCYPK-NH2
OSK1-Dpr
1130



16, D20, Y36-



amide





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCHCYPK-NH2
OSK1-Dab
1131



16, D20, Y36-



amide





GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACTPK-NH2
OSK1-
1132



O16, D20, A34-



amide





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACTPK-
OSK1-hLys
1133


NH2
16, D20, A34-



amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACTPK-
OSK1-hArg
1134


NH2
16, D20, A34-



amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACTPK-NH2
OSK1-Cit
1135



16, D20, A34-



amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCACTPK-
OSK1-hCit
1136


NH2
16, D20, A34-



amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACTPK-NH2
OSK1-Dpr
1137



16, D20, A34-



amide





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACTPK-NH2
OSK1-Dab
1138



16, D20, A34-



amide





GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-
1139



O16, D20, -



amide





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-
1140



hLys 16, D20-



amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-
1141



hArg 16, D20-



amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-Cit
1142



16, D20-amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-
1143



hCit16, D20-



amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-Dpr
1144



16, D20-amide





GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCAC-NH2
Δ36-38, OSK1-
1145



O16, D20, A34-



amide





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCAC-NH2
Δ36-38, OSK1-
1146



hLys 16, D20, A34-



amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCAC-NH2
Δ36-38, OSK1-
1147



hArg 16, D20, A34-



amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCAC-NH2
Δ36-38, OSK1-Cit
1148



16, D20, A34-



amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-
1149



hCit16, D20, A34





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCAC-NH2
Δ36-38, OSK1-Dpr
1150



16, D20, A34-



amide





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCAC-NH2
Δ36-38, OSK1-Dab
1151



16, D20, A34-



amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYGG-NH2
OSK1-
1152



O16, D20, Y36, G37, G38-



amide





GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCYGG-NH2
OSK1-
1153



O16, D20, Y36, G37, G38





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCYGG-
OSK1-hLys
1154


NH2
16, D20, Y36, G37, G38-



amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCYGG-
OSK1-hArg
1155


NH2
16, D20, Y36, G37, G38-



amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCYGG-NH2
OSK1-Cit
1156



16, D20, Y36, G37, G38-



amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCYGG-
OSK1-
1157


NH2
hCit16, D20, Y36, G37,



G38-amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCYGG-NH2
OSK1-Dpr
1158



16, D20, Y36, G37, G38-



amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCFGG-NH2
OSK1-
1159



K16, D20, F36, G37, G38-



amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCYG-NH2
Δ38-OSK1-
1160



K16, D20, Y36, G37-



amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYG-NH2
Δ38-OSK1-
1161



K16, D20, A34,



Y36, G37-amide





GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACYGG-NH2
OSK1-
1162



O16, D20, A34, Y36, G37,



G38-amide





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACYGG-
OSK1-hLys
1163


NH2
16, D20, A34, Y36, G37,



G38-amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACYGG-
OSK1-hArg
1164


NH2
16, D20, A34, Y36, G37,



G38-amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACYGG-NH2
OSK1-Cit
1165



16, D20, A34, Y36, G37,



G38





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCACYGG-
OSK1-
1166


NH2
hCit16, D20, A34, Y3,



G37, G38-amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACYGG-NH2
OSK1-Dpr
1167



16, D20, A34, Y36, G37,



G38-amide





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACYGG-NH2
OSK1-Dab
1168



16, D20, A34, Y36, G37,



G38-amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYGG-NH2
OSK1-
1169



K16, D20, A34, Y36, G37,



G38-amide





GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCHCGGG-NH2
OSK1-
1170



O16, D20, G36-38-



amide





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCHCGGG-
OSK1-hLys
1171


NH2
16, D20, G36-38-



amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCHCGGG-
OSK1-hArg
1172


NH2
16, D20, G36-38-



amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCHCGGG-NH2
OSK1-Cit
1173



16, D20, G36-38-



amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCHCGGG-
OSK1-
1174


NH2
hCit16, D20, G36-



38-amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCHCGGG-NH2
OSK1-Dpr
1175



16, D20, G36-38-



amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACGGG-NH2
OSK1-
1176



K16, D20, A34, G36-



38-amide





GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACFGG-NH2
OSK1-
1177



O16, D20, A34, F36,



G37-38-amide





GVIINVKCKISRQCLOPCKDAGMRFGKCMNGKCACGGG-NH2
OSK1-
1178



O16, D20, A34, G36-



38-amide





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMNGKCACGGG-
OSK1-hLys
1179


NH2
16, D20, A34, G36-



38-amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMNGKCACGGG-
OSK1-hArg
1180


NH2
16, D20, A34, G36-



38-amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMNGKCACGGG-NH2
OSK1-Cit
1181



16, D20, A34, G36-



38-amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMNGKCACGGG-
OSK1-
1182


NH2
hCit16, D20, A34, G36-



38-amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMNGKCACGGG-NH2
OSK1-Dpr
1183



16, D20, A34, G36-



38-amide





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMNGKCACGGG-NH2
OSK1-Dab
1184



16, D20, A34, G36-



38-amide





GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCTPK-NH2
OSK1-O16, E20-
1185



amide





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCTPK-
OSK1-hLys
1186


NH2
16, E20-amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCTPK-
OSK1-hArg
1187


NH2
16, E20-amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCTPK-NH2
OSK1-Cit
1188



16, E20-amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCTPK-
OSK1-
1189


NH2
hCit16, E20-



amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCTPK-NH2
OSK1-Dpr
1190



16, E20-amide





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCTPK-NH2
OSK1-Dab
1191



16, E20-amide





GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCYPK-NH2
OSK1-
1192



O16, E20, Y36-



amide





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCYPK-
OSK1-hLys
1193


NH2
16, E20, Y36-



amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCYPK-
OSK1-hArg
1194


NH2
16, E20, Y36-



amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCYPK-NH2
OSK1-Cit
1195



16, E20, Y36-



amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYPK-
OSK1-
1196


NH2
hCit16, E20, Y36-



amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCYPK-NH2
OSK1-Dpr
1197



16, E20, Y36-



amide





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCYPK-NH2
OSK1-Dab
1198



16, E20, Y36-



amide





GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACTPK-NH2
OSK1-
1199



O16, E20, A34-



amide





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACTPK-
OSK1-hLys
1200


NH2
16, E20, A34-



amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACTPK-
OSK1-hArg
1201


NH2
16, E20, A34-



amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACTPK-NH2
OSK1-Cit
1202



16, E20, A34-



amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCACTPK-
OSK1-
1203


NH2
hCit16, E20, A34-



amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACTPK-NH2
OSK1-Dpr
1204



16, E20, A34-



amide





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACTPK-NH2
OSK1-Dab
1205



16, E20, A34-



amide





GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-
1206



O16, E20, -



amide





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-
1207



hLys 16, E20-



amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-
1208



hArg 16, E20-



amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-Cit
1209



16, E20-amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-
1210



hCit16, E20-



amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-Dpr
1211



16, E20-amide





GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCAC-NH2
Δ36-38, OSK1-
1212



O16, E20, A34-



amide





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCAC-NH2
Δ36-38, OSK1-
1213



hLys16, E20, A34-



amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCAC-NH2
Δ36-38, OSK1-
1214



hArg16, E20, A34-



amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCAC-NH2
Δ36-38, OSK1-Cit
1215



16, E20, A34-



amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHC-NH2
Δ36-38, OSK1-
1216



hCit16, E20, A34-



amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCAC-NH2
Δ36-38, OSK1-Dpr
1217



16, E20, A34-



amide





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCAC-NH2
Δ36-38, OSK1-Dab
1218



16, E20, A34-



amide





GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCHCWGG-NH2
OSK1-
1219



O16, E20, W36, G37,



G38-amide





GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCYGG-NH2
OSK1-
1220



O16, E20, Y36, G37, G38-



amide





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCYGG-
OSK1-hLys
1221


NH2
16, E20, Y36, G37, G38-



amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCYGG-
OSK1-hArg
1222


NH2
16, E20, Y36, G37, G38-



amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCYGG-NH2
OSK1-Cit
1223



16, E20, Y36, G37, G38-



amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG-
OSK1-hCit
1224


NH2
16, E20, Y36, G37, G38-



amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCYGG-NH2
OSK1-Dpr
1225



16, E20, Y36, G37, G38-



amide





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCHCYGG-NH2
OSK1-Dpr
1226



16, E20, Y36, G37, G38-



amide





GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACYGG-NH2
OSK1-
1227



K16, E20, A34, Y36, G37,



G38-amide





GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACYGG-NH2
OSK1-
1228



O16, E20, A34, Y36, G37,



G38-amide





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACYGG-
OSK1-hLys
1229


NH2
16, E20, A34, Y36, G37,



G38-amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACYGG-
OSK1-hArg
1230


NH2
16, E20, A34, Y36, G37,



G38-amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACYGG-NH2
OSK1-Cit
1231



16, E20, A34, Y36, G37,



G38-amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG-
OSK1-hCit
1232


NH2
16, E20, A34, Y3, G37,



G38-amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACYGG-NH2
OSK1-Dpr
1233



16, E20, A34, Y36, G37,



G38-amide





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACYGG-NH2
OSK1-Dab
1234



16, E20, A34, Y36, G37,



G38-amide





GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCHCGGG-NH2
OSK1-
1235



O16, E20, G36-38-



amide





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCHCGGG-
OSK1-hLys
1236


NH2
16, E20, G36-38-



amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCHCGGG-
OSK1-hArg
1237


NH2
16, E20, G36-38-



amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCHCGGG-NH2
OSK1-Cit
1238



16, E20, G36-38-



amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCHCGGG-
OSK1-hCit
1239


NH2
16, E20, G36-38-



amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCHCGGG-NH2
OSK1-Dpr
1240



16, E20, G36-38-



amide





GVIINVKCKISRQCLOPCKEAGMRFGKCMNGKCACGGG-NH2
OSK1-
1241



O16, E20, A34, G36-



38-amide





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMNGKCACGGG-
OSK1-hLys
1242


NH2
16, E20, A34, G36-



38-amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMNGKCACGGG-
OSK1-hArq
1243


NH2
16, E20, A34, G36-



38-amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMNGKCACGGG-NH2
OSK1-Cit
1244



16, E20, A34, G36-



38-amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMNGKCACTP-NH2
Δ38 OSK1-hCit
1245



16, E20, A34-



amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMNGKCACGGG-NH2
OSK1-Dpr
1246



16, E20, A34, G36-



38-amide





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMNGKCACGGG-NH2
OSK1-Dab
1247



16, E20, A34, G36-



38-amide





GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMNGKCACYGG-NH2
OSK1-K
1248



16, CPA20, A34, Y36,



G37, G38-amide





GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMNGKCACGGG-NH2
OSK1-K
1249



16, CPA20, A34, G36-



38-amide





GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMNGKCACY-NH2
Δ37-38OSK1-K
1250



16, CPA20, A34, Y36-



amide





Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCACYGG-NH2
Acetyl-OSK1-K
1251



16, D20, A34, Y36, G37,



G38-amide





GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMNGKCACYGG-NH2
OSK1-K 16,
1252



Aad20, A34, Y36, G37,



G38-amide





GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMNGKCHCYGG-NH2
OSK1-K 16,
1253



Aad20, Y36, G37, G38-



amide





GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMNGKCACYGG
OSK1-K 16,
1254



Aad20, A34, Y36, G37,



G38





GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCACYGG-NH2
OSK1-H
1255



16, D20, A34, Y36, G37,



G38-amide





GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCACYGG
OSK1-H
1256



16, D20, A34, Y36, G37,



G38





GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCACY-NH2
Δ37-38-OSK1-H
1257



16, D20, A34, Y36-



amide





GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCHCYGG-NH2
OSK1-H
1258



16, D20, A34, Y36, G37,



G38-amide





GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCHCYGG
OSK1-H
1259



16, D20, A34, Y36, G37,



G38





GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCHCYPK
OSK1-H
1260



16, D20, A34, Y36,





GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCAC
Δ36-38 OSK1-H
1261



16, D20, A34, Y36,





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[1Nal]GG-
OSK1-K
1262


NH2
16, D20, A34, 1Nal36,



G37, G38-amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[1Nal]PK-
OSK1-K
1263


NH2
16, D20, A34, 1Nal36-



amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[2Nal]GG-
OSK1-K
1264


NH2
16, D20, A34, 2Nal36,



G37, G38-amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[Cha]GG-NH2
OSK1-K
1265



16, D20, A34, Cha36,



G37, G38-amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[MePhe]GG-
OSK1-K
1266


NH2
16, D20, A34,



MePhe36, G37, G38-



amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[BiPhA]GG-
OSK1-K
1267


NH2 16, D20, A34,



BiPhA36, G37, G38-



amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKC[Aib]CYGG-NH2
OSK1-K 16, D20,
1268



Aib34, Y36, G37, G38-



amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKC[Abu]CYGG-NH2
OSK1-K 16, D20,
1269



Abu34, Y36, G37, G38-



amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[1Nal]
Δ37-38 OSK1-H
1270



16, D20, A34, 1Nal36, -



amide





GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCAC[1Nal]GG-
OSK1-H
1271


NH2
16, D20, A34,



1Nal36, G37, G38-



amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCAC[4Bip]-NH2
Δ37-38 OSK1-H
1272



16, D20, A34, 4Bip



36, -amide





GVIINVKCKISRQCLHPCKDAGMRFGKCMNGKCAC[4Bip]GG-
OSK1-H
1273


NH2
16, D20, A34, 4Bip



36, G37, G38-



amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCGGG
OSK1-K16, E20, G36-
1274



38
















TABLE 7A







Additional useful OSK1 peptide analog sequences








SEQ



ID


NO
Sequence





1391

GVIINVKCKISAQCLKPCRDAGMRFGKCMNGKCACTPK






1392

GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK






1393

GVIINVKCKISPQCLKPCKDAGIRFGKCINGKCACTPK






1394

GVIINVKCKISRQCLKPCKEAGMRFGKCMNGKCACTPK






1395

GGGGSGVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK






1396

GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHC






1397

GVIINVKCKISPQCLOPCKEAGMRFGKCMNGKCHCTY[Nle]






1398

GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTY[Nle]






1399

GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCHCGGG






1400

AVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK






1401
GAIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1402
GVAINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1403
GVIANVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1404
GVIIAVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1405
GVIINAKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1406
GVIINVACKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1407
GVIINVKCAISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1408
GVIINVKCKASRQCLEPCKKAGMRFGKCMNGKCHCTPK





1409
GVIINVKCKIARQCLEPCKKAGMRFGKCMNGKCHCTPK





1410
GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK





1411
GVIINVKCKISRACLEPCKKAGMRFGKCMNGKCHCTPK





1412
GVIINVKCKISRQCAEPCKKAGMRFGKCMNGKCHCTPK





1413
GVIINVKCKISRQCLAPCKKAGMRFGKCMNGKCHCTPK





1414
GVIINVKCKISRQCLEACKKAGMRFGKCMNGKCHCTPK





1415
GVIINVKCKISRQCLEPCAKAGMRFGKCMNGKCHCTPK





1416
GVIINVKCKISRQCLEPCKAAGMRFGKCMNGKCHCTPK





1417
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1418
GVIINVKCKISRQCLEPCKKAAMRFGKCMNGKCHCTPK





1419
GVIINVKCKISRQCLEPCKKAGARFGKCMNGKCHCTPK





1420
GVIINVKCKISRQCLEPCKKAGMAFGKCMNGKCHCTPK





1421
GVIINVKCKISRQCLEPCKKAGMRAGKCMNGKCHCTPK





1422
GVIINVKCKISRQCLEPCKKAGMRFAKCMNGKCHCTPK





1423
GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK





1424
GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK





1425
GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK





1426
GVIINVKCKISRQCLEPCKKAGMRFGKCMNAKCHCTPK





1427
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGACHCTPK





1428
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK





1429
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCAPK





1430
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTAK





1431
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPA





1432

RVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK






1433
GRIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1434
GVRINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1435
GVIRNVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1436
GVIIRVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1437
GVIINRKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1438
GVIINVRCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1439
GVIINVKCRISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1440
GVIINVKCKRSRQCLEPCKKAGMRFGKCMNGKCHCTPK





1441
GVIINVKCKIRRQCLEPCKKAGMRFGKCMNGKCHCTPK





1442
GVIINVKCKISRRCLEPCKKAGMRFGKCMNGKCHCTPK





1443
GVIINVKCKISRQCREPCKKAGMRFGKCMNGKCHCTPK





1444
GVIINVKCKISRQCLRPCKKAGMRFGKCMNGKCHCTPK





1445
GVIINVKCKISRQCLERCKKAGMRFGKCMNGKCHCTPK





1446
GVIINVKCKISRQCLEPCRKAGMRFGKCMNGKCHCTPK





1447
GVIINVKCKISRQCLEPCKRAGMRFGKCMNGKCHCTPK





1448
GVIINVKCKISRQCLEPCKKRGMRFGKCMNGKCHCTPK





1449
GVIINVKCKISRQCLEPCKKARMRFGKCMNGKCHCTPK





1450
GVIINVKCKISRQCLEPCKKAGRRFGKCMNGKCHCTPK





1451
GVIINVKCKISRQCLEPCKKAGMRRGKCMNGKCHCTPK





1452
GVIINVKCKISRQCLEPCKKAGMRFRKCMNGKCHCTPK





1453
GVIINVKCKISRQCLEPCKKAGMRFGRCMNGKCHCTPK





1454
GVIINVKCKISRQCLEPCKKAGMRFGKCRNGKCHCTPK





1455
GVIINVKCKISRQCLEPCKKAGMRFGKCMRGKCHCTPK





1456
GVIINVKCKISRQCLEPCKKAGMRFGKCMNRKCHCTPK





1457
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGRCHCTPK





1458
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCRCTPK





1459
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCRPK





1460
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTRK





1461
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPR





1462

EVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK






1463
GEIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1464
GVEINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1465
GVIENVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1466
GVIIEVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1467
GVIINEKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1468
GVIINVECKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1469
GVIINVKCEISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1470
GVIINVKCKESRQCLEPCKKAGMRFGKCMNGKCHCTPK





1471
GVIINVKCKIERQCLEPCKKAGMRFGKCMNGKCHCTPK





1472
GVIINVKCKISEQCLEPCKKAGMRFGKCMNGKCHCTPK





1473
GVIINVKCKISRECLEPCKKAGMRFGKCMNGKCHCTPK





1474
GVIINVKCKISRQCEEPCKKAGMRFGKCMNGKCHCTPK





1475
GVIINVKCKISRQCLEECKKAGMRFGKCMNGKCHCTPK





1476
GVIINVKCKISRQCLEPCEKAGMRFGKCMNGKCHCTPK





1477
GVIINVKCKISRQCLEPCKEAGMRFGKCMNGKCHCTPK





1478
GVIINVKCKISRQCLEPCKKEGMRFGKCMNGKCHCTPK





1479
GVIINVKCKISRQCLEPCKKAEMRFGKCMNGKCHCTPK





1480
GVIINVKCKISRQCLEPCKKAGERFGKCMNGKCHCTPK





1481
GVIINVKCKISRQCLEPCKKAGMEFGKCMNGKCHCTPK





1482
GVIINVKCKISRQCLEPCKKAGMREGKCMNGKCHCTPK





1483
GVIINVKCKISRQCLEPCKKAGMRFEKCMNGKCHCTPK





1484
GVIINVKCKISRQCLEPCKKAGMRFGECMNGKCHCTPK





1485
GVIINVKCKISRQCLEPCKKAGMRFGKCENGKCHCTPK





1486
GVIINVKCKISRQCLEPCKKAGMRFGKCMEGKCHCTPK





1487
GVIINVKCKISRQCLEPCKKAGMRFGKCMNEKCHCTPK





1488
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGECHCTPK





1489
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCECTPK





1490
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCEPK





1491
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTEK





1492
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPE





1493

[1-Nal]VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK






1494
G[1-Nal]IINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1495
GV[1-Nal]INVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1496
GVI[1-Nal]NVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1497
GVII[1-Nal]VKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1498
GVIIN[1-Nal]KCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1499
GVIINV[1-Nal]CKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1500
GVIINVKC[1-Nal]ISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1501
GVIINVKCK[1-Nal]SRQCLEPCKKAGMRFGKCMNGKCHCTPK





1502
GVIINVKCKI[1-Nal]RQCLEPCKKAGMRFGKCMNGKCHCTPK





1503
GVIINVKCKIS[1-Nal]QCLEPCKKAGMRFGKCMNGKCHCTPK





1504
GVIINVKCKISR[1-Nal]CLEPCKKAGMRFGKCMNGKCHCTPK





1505
GVIINVKCKISRQC[1-Nal]EPCKKAGMRFGKCMNGKCHCTPK





1506
GVIINVKCKISRQCL[1-Nal]PCKKAGMRFGKCMNGKCHCTPK





1507
GVIINVKCKISRQCLE[1-Nal]CKKAGMRFGKCMNGKCHCTPK





1508
GVIINVKCKISRQCLEPC[1-Nal]KAGMRFGKCMNGKCHCTPK





1509
GVIINVKCKISRQCLEPCK[1-Nal]AGMRFGKCMNGKCHCTPK





1510
GVIINVKCKISRQCLEPCKK[1-Nal]GMRFGKCMNGKCHCTPK





1511
GVIINVKCKISRQCLEPCKKA[1-Nal]MRFGKCMNGKCHCTPK





1512
GVIINVKCKISRQCLEPCKKAG[1-Nal]RFGKCMNGKCHCTPK





1513
GVIINVKCKISRQCLEPCKKAGM[1-Nal]FGKCMNGKCHCTPK





1514
GVIINVKCKISRQCLEPCKKAGMR[1-Nal]GKCMNGKCHCTPK





1515
GVIINVKCKISRQCLEPCKKAGMRF[1-Nal]KCMNGKCHCTPK





1516
GVIINVKCKISRQCLEPCKKAGMRFG[1-Nal]CMNGKCHCTPK





1517
GVIINVKCKISRQCLEPCKKAGMRFGKC[1-Nal]NGKCHCTPK





1518
GVIINVKCKISRQCLEPCKKAGMRFGKCM[1-Nal]GKCHCTPK





1519
GVIINVKCKISRQCLEPCKKAGMRFGKCMN[1-Nal]KCHCTPK





1520
GVIINVKCKISRQCLEPCKKAGMRFGKCMNG[1-Nal]CHCTPK





1521
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKC[1-Nal]CTPK





1522
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC[1-Nal]PK





1523
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCT[1-Nal]K





1524
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTP[1-Nal]





1525
Ac-AVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1526
Ac-GAIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1527
Ac-GVAINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1528
Ac-GVIANVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1529
Ac-GVIIAVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1530
Ac-GVIINAKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1531
Ac-GVIINVACKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1532
Ac-GVIINVKCAISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1533
Ac-GVIINVKCKASRQCLEPCKKAGMRFGKCMNGKCHCTPK





1534
Ac-GVIINVKCKIARQCLEPCKKAGMRFGKCMNGKCHCTPK





1535
Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK





1536
Ac-GVIINVKCKISRACLEPCKKAGMRFGKCMNGKCHCTPK





1537
Ac-GVIINVKCKISRQCAEPCKKAGMRFGKCMNGKCHCTPK





1538
Ac-GVIINVKCKISRQCLAPCKKAGMRFGKCMNGKCHCTPK





1539
Ac-GVIINVKCKISRQCLEACKKAGMRFGKCMNGKCHCTPK





1540
Ac-GVIINVKCKISRQCLEPCAKAGMRFGKCMNGKCHCTPK





1541
Ac-GVIINVKCKISRQCLEPCKAAGMRFGKCMNGKCHCTPK





1542
Ac-GVIINVKCKISRQCLEPCKKAAMRFGKCMNGKCHCTPK





1543
Ac-GVIINVKCKISRQCLEPCKKAGARFGKCMNGKCHCTPK





1544
Ac-GVIINVKCKISRQCLEPCKKAGMAFGKCMNGKCHCTPK





1545
Ac-GVIINVKCKISRQCLEPCKKAGMRAGKCMNGKCHCTPK





1546
Ac-GVIINVKCKISRQCLEPCKKAGMRFAKCMNGKCHCTPK





1547
Ac-GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK





1548
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK





1549
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK





1550
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNAKCHCTPK





1551
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGACHCTPK





1552
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK





1553
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCAPK





1554
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTAK





1555
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPA





1556
Ac-RVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1557
Ac-GRIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1558
Ac-GVRINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1559
Ac-GVIRNVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1560
Ac-GVIIRVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1561
Ac-GVIINRKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1562
Ac-GVIINVRCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1563
Ac-GVIINVKCRISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1564
Ac-GVIINVKCKRSRQCLEPCKKAGMRFGKCMNGKCHCTPK





1565
Ac-GVIINVKCKIRRQCLEPCKKAGMRFGKCMNGKCHCTPK





1566
Ac-GVIINVKCKISRRCLEPCKKAGMRFGKCMNGKCHCTPK





1567
Ac-GVIINVKCKISRQCREPCKKAGMRFGKCMNGKCHCTPK





1568
Ac-GVIINVKCKISRQCLRPCKKAGMRFGKCMNGKCHCTPK





1569
Ac-GVIINVKCKISRQCLERCKKAGMRFGKCMNGKCHCTPK





1570
Ac-GVIINVKCKISRQCLEPCRKAGMRFGKCMNGKCHCTPK





1571
Ac-GVIINVKCKISRQCLEPCKRAGMRFGKCMNGKCHCTPK





1572
Ac-GVIINVKCKISRQCLEPCKKRGMRFGKCMNGKCHCTPK





1573
Ac-GVIINVKCKISRQCLEPCKKARMRFGKCMNGKCHCTPK





1574
Ac-GVIINVKCKISRQCLEPCKKAGRRFGKCMNGKCHCTPK





1575
Ac-GVIINVKCKISRQCLEPCKKAGMRRGKCMNGKCHCTPK





1576
Ac-GVIINVKCKISRQCLEPCKKAGMRFRKCMNGKCHCTPK





1577
Ac-GVIINVKCKISRQCLEPCKKAGMRFGRCMNGKCHCTPK





1578
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCRNGKCHCTPK





1579
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMRGKCHCTPK





1580
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNRKCHCTPK





1581
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGRCHCTPK





1582
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCRCTPK





1583
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCRPK





1584
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTRK





1585
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPR





1586
Ac-EVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1587
Ac-GEIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1588
Ac-GVEINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1589
Ac-GVIENVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1590
Ac-GVIIEVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1591
Ac-GVIINEKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1592
Ac-GVIINVECKISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1593
Ac-GVIINVKCEISRQCLEPCKKAGMRFGKCMNGKCHCTPK





1594
Ac-GVIINVKCKESRQCLEPCKKAGMRFGKCMNGKCHCTPK





1595
Ac-GVIINVKCKIERQCLEPCKKAGMRFGKCMNGKCHCTPK





1596
Ac-GVIINVKCKISEQCLEPCKKAGMRFGKCMNGKCHCTPK





1597
Ac-GVIINVKCKISRECLEPCKKAGMRFGKCMNGKCHCTPK





1598
Ac-GVIINVKCKISRQCEEPCKKAGMRFGKCMNGKCHCTPK





1599
Ac-GVIINVKCKISRQCLEECKKAGMRFGKCMNGKCHCTPK





1600
Ac-GVIINVKCKISRQCLEPCEKAGMRFGKCMNGKCHCTPK





1601
Ac-GVIINVKCKISRQCLEPCKEAGMRFGKCMNGKCHCTPK





1602
Ac-GVIINVKCKISRQCLEPCKKEGMRFGKCMNGKCHCTPK





1603
Ac-GVIINVKCKISRQCLEPCKKAEMRFGKCMNGKCHCTPK





1604
Ac-GVIINVKCKISRQCLEPCKKAGERFGKCMNGKCHCTPK





1605
Ac-GVIINVKCKISRQCLEPCKKAGMEFGKCMNGKCHCTPK





1606
Ac-GVIINVKCKISRQCLEPCKKAGMREGKCMNGKCHCTPK





1607
Ac-GVIINVKCKISRQCLEPCKKAGMRFEKCMNGKCHCTPK





1608
Ac-GVIINVKCKISRQCLEPCKKAGMRFGECMNGKCHCTPK





1609
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCENGKCHCTPK





1610
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMEGKCHCTPK





1611
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNEKCHCTPK





1612
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGECHCTPK





1613
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCECTPK





1614
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCEPK





1615
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTEK





1616
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPE





1617
Ac-[1-Nal]VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC



TPK





1618
Ac-G[1-Nal]IINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC



TPK





1619
Ac-GV[1-Nal]INVKCKISRQCLEPCKKAGMRFGKCMNGKCHC



TPK





1620
Ac-GVI[1-Nal]NVKCKISRQCLEPCKKAGMRFGKCMNGKCHC



TPK





1621
Ac-GVII[1-Nal]VKCKISRQCLEPCKKAGMRFGKCMNGKCHC



TPK





1622
Ac-GVIIN[1-Nal]KCKISRQCLEPCKKAGMRFGKCMNGKCHC



TPK





1623
Ac-GVIINV[1-Nal]CKISRQCLEPCKKAGMRFGKCMNGKCHC



TPK





1624
Ac-GVIINVKC[1-Nal]ISRQCLEPCKKAGMRFGKCMNGKCHC



TPK





1625
Ac-GVIINVKCK[1-Nal]SRQCLEPCKKAGMRFGKCMNGKCHC



TPK





1626
Ac-GVIINVKCKI[1-Nal]RQCLEPCKKAGMRFGKCMNGKCHC



TPK





1627
Ac-GVIINVKCKIS[1-Nal]QCLEPCKKAGMRFGKCMNGKCHC



TPK





1628
Ac-GVIINVKCKISR[1-Nal]CLEPCKKAGMRFGKCMNGKCHC



TPK





1629
Ac-GVIINVKCKISRQC[1-Nal]EPCKKAGMRFGKCMNGKCHC



TPK





1630
Ac-GVIINVKCKISRQCL[1-Nal]PCKKAGMRFGKCMNGKCHC



TPK





1631
Ac-GVIINVKCKISRQCLE[1-Nal]CKKAGMRFGKCMNGKCHC



TPK





1632
Ac-GVIINVKCKISRQCLEPC[1-Nal]KAGMRFGKCMNGKCHC



TPK





1633
Ac-GVIINVKCKISRQCLEPCK[1-Nal]AGMRFGKCMNGKCHC



TPK





1634
Ac-GVIINVKCKISRQCLEPCKK[1-Nal]GMRFGKCMNGKCHC



TPK





1635
Ac-GVIINVKCKISRQCLEPCKKA[1-Nal]MRFGKCMNGKCHC



TPK





1636
Ac-GVIINVKCKISRQCLEPCKKAG[1-Nal]RFGKCMNGKCHC



TPK





1637
Ac-GVIINVKCKISRQCLEPCKKAGM[1-Nal]FGKCMNGKCHC



TPK





1638
Ac-GVIINVKCKISRQCLEPCKKAGMR[1-Nal]GKCMNGKCHC



TPK





1639
Ac-GVIINVKCKISRQCLEPCKKAGMRF[1-Nal]KCMNGKCHC



TPK





1640
Ac-GVIINVKCKISRQCLEPCKKAGMRFG[1-Nal]CMNGKCHC



TPK





1641
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKC[1-Nal]NGKCHC



TPK





1642
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCM[1-Nal]GKCHC



TPK





1643
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMN[1-Nal]KCHC



TPK





1644
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNG[1-Nal]CHC



TPK





1645
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKC[1-Nal]C



TPK





1646
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC[1-Nal]



PK





1647
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCT[1-



Nal]K





1648
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTP[1-



Nal]





1649
AVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1650
GAIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1651
GVAINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1652
GVIANVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1653
GVIIAVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1654
GVIINAKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1655
GVIINVACKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1656
GVIINVKCAISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1657
GVIINVKCKASRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1658
GVIINVKCKIARQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1659
GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1660
GVIINVKCKISRACLEPCKKAGMRFGKCMNGKCHCTPK-amide





1661
GVIINVKCKISRQCAEPCKKAGMRFGKCMNGKCHCTPK-amide





1662
GVIINVKCKISRQCLAPCKKAGMRFGKCMNGKCHCTPK-amide





1663
GVIINVKCKISRQCLEACKKAGMRFGKCMNGKCHCTPK-amide





1664
GVIINVKCKISRQCLEPCAKAGMRFGKCMNGKCHCTPK-amide





1665
GVIINVKCKISRQCLEPCKAAGMRFGKCMNGKCHCTPK-amide





1666
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1667
GVIINVKCKISRQCLEPCKKAAMRFGKCMNGKCHCTPK-amide





1668
GVIINVKCKISRQCLEPCKKAGARFGKCMNGKCHCTPK-amide





1669
GVIINVKCKISRQCLEPCKKAGMAFGKCMNGKCHCTPK-amide





1670
GVIINVKCKISRQCLEPCKKAGMRAGKCMNGKCHCTPK-amide





1671
GVIINVKCKISRQCLEPCKKAGMRFAKCMNGKCHCTPK-amide





1672
GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK-amide





1673
GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK-amide





1674
GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK-amide





1675
GVIINVKCKISRQCLEPCKKAGMRFGKCMNAKCHCTPK-amide





1676
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGACHCTPK-amide





1677
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK-amide





1678
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCAPK-amide





1679
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTAK-amide





1680
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPA-amide





1681
RVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1682
GRIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1683
GVRINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1684
GVIRNVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1685
GVIIRVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1686
GVIINRKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1687
GVIINVRCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1688
GVIINVKCRISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1689
GVIINVKCKRSRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1690
GVIINVKCKIRRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1691
GVIINVKCKISRRCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1692
GVIINVKCKISRQCREPCKKAGMRFGKCMNGKCHCTPK-amide





1693
GVIINVKCKISRQCLRPCKKAGMRFGKCMNGKCHCTPK-amide





1694
GVIINVKCKISRQCLERCKKAGMRFGKCMNGKCHCTPK-amide





1695
GVIINVKCKISRQCLEPCRKAGMRFGKCMNGKCHCTPK-amide





1696
GVIINVKCKISRQCLEPCKRAGMRFGKCMNGKCHCTPK-amide





1697
GVIINVKCKISRQCLEPCKKRGMRFGKCMNGKCHCTPK-amide





1698
GVIINVKCKISRQCLEPCKKARMRFGKCMNGKCHCTPK-amide





1699
GVIINVKCKISRQCLEPCKKAGRRFGKCMNGKCHCTPK-amide





1700
GVIINVKCKISRQCLEPCKKAGMRRGKCMNGKCHCTPK-amide





1701
GVIINVKCKISRQCLEPCKKAGMRFRKCMNGKCHCTPK-amide





1702
GVIINVKCKISRQCLEPCKKAGMRFGRCMNGKCHCTPK-amide





1703
GVIINVKCKISRQCLEPCKKAGMRFGKCRNGKCHCTPK-amide





1704
GVIINVKCKISRQCLEPCKKAGMRFGKCMRGKCHCTPK-amide





1705
GVIINVKCKISRQCLEPCKKAGMRFGKCMNRKCHCTPK-amide





1706
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGRCHCTPK-amide





1707
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCRCTPK-amide





1708
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCRPK-amide





1709
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTRK-amide





1710
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPR-amide





1711
EVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1712
GEIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1713
GVEINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1714
GVIENVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1715
GVIIEVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1716
GVIINEKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1717
GVIINVECKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1718
GVIINVKCEISRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1719
GVIINVKCKESRQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1720
GVIINVKCKIERQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1721
GVIINVKCKISEQCLEPCKKAGMRFGKCMNGKCHCTPK-amide





1722
GVIINVKCKISRECLEPCKKAGMRFGKCMNGKCHCTPK-amide





1723
GVIINVKCKISRQCEEPCKKAGMRFGKCMNGKCHCTPK-amide





1724
GVIINVKCKISRQCLEECKKAGMRFGKCMNGKCHCTPK-amide





1725
GVIINVKCKISRQCLEPCEKAGMRFGKCMNGKCHCTPK-amide





1726
GVIINVKCKISRQCLEPCKEAGMRFGKCMNGKCHCTPK-amide





1727
GVIINVKCKISRQCLEPCKKEGMRFGKCMNGKCHCTPK-amide





1728
GVIINVKCKISRQCLEPCKKAEMRFGKCMNGKCHCTPK-amide





1729
GVIINVKCKISRQCLEPCKKAGERFGKCMNGKCHCTPK-amide





1730
GVIINVKCKISRQCLEPCKKAGMEFGKCMNGKCHCTPK-amide





1731
GVIINVKCKISRQCLEPCKKAGMREGKCMNGKCHCTPK-amide





1732
GVIINVKCKISRQCLEPCKKAGMRFEKCMNGKCHCTPK-amide





1733
GVIINVKCKISRQCLEPCKKAGMRFGECMNGKCHCTPK-amide





1734
GVIINVKCKISRQCLEPCKKAGMRFGKCENGKCHCTPK-amide





1735
GVIINVKCKISRQCLEPCKKAGMRFGKCMEGKCHCTPK-amide





1736
GVIINVKCKISRQCLEPCKKAGMRFGKCMNEKCHCTPK-amide





1737
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGECHCTPK-amide





1738
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCECTPK-amide





1739
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCEPK-amide





1740
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTEK-amide





1741
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPE-amide





1742
[1-Nal]VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1743
G[1-Nal]IINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1744
GV[1-Nal]INVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1745
GVI[1-Nal]NVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1746
GVII[1-Nal]VKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1747
GVIIN[1-Nal]KCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1748
GVIINV[1-Nal]CKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1749
GVIINVKC[1-Nal]ISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1750
GVIINVKCK[1-Nal]SRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1751
GVIINVKCKI[1-Nal]RQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1752
GVIINVKCKIS[1-Nal]QCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1753
GVIINVKCKISR[1-Nal]CLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1754
GVIINVKCKISRQC[1-Nal]EPCKKAGMRFGKCMNGKCHCTPK-



amide





1755
GVIINVKCKISRQCL[1-Nal]PCKKAGMRFGKCMNGKCHCTPK-



amide





1756
GVIINVKCKISRQCLE[1-Nal]CKKAGMRFGKCMNGKCHCTPK-



amide





1757
GVIINVKCKISRQCLEPC[1-Nal]KAGMRFGKCMNGKCHCTPK-



amide





1758
GVIINVKCKISRQCLEPCK[1-Nal]AGMRFGKCMNGKCHCTPK-



amide





1759
GVIINVKCKISRQCLEPCKK[1-Nal]GMRFGKCMNGKCHCTPK-



amide





1760
GVIINVKCKISRQCLEPCKKA[1-Nal]MRFGKCMNGKCHCTPK-



amide





1761
GVIINVKCKISRQCLEPCKKAG[1-Nal]RFGKCMNGKCHCTPK-



amide





1762
GVIINVKCKISRQCLEPCKKAGM[1-Nal]FGKCMNGKCHCTPK-



amide





1763
GVIINVKCKISRQCLEPCKKAGMR[1-Nal]GKCMNGKCHCTPK-



amide





1764
GVIINVKCKISRQCLEPCKKAGMRF[1-Nal]KCMNGKCHCTPK-



amide





1765
GVIINVKCKISRQCLEPCKKAGMRFG[1-Nal]CMNGKCHCTPK-



amide





1766
GVIINVKCKISRQCLEPCKKAGMRFGKC[1-Nal]NGKCHCTPK-



amide





1767
GVIINVKCKISRQCLEPCKKAGMRFGKCM[1-Nal]GKCHCTPK-



amide





1768
GVIINVKCKISRQCLEPCKKAGMRFGKCMN[1-Nal]KCHCTPK-



amide





1769
GVIINVKCKISRQCLEPCKKAGMRFGKCMNG[1-Nal]CHCTPK-



amide





1770
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKC[1-Nal]CTPK-



amide





1771
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC[1-Nal]PK-



amide





1772
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCT[1-Nal]K-



amide





1773
GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTP[1-Nal]-



amide





1774
Ac-AVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1775
Ac-GAIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1776
Ac-GVAINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1777
Ac-GVIANVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1778
Ac-GVIIAVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1779
Ac-GVIINAKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1780
Ac-GVIINVACKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1781
Ac-GVIINVKCAISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1782
Ac-GVIINVKCKASRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1783
Ac-GVIINVKCKIARQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1784
Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1785
Ac-GVIINVKCKISRACLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1786
Ac-GVIINVKCKISRQCAEPCKKAGMRFGKCMNGKCHCTPK-



amide





1787
Ac-GVIINVKCKISRQCLAPCKKAGMRFGKCMNGKCHCTPK-



amide





1788
Ac-GVIINVKCKISRQCLEACKKAGMRFGKCMNGKCHCTPK-



amide





1789
Ac-GVIINVKCKISRQCLEPCAKAGMRFGKCMNGKCHCTPK-



amide





1790
Ac-GVIINVKCKISRQCLEPCKAAGMRFGKCMNGKCHCTPK-



amide





1791
Ac-GVIINVKCKISRQCLEPCKKAAMRFGKCMNGKCHCTPK-



amide





1792
Ac-GVIINVKCKISRQCLEPCKKAGARFGKCMNGKCHCTPK-



amide





1793
Ac-GVIINVKCKISRQCLEPCKKAGMAFGKCMNGKCHCTPK-



amide





1794
Ac-GVIINVKCKISRQCLEPCKKAGMRAGKCMNGKCHCTPK-



amide





1795
Ac-GVIINVKCKISRQCLEPCKKAGMRFAKCMNGKCHCTPK-



amide





1796
Ac-GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK-



amide





1797
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK-



amide





1798
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK-



amide





1799
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNAKCHCTPK-



amide





1800
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGACHCTPK-



amide





1801
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCACTPK-



amide





1802
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCAPK-



amide





1803
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTAK-



amide





1804
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPA-



amide





1805
Ac-RVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1806
Ac-GRIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1807
Ac-GVRINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1808
Ac-GVIRNVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1809
Ac-GVIIRVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1810
Ac-GVIINRKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1811
Ac-GVIINVRCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1812
Ac-GVIINVKCRISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1813
Ac-GVIINVKCKRSRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1814
Ac-GVIINVKCKIRRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1815
Ac-GVIINVKCKISRRCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1816
Ac-GVIINVKCKISRQCREPCKKAGMRFGKCMNGKCHCTPK-



amide





1817
Ac-GVIINVKCKISRQCLRPCKKAGMRFGKCMNGKCHCTPK-



amide





1818
Ac-GVIINVKCKISRQCLERCKKAGMRFGKCMNGKCHCTPK-



amide





1819
Ac-GVIINVKCKISRQCLEPCRKAGMRFGKCMNGKCHCTPK-



amide





1820
Ac-GVIINVKCKISRQCLEPCKRAGMRFGKCMNGKCHCTPK-



amide





1821
Ac-GVIINVKCKISRQCLEPCKKRGMRFGKCMNGKCHCTPK-



amide





1822
Ac-GVIINVKCKISRQCLEPCKKARMRFGKCMNGKCHCTPK-



amide





1823
Ac-GVIINVKCKISRQCLEPCKKAGRRFGKCMNGKCHCTPK-



amide





1824
Ac-GVIINVKCKISRQCLEPCKKAGMRRGKCMNGKCHCTPK-



amide





1825
Ac-GVIINVKCKISRQCLEPCKKAGMRFRKCMNGKCHCTPK-



amide





1826
Ac-GVIINVKCKISRQCLEPCKKAGMRFGRCMNGKCHCTPK-



amide





1827
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCRNGKCHCTPK-



amide





1828
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMRGKCHCTPK-



amide





1829
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNRKCHCTPK-



amide





1830
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGRCHCTPK-



amide





1831
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCRCTPK-



amide





1832
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCRPK-



amide





1833
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTRK-



amide





1834
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPR-



amide





1835
Ac-EVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1836
Ac-GEIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1837
Ac-GVEINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1838
Ac-GVIENVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1839
Ac-GVIIEVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1840
Ac-GVIINEKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1841
Ac-GVIINVECKISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1842
Ac-GVIINVKCEISRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1843
Ac-GVIINVKCKESRQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1844
Ac-GVIINVKCKIERQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1845
Ac-GVIINVKCKISEQCLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1846
Ac-GVIINVKCKISRECLEPCKKAGMRFGKCMNGKCHCTPK-



amide





1847
Ac-GVIINVKCKISRQCEEPCKKAGMRFGKCMNGKCHCTPK-



amide





1848
Ac-GVIINVKCKISRQCLEECKKAGMRFGKCMNGKCHCTPK-



amide





1849
Ac-GVIINVKCKISRQCLEPCEKAGMRFGKCMNGKCHCTPK-



amide





1850
Ac-GVIINVKCKISRQCLEPCKEAGMRFGKCMNGKCHCTPK-



amide





1851
Ac-GVIINVKCKISRQCLEPCKKEGMRFGKCMNGKCHCTPK-



amide





1852
Ac-GVIINVKCKISRQCLEPCKKAEMRFGKCMNGKCHCTPK-



amide





1853
Ac-GVIINVKCKISRQCLEPCKKAGERFGKCMNGKCHCTPK-



amide





1854
Ac-GVIINVKCKISRQCLEPCKKAGMEFGKCMNGKCHCTPK-



amide





1855
Ac-GVIINVKCKISRQCLEPCKKAGMREGKCMNGKCHCTPK-



amide





1856
Ac-GVIINVKCKISRQCLEPCKKAGMRFEKCMNGKCHCTPK-



amide





1857
Ac-GVIINVKCKISRQCLEPCKKAGMRFGECMNGKCHCTPK-



amide





1858
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCENGKCHCTPK-



amide





1859
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMEGKCHCTPK-



amide





1860
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNEKCHCTPK-



amide





1861
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGECHCTPK-



amide





1862
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCECTPK-



amide





1863
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCEPK-



amide





1864
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTEK-



amide





1865
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPE-



amide





1866
Ac-[1-Nal]VIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC



TPK-amide





1867
Ac-G[1-Nal]IINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC



TPK-amide





1868
Ac-GV[1-Nal]INVKCKISRQCLEPCKKAGMRFGKCMNGKCHC



TPK-amide





1869
Ac-GVI[1-Nal]NVKCKISRQCLEPCKKAGMRFGKCMNGKCHC



TPK-amide





1870
Ac-GVII[1-Nal]VKCKISRQCLEPCKKAGMRFGKCMNGKCHC



TPK-amide





1871
Ac-GVIIN[1-Nal]KCKISRQCLEPCKKAGMRFGKCMNGKCHC



TPK-amide





1872
Ac-GVIINV[1-Nal]CKISRQCLEPCKKAGMRFGKCMNGKCHC



TPK-amide





1873
Ac-GVIINVKC[1-Nal]ISRQCLEPCKKAGMRFGKCMNGKCHC



TPK-amide





1874
Ac-GVIINVKCK[1-Nal]SRQCLEPCKKAGMRFGKCMNGKCHC



TPK-amide





1875
Ac-GVIINVKCKI[1-Nal]RQCLEPCKKAGMRFGKCMNGKCHC



TPK-amide





1876
Ac-GVIINVKCKIS[1-Nal]QCLEPCKKAGMRFGKCMNGKCHC



TPK-amide





1877
Ac-GVIINVKCKISR[1-Nal]CLEPCKKAGMRFGKCMNGKCHC



TPK-amide





1878
Ac-GVIINVKCKISRQC[1-Nal]EPCKKAGMRFGKCMNGKCHC



TPK-amide





1879
Ac-GVIINVKCKISRQCL[1-Nal]PCKKAGMRFGKCMNGKCHC



TPK-amide





1880
Ac-GVIINVKCKISRQCLE[1-Nal]CKKAGMRFGKCMNGKCHC



TPK-amide





1881
Ac-GVIINVKCKISRQCLEPC[1-Nal]KAGMRFGKCMNGKCHC



TPK-amide





1882
Ac-GVIINVKCKISRQCLEPCK[1-Nal]AGMRFGKCMNGKCHC



TPK-amide





1883
Ac-GVIINVKCKISRQCLEPCKK[1-Nal]GMRFGKCMNGKCHC



TPK-amide





1884
Ac-GVIINVKCKISRQCLEPCKKA[1-Nal]MRFGKCMNGKCHC



TPK-amide





1885
Ac-GVIINVKCKISRQCLEPCKKAG[1-Nal]RFGKCMNGKCHC



TPK-amide





1886
Ac-GVIINVKCKISRQCLEPCKKAGM[1-Nal]FGKCMNGKCHC



TPK-amide





1887
Ac-GVIINVKCKISRQCLEPCKKAGMR[1-Nal]GKCMNGKCHC



TPK-amide





1888
Ac-GVIINVKCKISRQCLEPCKKAGMRF[1-Nal]KCMNGKCHC



TPK-amide





1889
Ac-GVIINVKCKISRQCLEPCKKAGMRFG[1-Nal]CMNGKCHC



TPK-amide





1890
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKC[1-Nal]NGKCHC



TPK-amide





1891
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCM[1-Nal]GKCHC



TPK-amide





1892
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMN[1-Nal]KCHC



TPK-amide





1893
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNG[1-Nal]CHC



TPK-amide





1894
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKC[1-Nal]C



TPK-amide





1895
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHC[1-Nal]



PK-amide





1896
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCT[1-



Nal]K-amide





1897
Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTP[1-



Nal]-amide
















TABLE 7B







Additional useful OSK1 peptide analog


sequences: Ala-12 Substituted Series









SEQ



ID


Sequence/structure
NO:





GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK
1898





GVIINVSCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK
1899





GVIINVKCKISAQCLKPCKKAGMRFGKCMNGKCHCTPK
1900





GVIINVKCKISAQCLEPCKDAGMRFGKCMNGKCHCTPK
1901





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1902





GVIINVSCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1903





GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK
1904





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCYPK
1905





Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK
1906





GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK-amide
1907





Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCHCTPK-
1908


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCYPK-amide
1909





Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCYPK
1910





Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCYPK-
1911


amide





GVIINVKCKISAQCLKPCKKAGMRFGKCMNGKCHCTPK-amide
1912





Ac-GVIINVKCKISAQCLKPCKKAGMRFGKCMNGKCHCTPK
1913





Ac-GVIINVKCKISAQCLKPCKKAGMRFGKCMNGKCHCTPK-
1914


amide





Ac-GVIINVKCKISAQCLEPCKDAGMRFGKCMNGKCHCTPK
1915





GVIINVKCKISAQCLEPCKDAGMRFGKCMNCKCHCTPK-amide
1916





Ac-GVIINVKCKISAQCLEPCKDAGMRFGKCMNGKCHCTPK-
1917


amide





GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK-amide
1918





Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK
1919





Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK-
1920


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide
1921





Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1922





Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-
1923


amide





VIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK
1924





Ac-VIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK
1925





VIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK-amide
1926





Ac-VIINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK-
1927


amide





GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK
1928





Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK
1929





GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK-amide
1930





Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK-
1931


amide





VIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1932





Ac-VIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1933





VIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide
1934





Ac-VIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-
1935


amide





NVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1936





Ac-NVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1937





NVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide
1938





Ac-NVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide
1939





KCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1940





Ac-KCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1941





KCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide
1942





Ac-KCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide
1943





CKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1944





Ac-CKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1945





CKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide
1946





Ac-CKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide
1947





GVIINVKCKISAQCLKPCKDAGMRNGKCMNGKCHCTPK
1948





GVIINVKCKISAQCLKPCKDAGMRNGKCMNGKCHCTPK-amide
1949





Ac-GVIINVKCKISAQCLKPCKDAGMRNGKCMNGKCHCTPK
1950





Ac-GVIINVKCKISAQCLKPCKDAGMRNGKCMNGKCHCTPK-
1951


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK
1952





GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK-amide
1953





Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK
1954





Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK-
1955


amide





GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK
1956





Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK
1957





GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK-amide
1958





Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMNGKCHCTPK-
1959


amide





TIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1960





Ac-TIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1961





TIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide
1962





Ac-TIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-
1963


amide





GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK
1964





Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK
1965





GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK-amide
1966





Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCHCTPK-
1967


amide





GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK
1968





GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1969





GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCACTPK
1970





Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK
1971





GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK-amide
1972





Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMNGKCACTPK-
1973


amide





Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCACTPK
1974





GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCACTPK-amide
1975





Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCACTPK-
1976


amide





Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK
1977





GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-amide
1978





Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCTPK-
1979


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCT
1980





GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCTPK
1981





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCTPK
1982





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCTPK
1983





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCTPK
1984





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK
1985





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCTPK
1986





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCHCTPK
1987





GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCYPK
1988





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCYPK
1989





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCYPK
1990





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCYPK
1991





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCYPK
1992





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCYPK
1993





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCHCYPK
1994





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYPK
1995





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCGCYPK
1996





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACFPK
1997





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACWPK
1998





GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCACYPK
1999





GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACTPK
2000





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCACTPK
2001





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCACTPK
2002





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCACTPK
2003





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK
2004





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCACTPK
2005





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCACTPK
2006





GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHC
2007





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHC
2008





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHC
2009





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHC
2010





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHC
2011





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHC
2012





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCHC
2013





GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCAC
2014





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCAC
2015





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCAC
2016





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCAC
2017





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHC
2018





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCAC
2019





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCAC
2020





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCGCYGG
2021





GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCYGG
2022





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCYGG
2023





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCYGG
2024





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCYGG
2025





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCYGG
2026





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCYGG
2027





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYGG
2028





GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACYGG
2029





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCACYGG
2030





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCACYGG
2031





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCACYGG
2032





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCYGG
2033





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCACYGG
2034





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCACYGG
2035





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYG
2036





GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCGGG
2037





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCGGG
2038





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCGGG
2039





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCGGG
2040





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCGGG
2041





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCGGG
2042





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACFGG
2043





GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACGGG
2044





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCACGGG
2045





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCACGGG
2046





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCACGGG
2047





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCACGGG
2048





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCACGGG
2049





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCACGGG
2050





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACGG
2051





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYG
2052





GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACGG
2053





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCTPK
2054





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCTPK
2055





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCTPK
2056





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCTPK
2057





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCTPK
2058





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCHCTPK
2059





GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCYPK
2060





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCYPK
2061





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCYPK
2062





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCYPK
2063





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCYPK
2064





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCYPK
2065





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCHCYPK
2066





GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCACTPK
2067





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCACTPK
2068





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCACTPK
2069





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCACTPK
2070





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCTPK
2071





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCACTPK
2072





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCACTPK
2073





GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHC
2074





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHC
2075





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHC
2076





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHC
2077





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHC
2078





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHC
2079





GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCAC
2080





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCAC
2081





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCAC
2082





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCAC
2083





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHC
2084





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCAC
2085





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCAC
2086





GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCHCYGG
2087





GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCYGG
2088





GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCHCYG
2089





GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCACYG
2090





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCYGG
2091





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCYGG
2092





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCYGG
2093





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG
2094





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCYGG
2095





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCHCYGG
2096





GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCACYG
2097





GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCACYGG
2098





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCACYGG
2099





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCACYGG
2100





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCACYGG
2101





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG
2102





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCACYGG
2103





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCACYGG
2104





GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCACFGG
2105





GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCGGG
2106





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCGGG
2107





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCGGG
2108





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCGGG
2109





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCGGG
2110





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCGGG
2111





GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCACGGG
2112





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCACGGG
2113





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCACGGG
2114





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCACGGG
2115





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCACTP
2116





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCACTP
2117





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCACTP
2118





GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCTPK-amide
2119





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCTPK-
2120


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCTPK-
2121


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCTPK-
2122


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCTPK-
2123


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCTPK-
2124


amide





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCHCTPK-
2125


amide





GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCYPK-amide
2126





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCYPK-
2127


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCYPK-
2128


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCYPK-
2129


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCYPK-
2130


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCYPK-
2131


amide





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCHCYPK-
2132


amide





GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACTPK-amide
2133





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCACTPK-
2134


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCACTPK-
2135


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCACTPK-
2136


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCACTPK-
2137


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCACTPK-
2138


amide





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCACTPK-
2139


amide





GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHC-amide
2140





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHC-
2141


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHC-
2142


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHC-
2143


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHC-
2144


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHC-
2145


amide





GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCAC-amide
2146





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCAC-
2147


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCAC-
2148


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCAC-
2149


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHC-
2150


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCAC-
2151


amide





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCAC-
2152


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCYGG-amide
2153





GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCYGG-amide
2154





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCYGG-
2155


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCYGG-
2156


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCYGG-
2157


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCYGG-
2158


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCYGG-
2159


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCFGG-amide
2160





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCYG-amide
2161





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYG-amide
2162





GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACYGG-amide
2163





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCACYGG-
2164


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCACYGG-
2165


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCACYGG-
2166


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCACYGG-
2167


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCACYGG-
2168


amide





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCACYGG-
2169


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYGG-amide
2170





GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCHCGGG-amide
2171





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCHCGGG-
2172


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCHCGGG-
2173


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCHCGGG-
2174


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCHCGGG-
2175


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCHCGGG-
2176


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACGGG-amide
2177





GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACFGG-amide
2178





GVIINVKCKISAQCLOPCKDAGMRFGKCMNGKCACGGG-amide
2179





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMNGKCACGGG-
2180


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMNGKCACGGG-
2181


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMNGKCACGGG-
2182


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMNGKCACGGG-
2183


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMNGKCACGGG-
2184


amide





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMNGKCACGGG-
2185


amide





GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCTPK-amide
2186





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCTPK-
2187


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCTPK-
2188


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCTPK-
2189


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCTPK-
2190


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCTPK-
2191


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCHCTPK-
2192


amide





GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCYPK-amide
2193





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCYPK-
2194


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCYPK-
2195


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCYPK-
2196


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCYPK-
2197


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCYPK-
2198


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCHCYPK-
2199


amide





GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCACTPK-amide
2200





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCACTPK-
2201


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCACTPK-
2202


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCACTPK-
2203


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCACTPK-
2204


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCACTPK-
2205


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCACTPK-
2206


amide





GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHC-amide
2207





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHC-
2208


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHC-
2209


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHC-
2210


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHC-
2211


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHC-
2212


amide





GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCAC-amide
2213





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCAC-
2214


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCAC-
2215


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCAC-
2216


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHC-
2217


amnide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCAC-
2218


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCAC-
2219


amide





GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCHCWGG-amide
2220





GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCYGG-amide
2221





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCYGG-
2222


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCYGG-
2223


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCYGG-
2224


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG-
2225


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCYGG-
2226


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCHCYGG-
2227


amide





GVIINVKCKISAQCLKPCKEAGMRFGKCMNGKCACYGG-amide
2228





GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCACYGG-amide
2229





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCACYGG-
2230


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCACYGG-
2231


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCACYGG-
2232


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCYGG-
2233


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCACYGG-
2234


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCACYGG-
2235


amide





GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCHCGGG-amide
2236





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCHCGGG-
2237


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCHCGGG-
2238


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCHCGGG-
2239


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCHCGGG-
2240


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCHCGGG-
2241


amide





GVIINVKCKISAQCLOPCKEAGMRFGKCMNGKCACGGG-amide
2242





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMNGKCACGGG-
2243


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMNGKCACGGG-
2244


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMNGKCACGGG-
2245


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMNGKCACTP-
2246


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMNGKCACGGG-
2247


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMNGKCACGGG-
2248


amide





GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCMNGKCACYGG-
2249


amide





GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCMNGKCACGGG-
2250


amide





GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCMNGKCACY-
2251


amide





Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCACYGG-
2252


amide





GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCMNGKCACYGG-
2253


amide





GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCMNGKCHCYGG-
2254


amide





GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCMNGKCACYGG
2255





GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCACYGG-amide
2256





GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCACYGG
2257





GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCACY-amide
2258





GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCHCYGG-
2259


amicie





GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCHCYGG
2260





GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCHCYPK
2261





GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCAC
2262





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[1Nal]GG-
2263


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[1Nal]PK-
2264


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[2Nal]GG-
2265


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[Cha]GG-
2266


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[MePhe]GG-
2267


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[BiPhA]GG-
2268


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKC[Aib]CYGG-
2269


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKC[Abu]CYGG-
2270


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[1Nal]
2271





GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCAC[1Nal]GG-
2272


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCAC[4Bip]-
2273


amide





GVIINVKCKISAQCLHPCKDAGMRFGKCMNGKCAC[4Bip]GG-
2274


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCHCGGG
2275
















TABLE 7C







Additional useful OSK1 peptide analog


sequences: Ala-12 & Ala27


Substituted Series









SEQ



ID


Sequence/structure
NO:





GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK
2276





GVIINVSCKISAQCLEPCKKAGMRFGACMNGKCHCTPK
2277





GVIINVKCKISAQCLKPCKKAGMRFGACMNGKCHCTPK
2278





GVIINVKCKISAQCLEPCKDAGMRFGACMNGKCHCTPK
2279





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2280





GVIINVSCKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2281





GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK
2282





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCYPK
2283





Ac-GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK
2284





GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK-amide
2285





Ac-GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK-
2286


amide





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCYPK-amide
2287





Ac-GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCYPK
2288





Ac-GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCYPK-
2289


amide





GVIINVKCKISAQCLKPCKKAGMRFGACMNGKCHCTPK-amide
2290





Ac-GVIINVKCKISAQCLKPCKKAGMRFGACMNGKCHCTPK
2291





Ac-GVIINVKCKISAQCLKPCKKAGMRFGACMNGKCHCTPK-
2292


amide





Ac-GVIINVKCKISAQCLEPCKDAGMRFGACMNGKCHCTPK
2293





GVIINVKCKISAQCLEPCKDAGMRFGACMNGKCHCTPK-amide
2294





Ac-GVIINVKCKISAQCLEPCKDAGMRFGACMNGKCHCTPK-
2295


amide





GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK-amide
2296





Ac-GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK
2297





Ac-GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK-
2298


amide





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide
2299





Ac-GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2300





Ac-GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-
2301


amide





VIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK
2302





Ac-VIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK
2303





VIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK-amide
2304





Ac-VIINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK-
2305


amide





GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK
2306





Ac-GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK
2307





GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK-amide
2308





Ac-GVIINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK-
2309


amide





VIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2310





Ac-VIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2311





VIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide
2312





Ac-VIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-
2313


amide





NVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2314





Ac-NVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2315





NVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide
2316





Ac-NVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide
2317





KCKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2318





Ac-KCKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2319





KCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide
2320





Ac-KCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide
2321





CKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2322





Ac-CKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2323





CKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide
2324





Ac-CKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide
2325





GVIINVKCKISAQCLKPCKDAGMRNGACMNGKCHCTPK
2326





GVIINVKCKISAQCLKPCKDAGMRNGACMNGKCHCTPK-amide
2327





Ac-GVIINVKCKISAQCLKPCKDAGMRNGACMNGKCHCTPK
2328





Ac-GVIINVKCKISAQCLKPCKDAGMRNGACMNGKCHCTPK-
2329


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK
2330





GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK-amide
2331





Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK
2332





Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK-
2333


amide





GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK
2334





Ac-GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK
2335





GVIINVKCKISKQCLKPCGDAGMRFGACMNGKCHCTPK-amide
2336





Ac-GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK-
2337


amide





TIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2338





Ac-TIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2339





TIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide
2340





Ac-TIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-
2341


amide





GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK
2342





Ac-GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK
2343





GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK-amide
2344





Ac-GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCHCTPK-
2345


amide





GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK
2346





GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2347





GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCACTPK
2348





Ac-GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK
2349





GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK-amide
2350





Ac-GVKINVKCKISAQCLEPCKKAGMRFGACMNGKCACTPK-
2351


amide





Ac-GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCACTPK
2352





GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCACTPK-amide
2353





Ac-GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCACTPK-
2354


amide





Ac-GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK
2355





GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-amide
2356





Ac-GVKINVKCKISAQCLKPCKDAGMRFGACMNGKCHCTPK-
2357


amide





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCT
2358





GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCTPK
2359





GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCTPK
2360





GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCTPK
2361





GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCTPK
2362





GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCTPK
2363





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCTPK
2364





GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCHCTPK
2365





GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCYPK
2366





GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCYPK
2367





GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCYPK
2368





GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCYPK
2369





GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCYPK
2370





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCYPK
2371





GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCHCYPK
2372





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYPK
2373





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCGCYPK
2374





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACFPK
2375





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACWPK
2376





GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCACYPK
2377





GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACTPK
2378





GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCACTPK
2379





GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCACTPK
2380





GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCACTPK
2381





GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCTPK
2382





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCACTPK
2383





GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCACTPK
2384





GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHC
2385





GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHC
2386





GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHC
2387





GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHC
2388





GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHC
2389





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHC
2390





GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCHC
2391





GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCAC
2392





GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCAC
2393





GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCAC
2394





GVIINVKCKTTAQCL[Cit]PCKDAGMRFGACMNGKCAC
2395





GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHC
2396





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCAC
2397





GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCAC
2398





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCGCYGG
2399





GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCYGG
2400





GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCYGG
2401





GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCYGG
2402





GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCYGG
2403





GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCYGG
2404





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCYGG
2405





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYGG
2406





GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACYGG
2407





GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCACYGG
2408





GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCACYGG
2409





GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCACYGG
2410





GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCYGG
2411





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCACYGG
2412





GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCACYGG
2413





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYG
2415





GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCGGG
2416





GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCGGG
2417





GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCGGG
2418





GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCGGG
2419





GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCGGG
2420





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCGGG
2421





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACFGG
2422





GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACGGG
2423





GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCACGGG
2424





GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCACGGG
2425





GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCACGGG
2426





GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCACGGG
2427





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCACGGG
2428





GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCACGGG
2429





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACGG
2430





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYG
2431





GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACGG
2432





GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCTPK
2433





GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCTPK
2434





GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCTPK
2435





GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCTPK
2436





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCTPK
2437





GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCHCTPK
2438





GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCYPK
2439





GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCYPK
2440





GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCYPK
2441





GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCYPK
2442





GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCYPK
2443





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCYPK
2444





GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCHCYPK
2445





GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCACTPK
2446





GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCACTPK
2447





GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCACTPK
2448





GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCACTPK
2449





GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCTPK
2450





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCACTPK
2451





GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCACTPK
2452





GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHC
2453





GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHC
2454





GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHC
2455





GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHC
2456





GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHC
2457





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHC
2458





GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCAC
2459





GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCAC
2460





GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCAC
2461





GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCAC
2462





GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHC
2463





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCAC
2464





GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCAC
2465





GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCHCYGG
2466





GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCYGG
2467





GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCHCYG
2468





GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCACYG
2469





GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCYGG
2470





GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCYGG
2471





GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCYGG
2472





GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCYGG
2473





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCYGG
2474





GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCHCYGG
2475





GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCACYG
2476





GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCACYGG
2477





GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCACYGG
2478





GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCACYGG
2479





GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCACYGG
2480





GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCYGG
2481





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCACYGG
2482





GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCACYGG
2483





GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCACFGG
2484





GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCGGG
2485





GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCGGG
2486





GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCGGG
2487





GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCGGG
2488





GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCGGG
2489





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCGGG
2490





GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCACGGG
2491





GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCACGGG
2492





GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCACGGG
2493





GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCACGGG
2494





GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCACTP
2495





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCACTP
2496





GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCACTP
2497





GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCTPK-amide
2498





GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCTPK-
2499


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCTPK-
2500


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCTPK-
2501


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCTPK-
2502


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCTPK-
2503


amide





GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCHCTPK-
2504


amide





GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCYPK-amide
2505





GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCYPK-
2506


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCYPK-
2507


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCYPK-
2508


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCYPK-
2509


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCYPK-
2510


amide





GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCHCYPK-
2511


amide





GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACTPK-amide
2512





GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCACTPK-
2513


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCACTPK-
2514


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCACTPK-
2515


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCACTPK-
2516


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCACTPK-
2517


amide





GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCACTPK-
2518


amide





GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHC-amide
2519





GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHC-
2520


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHC-
2521


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHC-amide
2522





GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHC-
2523


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHC-
2524


amide





GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCAC-amide
2525





GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCAC-
2526


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCAC-
2527


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCAC-amide
2528





GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHC-
2529


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCAC-amide
2530





GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCAC-amide
2531





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCYGG-amide
2532





GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCYGG-amide
2533





GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCYGG-
2534


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCYGG-
2535


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCYGG-
2536


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCYGG-
2537


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCYGG-
2538


amide





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCFGG-amide
2539





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCYG-amide
2540





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYG-amide
2541





GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACYGG-amide
2542





GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCACYGG-
2543


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCACYGG-
2544


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCACYGG-
2545


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCACYGG-
2546


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCACYGG-
2547


amide





GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCACYGG-
2548


amide





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYGG-amide
2549





GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCHCGGG-amide
2550





GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCHCGGG-
2551


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCHCGGG-
2552


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCHCGGG-
2553


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCHCGGG-
2554


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCHCGGG-
2555


amide





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACGGG-amide
2556





GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACFGG-amide
2557





GVIINVKCKISAQCLOPCKDAGMRFGACMNGKCACGGG-amide
2558





GVIINVKCKISAQCL[hLys]PCKDAGMRFGACMNGKCACGGG-
2559


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGACMNGKCACGGG-
2560


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGACMNGKCACGGG-
2561


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGACMNGKCACGGG-
2562


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGACMNGKCACGGG-
2563


amide





GVIINVKCKISAQCL[Dab]PCKDAGMRFGACMNGKCACGGG-
2564


amide





GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCTPK-amide
2565





GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCTPK-
2566


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCTPK-
2567


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCTPK-
2568


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCTPK-
2569


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCTPK-
2570


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCHCTPK-
2571


amide





GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCYPK-amide
2572





GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCYPK-
2573


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCYPK-
2574


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCYPK-
2575


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCYPK-
2576


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCYPK-
2577


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCHCYPK-
2578


amide





GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCACTPK-amide
2579





GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCACTPK-
2580


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCACTPK-
2581


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCACTPK-
2582


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCACTPK-
2583


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCACTPK-
2584


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCACTPK-
2585


amide





GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHC-amide
2586





GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHC-
2587


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHC-
2588


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHC-amide
2589





GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHC-
2590


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHC-amide
2591





GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCAC-amide
2592





GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCAC-
2593


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCAC-
2594


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCAC-amide
2595





GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHC-
2596


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCAC-
2597


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCAC-amide
2598





GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCHCWGG-amide
2599





GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCYGG-amide
2600





GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCYGG-
2601


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCYGG-
2602


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCYGG-
2603


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCYGG-
2604


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCYGG-
2605


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCHCYGG-
2606


amide





GVIINVKCKISAQCLKPCKEAGMRFGACMNGKCACYGG-amide
2607





GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCACYGG-amide
2608





GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCACYGG-
2609


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCACYGG-
2610


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCACYGG-
2611


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCYGG-
2612


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCACYGG-
2613


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCACYGG-
2614


amide





GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCHCGGG-amide
2615





GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCHCGGG-
2616


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCHCGGG-
2617


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCHCGGG-
2618


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCHCGGG-
2619


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCHCGGG-
2620


amide





GVIINVKCKISAQCLOPCKEAGMRFGACMNGKCACGGG-amide
2621





GVIINVKCKISAQCL[hLys]PCKEAGMRFGACMNGKCACGGG-
2622


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGACMNGKCACGGG-
2623


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGACMNGKCACGGG-
2624


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGACMNGKCACTP-
2625


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGACMNGKCACGGG-
2626


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGACMNGKCACGGG-
2627


amide





GVIINVKCKISAQCLKPCK[Cpa]AGMRFGACMNGKCACYGG-
2628


amide





GVIINVKCKISAQCLKPCK[Cpa]AGMRFGACMNGKCACGGG-
2629


amide





GVIINVKCKISAQCLKPCK[Cpa]AGMRFGACMNGKCACY-
2630


amide





Ac-GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCACYGG-
2631


amide





GVIINVKCKISAQCLKPCK[Aad]AGMRFGACMNGKCACYGG-
2632


amide





GVIINVKCKISAQCLKPCK[Aad]AGMRFGACMNGKCHCYGG-
2633


amide





GVIINVKCKISAQCLKPCK[Aad]AGMRFGACMNGKCACYGG
2634





GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCACYGG-amide
2635





GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCACYGG
2636





GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCACY-amide
2637





GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCHCYGG-amide
2638





GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCHCYGG
2639





GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCHCYPK
2640





GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCAC
2641





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[1Nal]GG-
2642


amide





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[1Nal]PK-
2643


amide





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[2Nal]GG-
2644


amide





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[Cha]GG-
2645


amide





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[MePhe]GG-
2646


amide





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[BiPhA]GG-
2647


amide





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKC[Aib]CYGG-
2648


amide





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKC[Abu]CYGG-
2649


amide





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[1Nal]
2650





GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCAC[1Nal]GG-
2651


amide





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCAC[4Bip]-
2652


amide





GVIINVKCKISAQCLHPCKDAGMRFGACMNGKCAC[4Bip]GG-
2653


amide





GVIINVKCKISAQCLKPCKDAGMRFGACMNGKCHCGGG
2654
















TABLE 7D







Additional useful OSK1 peptide analog


sequences: Ala-12 & Ala29


Substituted Series









SEQ



ID


Sequence/structure
NO:





GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK
2655





GVIINVSCKISAQCLEPCKKAGMRFGKCANGKCHCTPK
2656





GVIINVKCKISAQCLKPCKKAGMRFGKCANGKCHCTPK
2657





GVIINVKCKISAQCLEPCKDAGMRFGKCANGKCHCTPK
2658





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2659





GVIINVSCKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2660





GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK
2661





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCYPK
2662





Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK
2663





GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK-amide
2664





Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK-
2665


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCYPK-amide
2666





Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCYPK
2667





Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCYPK-
2668


amide





GVIINVKCKISAQCLKPCKKAGMRFGKCANGKCHCTPK-amide
2669





Ac-GVIINVKCKISAQCLKPCKKAGMRFGKCANGKCHCTPK
2670





Ac-GVIINVKCKISAQCLKPCKKAGMRFGKCANGKCHCTPK-
2671


amide





Ac-GVIINVKCKISAQCLEPCKDAGMRFGKCANGKCHCTPK
2672





GVIINVKCKISAQCLEPCKDAGMRFGKCANGKCHCTPK-amide
2673





Ac-GVIINVKCKISAQCLEPCKDAGMRFGKCANGKCHCTPK-
2674


amide





GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK-amide
2675





Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK
2676





Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK-
2677


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide
2678





Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2679





Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-
2680


amide





VIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK
2681





Ac-VTINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK
2682





VIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK-amide
2683





Ac-VIINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK-
2684


amide





GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK
2685





Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK
2686





GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK-amide
2687





Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK-
2688


amide





VIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2689





Ac-VIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2690





VIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide
2691





Ac-VIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-
2692


amide





NVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2693





Ac-NVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2694





NVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide
2695





Ac-NVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide
2696





KCKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2697





Ac-KCKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2698





KCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide
2699





Ac-KCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide
2700





CKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2701





Ac-CKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2702





CKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide
2703





Ac-CKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide
2704





GVIINVKCKISAQCLKPCKDAGMRNGKCANGKCHCTPK
2705





GVIINVKCKISAQCLKPCKDAGMRNGKCANGKCHCTPK-amide
2706





Ac-GVIINVKCKISAQCLKPCKDAGMRNGKCANGKCHCTPK
2707





Ac-GVIINVKCKISAQCLKPCKDAGMRNGKCANGKCHCTPK-
2708


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK
2709





GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK-amide
2710





Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK
2711





Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK-
2712


amide





GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK
2713





Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK
2714





GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK-amide
2715





Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK-
2716


amide





TIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2717





Ac-TIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2718





TIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide
2719





Ac-TIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-
2720


amide





GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK
2721





Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK
2722





GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK-amide
2723





Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCHCTPK-
2724


amide





GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK
2725





GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2726





GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCACTPK
2727





Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK
2728





GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK-amide
2729





Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCANGKCACTPK-
2730


amide





Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCACTPK
2731





GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCACTPK-amide
2732





Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCACTPK-
2733


amide





Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK
2734





GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-amide
2735





Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCANGKCHCTPK-
2736


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCT
2737





GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCTPK
2738





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCTPK
2739





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCTPK
2740





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCTPK
2741





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCTPK
2742





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCTPK
2743





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCHCTPK
2744





GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCYPK
2745





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCYPK
2746





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCYPK
2747





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCYPK
2748





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCYPK
2749





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCYPK
2750





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCHCYPK
2751





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYPK
2752





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCGCYPK
2753





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACFPK
2754





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACWPK
2755





GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCACYPK
2756





GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACTPK
2757





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCACTPK
2758





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCACTPK
2759





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCACTPK
2760





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCTPK
2761





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCACTPK
2762





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCACTPK
2763





GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHC
2764





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHC
2765





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHC
2766





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHC
2767





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHC
2768





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHC
2769





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCHC
2770





GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCAC
2771





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCAC
2772





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCAC
2773





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCAC
2774





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHC
2775





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCAC
2776





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCAC
2777





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCGCYGG
2778





GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCYGG
2779





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCYGG
2780





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCYGG
2781





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCYGG
2782





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCYGG
2783





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCYGG
2784





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYGG
2785





GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACYGG
2786





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCACYGG
2787





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCACYGG
2788





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCACYGG
2789





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCYGG
2790





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCACYGG
2791





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCACYGG
2792





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYG
2793





GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCGGG
2794





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCGGG
2795





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCGGG
2796





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCGGG
2797





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCGGG
2798





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCGGG
2799





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACFGG
2800





GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACGGG
2801





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCACGGG
2802





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCACGGG
2803





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCACGGG
2804





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCACGGG
2805





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCACGGG
2806





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCACGGG
2807





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACGG
2808





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYG
2809





GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACGG
2810





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCTPK
2811





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCTPK
2812





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCTPK
2813





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCTPK
2814





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCTPK
2815





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCHCTPK
2816





GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCYPK
2817





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCYPK
2818





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCYPK
2819





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCYPK
2820





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCYPK
2821





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCYPK
2822





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCHCYPK
2823





GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCACTPK
2824





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCACTPK
2825





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCACTPK
2826





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCACTPK
2827





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCTPK
2828





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCACTPK
2829





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCACTPK
2830





GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHC
2831





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHC
2832





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHC
2833





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHC
2834





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHC
2835





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHC
2836





GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCAC
2837





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCAC
2838





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCAC
2839





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCAC
2840





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHC
2841





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCAC
2842





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCAC
2843





GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCHCYGG
2844





GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCYGG
2845





GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCHCYG
2846





GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCACYG
2847





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCYGG
2848





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCYGG
2849





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCYGG
2850





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCYGG
2851





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCYGG
2852





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCHCYGG
2853





GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCACYG
2854





GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCACYGG
2855





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCACYGG
2856





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCACYGG
2857





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCACYGG
2858





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCYGG
2859





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCACYGG
2860





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCACYGG
2861





GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCACFGG
2862





GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCGGG
2863





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCGGG
2864





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCGGG
2865





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCGGG
2866





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCGGG
2867





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCGGG
2868





GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCACGGG
2869





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCACGGG
2870





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCACGGG
2871





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCACGGG
2872





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCACTP
2873





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCACTP
2874





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCACTP
2875





GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCTPK-amide
2876





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCTPK-
2877


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCTPK-
2878


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCTPK-
2879


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCTPK-
2880


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCTPK-
2881


amide





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCHCTPK-
2882


amide





GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCYPK-amide
2883





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCYPK-
2884


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCYPK-
2885


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCYPK-
2886


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCYPK-
2887


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCYPK-
2888


amide





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCHCYPK-
2889


amide





GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACTPK-amide
2890





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCACTPK-
2891


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCACTPK-
2892


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCACTPK-
2893


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCACTPK-
2894


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCACTPK-
2895


amide





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCACTPK-
2896


amide





GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHC-amide
2897





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHC-
2898


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHC-
2899


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHC-amide
2900





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHC-
2901


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHC-
2902


amide





GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCAC-amide
2903





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCAC-
2904


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCAC-
2905


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCAC-amide
2906





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHC-
2907


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCAC-
2908


amide





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCAC-amide
2909





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCYGG-amide
2910





GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCYGG-amide
2911





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCYGG-
2912


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCYGG-
2913


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCYGG-
2914


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCYGG-
2915


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCYGG-
2916


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCFGG-amide
2917





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCYG-amide
2918





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYG-amide
2919





GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACYGG-amide
2920





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCACYGG-
2921


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCACYGG-
2922


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCACYGG-
2923


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCACYGG-
2924


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCACYGG-
2925


amide





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCACYGG-
2926


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYGG-amide
2927





GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCHCGGG-amide
2928





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCHCGGG-
2929


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCHCGGG-
2930


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCHCGGG-
2931


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCHCGGG-
2932


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCHCGGG-
2933


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACGGG-amide
2934





GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACFGG-amide
2935





GVIINVKCKISAQCLOPCKDAGMRFGKCANGKCACGGG-amide
2936





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCANGKCACGGG-
2937


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCANGKCACGGG-
2938


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCANGKCACGGG-
2939


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCANGKCACGGG-
2940


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCANGKCACGGG-
2941


amide





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCANGKCACGGG-
2942


amide





GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCTPK-amide
2943





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCTPK-
2944


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCTPK-
2945


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCTPK-
2946


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCTPK-
2947


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCTPK-
2948


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCHCTPK-
2949


amide





GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCYPK-amide
2950





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCYPK-
2951


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCYPK-
2952


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCYPK-
2953


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCYPK-
2954


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCYPK-
2955


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCHCYPK-
2956


amide





GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCACTPK-amide
2957





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCACTPK-
2958


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCACTPK-
2959


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCACTPK-
2960


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCACTPK-
2961


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCACTPK-
2962


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCACTPK-
2963


amide





GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHC-amide
2964





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHC-
2965


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHC-
2966


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHC-amide
2967





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHC-
2968


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHC-amide
2969





GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCAC-amide
2970





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCAC-
2971


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCAC-
2972


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCAC-amide
2973





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHC-
2974


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCAC-amide
2975





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCAC-amide
2976





GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCHCWGG-amide
2977





GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCYGG-amide
2978





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCYGG-
2979


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCYGG-
2980


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCYGG-
2981


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCYGG-
2982


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCYGG-
2983


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCHCYGG-
2984


amide





GVIINVKCKISAQCLKPCKEAGMRFGKCANGKCACYGG-amide
2985





GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCACYGG-amide
2986





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCACYGG-
2987


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCACYGG-
2988


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCACYGG-
2989


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCYGG-
2990


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCACYGG-
2991


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCACYGG-
2992


amide





GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCHCGGG-amide
2993





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCHCGGG-
2994


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCHCGGG-
2995


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCHCGGG-
2996


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCHCGGG-
2997


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCHCGGG-
2998


amide





GVIINVKCKISAQCLOPCKEAGMRFGKCANGKCACGGG-amide
2999





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCANGKCACGGG-
3000


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCANGKCACGGG-
3001


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCANGKCACGGG-
3002


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCANGKCACTP-
3003


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCANGKCACGGG-
3004


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCANGKCACGGG-
3005


amide





GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCANGKCACYGG-
3006


amide





GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCANGKCACGGG-
3007


amide





GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCANGKCACY-
3008


amide





Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCACYGG-
3009


amide





GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCANGKCACYGG-
3010


amide





GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCANGKCHCYGG-
3011


amide





GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCANGKCACYGG
3012





GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCACYGG-amide
3013





CVIINVKCKISAQCLHPCKDAGMRFGKCANGKCACYGG
3014





GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCACY-amide
3015





GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCHCYGG-amide
3016





GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCHCYGG
3017





GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCHCYPK
3018





GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCAC
3019





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[1Nal]GG-
3020


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[1Nal]PK-
3021


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[2Nal]GG-
3022


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[Cha]GG-
3023


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[MePhe]GG-
3024


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[BiPhA]GG-
3025


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKC[Aib]CYGG-
3026


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKC[Abu]CYGG-
3027


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[1Nal]
3028





GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCAC[1Nal]GG-
3029


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCAC[4Bip]-
3030


amide





GVIINVKCKISAQCLHPCKDAGMRFGKCANGKCAC[4Bip]GG-
3031


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCHCGGG
3032
















TABLE 7E







Additional useful OSK1 peptide


analogs: Ala-12 & Ala30


Substituted Series









SEQ



ID


Sequence/structure
NO:





GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK
3033





GVIINVSCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK
3034





GVIINVKCKISAQCLKPCKKAGMRFGKCMAGKCHCTPK
3035





GVIINVKCKISAQCLEPCKDAGMRFGKCMAGKCHCTPK
3036





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3037





GVIINVSCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3038





GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK
3039





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCYPK
3040





Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK
3041





GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
3042





Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK-
3043


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCYPK-amide
3044





Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCYPK
3045





Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCYPK-
3046


amide





GVIINVKCKISAQCLKPCKKAGMRFGKCMAGKCHCTPK-amide
3047





Ac-GVIINVKCKISAQCLKPCKKAGMRFGKCMAGKCHCTPK
3048





Ac-GVIINVKCKISAQCLKPCKKAGMRFGKCMAGKCHCTPK-
3049


amide





Ac-GVIINVKCKISAQCLEPCKDAGMRFGKCMAGKCHCTPK
3050





GVIINVKCKISAQCLEPCKDAGMRFGKCMAGKCHCTPK-amide
3051





Ac-GVIINVKCKISAQCLEPCKDAGMRFGKCMAGKCHCTPK-
3052


amide





GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK-amide
3053





Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK
3054





Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK-
3055


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
3056





Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3057





Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-
3058


amide





VIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK
3059





Ac-VIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK
3060





VIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK-amide
3061





Ac-VIINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK-
3062


amide





GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK
3063





Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK
3064





GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK-amide
3065





Ac-GVIINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK-
3066


amide





VIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3067





Ac-VIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3068





VIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
3069





Ac-VIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-
3070


amide





NVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3071





Ac-NVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3072





NVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
3073





Ac-NVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
3074





KCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3075





Ac-KCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3076





KCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
3077





Ac-KCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
3078





CKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3079





Ac-CKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3080





CKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
3081





Ac-CKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
3082





GVIINVKCKISAQCLKPCKDAGMRNGKCMAGKCHCTPK
3083





GVIINVKCKISAQCLKPCKDAGMRNGKCMAGKCHCTPK-amide
3084





Ac-GVIINVKCKISAQCLKPCKDAGMRNGKCMAGKCHCTPK
3085





Ac-GVIINVKCKISAQCLKPCKDAGMRNGKCMAGKCHCTPK-
3086


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK
3087





GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK-amide
3088





Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK
3089





Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMNRKCHCTPK-
3090


amide





GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK
3091





Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK
3092





GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK-amide
3093





Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK-
3094


amide





TIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3095





Ac-TIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3096





TIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
3097





Ac-TIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-
3098


amide





GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK
3099





Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK
3100





GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK-amide
3101





Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCHCTPK-
3102


amide





GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK
3103





GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3104





GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCACTPK
3105





Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK
3106





GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK-amide
3107





Ac-GVKINVKCKISAQCLEPCKKAGMRFGKCMAGKCACTPK-
3108


amide





Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCACTPK
3109





GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCACTPK-amide
3110





Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCACTPK-
3111


amide





Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK
3112





GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
3113





Ac-GVKINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCTPK-
3114


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCT
3115





GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCTPK
3116





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCTPK
3117





GVIINVKCKISAQCL[hArq]PCKDAGMRFGKCMAGKCHCTPK
3118





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCTPK
3119





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCTPK
3120





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCTPK
3121





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCHCTPK
3122





GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCYPK
3123





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCYPK
3124





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCYPK
3125





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCYPK
3126





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCYPK
3127





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCYPK
3128





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCHCYPK
3129





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYPK
3130





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCGCYPK
3131





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACFPK
3132





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACWPK
4920





GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCACYPK
4921





GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACTPK
4922





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCACTPK
4923





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCACTPK
4924





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCACTPK
4925





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCTPK
4926





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCACTPK
4927





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCACTPK
4928





GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHC
4929





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHC
3133





GVIINVKCKISAQCL[hArq]PCKDAGMRFGKCMAGKCHC
3134





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHC
3135





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHC
3136





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHC
3137





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCHC
3138





GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCAC
3139





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCAC
3140





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCAC
3141





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCAC
3142





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHC
3143





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCAC
3144





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCAC
3145





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCGCYGG
3146





GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCYGG
3147





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCYGG
3148





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCYGG
3149





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCYGG
3150





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCYGG
3151





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCYGG
3152





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYGG
3153





GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACYGG
3154





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCACYGG
3155





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCACYGG
3156





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCACYGG
3157





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCYGG
3158





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCACYGG
3159





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCACYGG
3160





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYG
3161





GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCGGG
3162





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCGGG
3163





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCGGG
3164





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCGGG
3165





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCGGG
3166





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCGGG
3167





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACFGG
3168





GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACGGG
3169





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCACGGG
3170





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCACGGG
3171





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCACGGG
3172





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCACGGG
3173





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCACGGG
3174





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCACGGG
3175





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACGG
3176





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYG
3177





GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACGG
3178





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCTPK
3179





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCTPK
3180





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCTPK
3181





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCTPK
3182





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCTPK
3183





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCHCTPK
3184





GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCYPK
3185





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCYPK
3186





GVIINVKCKISAQCL[hArq]PCKEAGMRFGKCMAGKCHCYPK
3187





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCYPK
3188





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCYPK
3189





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCYPK
3190





GVIINVKCKISAQCL[Dab]PCKEAGMREGKCMAGKCHCYPK
3191





GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCACTPK
3192





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCACTPK
3193





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCACTPK
3194





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCACTPK
3195





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCTPK
3196





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCACTPK
3197





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCACTPK
3198





GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHC
3199





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHC
3200





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHC
3201





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHC
3202





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHC
3203





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHC
3204





GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCAC
3205





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCAC
3206





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCAC
3207





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCAC
3208





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHC
3209





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCAC
3210





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCAC
3211





GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCHCYGG
3212





GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCYGG
3213





GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCHCYG
3214





GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCACYG
3215





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCYGG
3216





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCYGG
3217





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCYGG
3218





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG
3219





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCYGG
3220





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCHCYGG
3221





GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCACYG
3222





GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCACYGG
3223





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCACYGG
3224





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCACYGG
3225





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCACYGG
3226





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG
3227





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCACYGG
3228





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCACYGG
3229





GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCACFGG
3230





GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCGGG
3231





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCGGG
3232





GVIINVKCKISAQCL[hArq]PCKEAGMRFGKCMAGKCHCGGG
3233





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCGGG
3234





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCGGG
3235





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCGGG
3236





GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCACGGG
3237





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCACGGG
3238





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCACGGG
3239





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCACGGG
3240





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCACTP
3241





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCACTP
3242





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCACTP
3243





GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCTPK-amide
3244





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCTPK-
3245


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCTPK-
3246


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCTPK-
3247


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCTPK-
3248


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCTPK-
3249


amide





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCHCTPK-
3250


amide





GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCYPK-amide
3251





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCYPK-
3252


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCYPK-
3253


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCYPK-
3254


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCYPK-
3255


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCYPK-
3256


amide





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCHCYPK-
3257


amide





GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACTPK-amide
3258





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCACTPK-
3259


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCACTPK-
3260


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCACTPK-
3261


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCACTPK-
3262


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCACTPK-
3263


amide





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCACTPK-
3264


amide





GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHC-amide
3265





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHC-
3266


amide





GVIINVKCKISAQCL[hArq]PCKDAGMRFGKCMAGKCHC-
3267


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHC-amide
3268





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHC-
3269


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHC-amide
3270





GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCAC-amide
3271





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCAC-
3272


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCAC-
3273


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCAC-amide
3274





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHC-
3275


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCAC-
3276


amide





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCAC-
3277


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCYGG-amide
3278





GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCYGG-amide
3279





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCYGG-
3280


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCYGG-
3281


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCYGG-
3282


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCYGG-
3283


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCYGG-
3284


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCFGG-amide
3285





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCYG-amide
3286





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYG-amide
3287





GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACYGC-amide
3288





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCACYGG-
3289


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCACYGG-
3290


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCACYGG-
3291


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCACYGG-
3292


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCACYGG-
3293


amide





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCACYGG-
3294


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYGG-amide
3295





GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCHCGGG-amide
3296





GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCHCGGG-
3297


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCHCGGG-
3298


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCHCGGG-
3299


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCHCGGG-
3300


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCHCGGG-
3301


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACGGG-amide
3302





GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACFGG-amide
3303





GVIINVKCKISAQCLOPCKDAGMRFGKCMAGKCACGGG-amide
3304


GVIINVKCKISAQCL[hLys]PCKDAGMRFGKCMAGKCACGGG-
3305


amide





GVIINVKCKISAQCL[hArg]PCKDAGMRFGKCMAGKCACGGG-
3306


amide





GVIINVKCKISAQCL[Cit]PCKDAGMRFGKCMAGKCACGGG-
3307


amide





GVIINVKCKISAQCL[hCit]PCKDAGMRFGKCMAGKCACGGG-
3308


amide





GVIINVKCKISAQCL[Dpr]PCKDAGMRFGKCMAGKCACGGG-
3309


amide





GVIINVKCKISAQCL[Dab]PCKDAGMRFGKCMAGKCACGGG-
3310


amide





GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCTPK-amide
3311





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCTPK-
3312


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCTPK-
3313


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCTPK-
3314


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCTPK-
3315


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCTPK-
3316


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCHCTPK-
3317


amide





GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCYPK-amide
3318





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCYPK-
3319


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCYPK-
3320


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCYPK-
3321


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCYPK-
3322


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCYPK-
3323


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCHCYPK-
3324


amide





GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCACTPK-amide
3325





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCACTPK-
3326


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCACTPK-
3327


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCACTPK-
3328


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCACTPK-
3329


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCACTPK-
3330


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCACTPK-
3331


amide





GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHC-amide
3332





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHC-
3333


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHC-
3334


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHC-amide
3335





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHC-
3336


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHC-amide
3337





GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCAC-amide
3338





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCAC-
3339


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCAC-
3340


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCAC-amide
3341





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHC-
3342


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCAC-
3343


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCAC-
3344


amide





GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCHCWGG-amide
3345





GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCYGG-amide
3346





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCYGG-
3347


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCYGG-
3348


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCYGG-
3349


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG-
3350


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCYGG-
3351


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCHCYGG-
3352


amide





GVIINVKCKISAQCLKPCKEAGMRFGKCMAGKCACYGG-amide
3353





GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCACYGG-amide
3354





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCACYGG-
3355


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCACYGG-
3356


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCACYGG-
3357


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG-
3358


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCACYGG-
3359


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCACYGG-
3360


amide





GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCHCGGG-amide
3361





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCHCGGG-
3362


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCHCGGG-
3363


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCHCGGG-
3364


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCHCGGG-
3365


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCHCGGG-
3366


amide





GVIINVKCKISAQCLOPCKEAGMRFGKCMAGKCACGGG-amide
3367





GVIINVKCKISAQCL[hLys]PCKEAGMRFGKCMAGKCACGGG-
3368


amide





GVIINVKCKISAQCL[hArg]PCKEAGMRFGKCMAGKCACGGG-
3369


amide





GVIINVKCKISAQCL[Cit]PCKEAGMRFGKCMAGKCACGGG-
3370


amide





GVIINVKCKISAQCL[hCit]PCKEAGMRFGKCMAGKCACTP-
3371


amide





GVIINVKCKISAQCL[Dpr]PCKEAGMRFGKCMAGKCACGGG-
3372


amide





GVIINVKCKISAQCL[Dab]PCKEAGMRFGKCMAGKCACGGG-
3373


amide





GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCMAGKCACYGG-
3374


amide





GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCMAGKCACGGG-
3375


amide





GVIINVKCKISAQCLKPCK[Cpa]AGMRFGKCMAGKCACY-
3376


amide





Ac-GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCACYGG-
3377


amide





GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCMAGKCACYGG-
3378


amide





GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCMAGKCHCYGG-
3379


amide





GVIINVKCKISAQCLKPCK[Aad]AGMRFGKCMAGKCACYGG
3380





GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCACYGG-amide
3381





GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCACYGG
3382





GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCACY-amide
3383





GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCHCYGG-amide
3384





GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCHCYGG
3385





GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCHCYPK
3386





GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCAC
3387





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[1Nal]GG-
3388


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[1Nal]PK-
3389


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[2Nal]GG-
3390


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[Cha]GG-
3391


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[MePhe]GG-
3392


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[BiPhA]GG-
3393


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKC[Aib]CYGG-
3394


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKC[Abu]CYGG-
3395


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[1Nal]
3396





GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCAC[1Nal]GG-
3397


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCAC[4Bip]-
3398


amide





GVIINVKCKISAQCLHPCKDAGMRFGKCMAGKCAC[4Bip]GG-
3399


amide





GVIINVKCKISAQCLKPCKDAGMRFGKCMAGKCHCGGG
3400
















TABLE 7F







Addit6ional useful OSK1 peptide


analogs: Ala27Substituted Series









SEQ



ID


Sequence/structure
NO:





GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK
3401





GVIINVSCKISRQCLEPCKKAGMRFGACMNGKCHCTPK
3402





GVIINVKCKISRQCLKPCKKAGMRFGACMNGKCHCTPK
3403





GVIINVKCKISRQCLEPCKDAGMRFGACMNGKCHCTPK
3404





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3405





GVIINVSCKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3406





GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK
3407





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCYPK
3408





Ac-GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK
3409





GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK-amide
3410





Ac-GVIINVKCKISPQCLKPCKDAGMRFGACMNGKCHCTPK-
3411


amide





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCYPK-amide
3412





Ac-GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCYPK
3413





Ac-GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCYPK-
3414


amide





GVIINVKCKISRQCLKPCKKAGMRFGACMNGKCHCTPK-amide
3415





Ac-GVIINVKCKISRQCLKPCKKAGMRFGACMNGKCHCTPK
3416





Ac-GVIINVKCKISRQCLKPCKKAGMRFGACMNGKCHCTPK-
3417


amide





Ac-GVIINVKCKISRQCLEPCKDAGMRFGACMNGKCHCTPK
3418





GVIINVKCKISRQCLEPCKDAGMRFGACMNGKCHCTPK-amide
3419





Ac-GVIINVKCKISRQCLEPCKDAGMRFGACMNGKCHCTPK-
3420


amide





GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK-amide
3421





Ac-GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK
3422





Ac-GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK-
3423


amide





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide
3424





Ac-GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3425





Ac-GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-
3426


amide





VIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK
3427





Ac-VIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK
3428





VIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK-amide
3429





Ac-VIINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK-
3430


amide





GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK
3431





Ac-GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK
3432





GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK-amide
3433





Ac-GVIINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK-
3434


amide





VIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3435





Ac-VIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3436





VIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide
3437





Ac-VIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-
3438


amide





NVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3439





Ac-NVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3440





NVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide
3441





Ac-NVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide
3442





KCKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3443





Ac-KCKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3444





KCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide
3445





Ac-KCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide
3446





CKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3447





Ac-CKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3448





CKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide
3449





Ac-CKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide
3450





GVIINVKCKISRQCLKPCKDAGMRNGACMNGKCHCTPK
3451





GVIINVKCKISRQCLKPCKDAGMRNGACMNGKCHCTPK-amide
3452





Ac-GVIINVKCKISRQCLKPCKDAGMRNGACMNGKCHCTPK
3453





Ac-GVIINVKCKISRQCLKPCKDAGMRNGACMNGKCHCTPK-
3454


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK
3455





GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-amide
3456





Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK
3457





Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-
3458


amide





GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK
3459





Ac-GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK
3460





GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK-amide
3461





Ac-GVIINVKCKISKQCLKPCRDAGMRFGACMNGKCHCTPK-
3462


amide





TIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3463





Ac-TIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3464





TIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide
3465





Ac-TIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-
3466


amide





GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK
3467





Ac-GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK
3468





GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK-amide
3469





Ac-GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCHCTPK-
3470


amide





GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK
3471





GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3472





GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCACTPK
3473





Ac-GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK
3474





GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK-amide
3475





Ac-GVKINVKCKISRQCLEPCKKAGMRFGACMNGKCACTPK-
3476


amide





Ac-GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCACTPK
3477





GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCACTPK-amide
3478





Ac-GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCACTPK-
3479


amide





Ac-GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK
3480





GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-amide
3481





Ac-GVKINVKCKISRQCLKPCKDAGMRFGACMNGKCHCTPK-
3482


amide





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCT
3483





GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCTPK
3484





GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCTPK
3485





GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCTPK
3486





GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCTPK
3487





GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCTPK
3488





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCTPK
3489





GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCHCTPK
3490





GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCYPK
3491





GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCYPK
3492





GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCYPK
3493





GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCYPK
3494





GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCYPK
3495





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCYPK
3496





GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCHCYPK
3497





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYPK
3498





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCGCYPK
3499





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACFPK
3500





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACWPK
3501





GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCACYPK
3502





GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACTPK
3503





GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCACTPK
3504





GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCACTPK
3505





GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCACTPK
3506





GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCTPK
3507





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCACTPK
3508





GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCACTPK
3509





GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHC
3510





GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHC
3511





GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHC
3512





GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHC
3513





GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHC
3514





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHC
3515





GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCHC
3516





GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCAC
3517





GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCAC
3518





GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCAC
3519





GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCAC
3520





GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHC
3521





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCAC
3522





GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCAC
3523





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCGCYGG
3524





GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCYGG
3525





GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCYGG
3526





GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCYGG
3527





GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCYGG
3528





GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCYGG
3529





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCYGG
3530





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYGG
3531





GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACYGG
3532





GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCACYGG
3533





GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCACYGG
3534





GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCACYGG
3535





GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCYGG
3536





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCACYGG
3537





GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCACYGG
3538





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYG
3539





GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCGGG
3540





GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCGGG
3541





GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCGGG
3542





GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCGGG
3543





GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCGGG
3544





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCGGG
3545





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACFGG
3546





GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACGGG
3547





GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCACGGG
3548





GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCACGGG
3549





GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCACGGG
3550





GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCACGGG
3551





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCACGGG
3552





GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCACGGG
3553





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACGG
3554





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYG
3555





GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACGG
3556





GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCTPK
3557





GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCTPK
3558





GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCTPK
3559





GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCTPK
3560





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCTPK
3561





GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCHCTPK
3562





GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCYPK
3563





GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCYPK
3564





GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCYPK
3565





GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCYPK
3566





GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCYPK
3567





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCYPK
3568





GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCHCYPK
3569





GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCACTPK
3570





GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCACTPK
3571





GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCACTPK
3572





GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCACTPK
3573





GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCTPK
3574





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCACTPK
3575





GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCACTPK
3576





GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHC
3577





GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHC
3578





GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHC
3579





GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHC
3580





GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHC
3581





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHC
3582





GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCAC
3583





GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCAC
3584





GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCAC
3585





GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCAC
3586





GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHC
3587





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCAC
3588





GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCAC
3589





GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCHCYGG
3590





GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCYGG
3591





GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCHCYG
3592





GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCACYG
3593





GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCYGG
3594





GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCYGG
3595





GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCYGG
3596





GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCYGG
3597





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCYGG
3598





GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCHCYGG
3599





GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCACYG
3600





GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCACYGG
3601





GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCACYGG
3602





GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCACYGG
3603





GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCACYGG
3604





GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCYGG
3605





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCACYGG
3606





GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCACYGG
3607





GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCACFGG
3608





GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCGGG
3609





GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCGGG
3610





GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCGGG
3611





GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCGGG
3612





GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCGGG
3613





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCGGG
3614





GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCACGGG
3615





GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCACGGG
3616





GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCACGGG
3617





GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCACGGG
3618





GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCACTP
3619





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCACTP
3620





GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCACTP
3621





GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCTPK-amide
3622





GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCTPK-
3623


amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCTPK-
3624


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCTPK-
3625


amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCTPK-
3626


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCTPK-
3627


amide





GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCHCTPK-
3628


amide





GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCYPK-amide
3629





GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCYPK-
3630


amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCYPK-
3631


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCYPK-
3632


amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCYPK-
3633


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCYPK-
3634


amide





GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCHCYPK-
3635


amide





GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACTPK-amide
3636





GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCACTPK-
3637


amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCACTPK-
3638


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCACTPK-
3639


amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCACTPK-
3640


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCACTPK-
3641


amide





GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCACTPK-
3642


amide





GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHC-amide
3643





GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHC-
3644


amide





GVIINVKCKISRQCL[hArg]PCKDAGsMRFGACMNGKCHC-
3645


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHC-amide
3646





GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHC-
3647


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHC-amide
3648





GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCAC-amide
3649





GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCAC-
3650


amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCAC-
3651


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCAC-amide
3652





GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHC-
3653


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCAC-amide
3654





GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCAC-amide
3655





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCYGG-amide
3656





GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCYGG-amide
3657





GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCYGG-
3658


amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCYGG-
3659


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCYGG-
3660


amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCYGG-
3661


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCYGG-
3662


amide





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCFGG-amide
3663





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCYG-amide
3664





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYG-amide
3665





GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACYGG-amide
3666





GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCACYGG-
3667


amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCACYGG-
3668


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCACYGG-
3669


amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCACYGG-
3670


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCACYGG-
3671


amide





GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCACYGG-
3672


amide





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYGG-amide
3673





GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCHCGGG-amide
3674





GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCHCGGG-
3675


amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCHCGGG-
3676


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCHCGGG-
3677


amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCHCGGG-
3678


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCHCGGG-
3679


amide





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACGGG-amide
3680





GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACFGG-amide
3681





GVIINVKCKISRQCLOPCKDAGMRFGACMNGKCACGGG-amide
3682





GVIINVKCKISRQCL[hLys]PCKDAGMRFGACMNGKCACGGG-
3683


amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGACMNGKCACGGG-
3684


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGACMNGKCACGGG-
3685


amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGACMNGKCACGGG-
3686


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGACMNGKCACGGG-
3687


amide





GVIINVKCKISRQCL[Dab]PCKDAGMRFGACMNGKCACGGG-
3688


amide





GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCTPK-amide
3689





GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCTPK-
3690


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCTPK-
3691


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCTPK-
3692


amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCTPK-
3693


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCTPK-
3694


amide





GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCHCTPK-
3695


amide





GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCYPK-amide
3696





GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCYPK-
3697


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCYPK-
3698


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCYPK-
3699


amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCYPK-
3700


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCYPK-
3701


amide





GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCHCYPK-
3702


amide





GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCACTPK-amide
3703





GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCACTPK-
3704


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCACTPK-
3705


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCACTPK-
3706


amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCACTPK-
3707


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCACTPK-
3708


amide





GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCACTPK-
3709


amide





GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHC-amide
3710





GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHC-
3711


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHC-
3712


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHC-amide
3713





GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHC-
3714


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHC-amide
3715





GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCAC-amide
3716





GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCAC-
3717


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCAC-
3718


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCAC-amide
3719





GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHC-
3720


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCAC-amide
3721





GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCAC-amide
3722





GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCHCWGG-amide
3723





GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCYGG-amide
3724





GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCYGG-
3725


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCYGG-
3726


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCYGG-
3727


amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCYGG-
3728


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCYGG-
3729


amide





GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCHCYGG-
3730


amide





GVIINVKCKISRQCLKPCKEAGMRFGACMNGKCACYGG-amide
3731





GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCACYGG-amide
3732





GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCACYGG-
3733


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCACYGG-
3734


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCACYGG-
3735


amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCYGG-
3736


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCACYGG-
3737


amide





GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCACYGG-
3738


amide





GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCHCGGG-amide
3739





GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCHCGGG-
3740


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCHCGGG-
3741


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCHCGGG-
3742


amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCHCGGG-
3743


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCHCGGG-
3744


amide





GVIINVKCKISRQCLOPCKEAGMRFGACMNGKCACGGG-amide
3745





GVIINVKCKISRQCL[hLys]PCKEAGMRFGACMNGKCACGGG-
3746


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGACMNGKCACGGG-
3747


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGACMNGKCACGGG-
3748


amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGACMNGKCACTP-
3749


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGACMNGKCACGGG-
3750


amide





GVIINVKCKISRQCL[Dab]PCKEAGMRFGACMNGKCACGGG-
3751


amide





GVIINVKCKISRQCLKPCK[Cpa]AGMRFGACMNGKCACYGG-
3752


amide





GVIINVKCKISRQCLKPCK[Cpa]AGMRFGACMNGKCACGGG-
3753


amide





GVIINVKCKISRQCLKPCK[Cpa]AGMRFGACMNGKCACY-
3754


amide





Ac-GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCACYGG-
3755


amide





GVIINVKCKISRQCLKPCK[Aad]AGMRFGACMNGKCACYGG-
3756


amide





GVIINVKCKISRQCLKPCK[Aad]AGMRFGACMNGKCHCYGG-
3757


amide





GVIINVKCKISRQCLKPCK[Aad]AGMRFGACMNGKCACYGG
3758





GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCACYGG-amide
3759





GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCACYGG
3760





GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCACY-amide
3761





GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCHCYGG-amide
3762





GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCHCYGG
3763





GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCHCYPK
3764





GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCAC
3765





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[1Nal]GG-
3766


amide





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[1Nal]PK-
3767


amide





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[2Nal]GG-
3768


amide





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[Cha]GG-
3769


amide





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[MePhe]GG-
3770


amide





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[BiPhA]GG-
3771


amide





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKC[Aib]CYGG-
3772


amide





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKC[Abu]CYGG-
3773


amide





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[1Nal]
3774





GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCAC[1Nal]GG-
3775


amide





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCAC[4Bip]-
3776


amide





GVIINVKCKISRQCLHPCKDAGMRFGACMNGKCAC[4Bip]GG-
3777


amide





GVIINVKCKISRQCLKPCKDAGMRFGACMNGKCHCGGG
3778
















TABLE 7G







Additional useful OSK1 peptide


analogs: Ala 29 Substituted Series









SEQ



ID


Sequence/structure
NO:





GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK
3779





GVIINVSCKISRQCLEPCKKAGMRFGKCANGKCHCTPK
3780





GVIINVKCKISRQCLKPCKKAGMRFGKCANGKCHCTPK
3781





GVIINVKCKISRQCLEPCKDAGMRFGKCANGKCHCTPK
3782





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3783





GVIINVSCKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3784





GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK
3785





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCYPK
3786





Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK
3787





GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK-amide
3788





Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCANGKCHCTPK-
3789


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCYPK-amide
3790





Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCYPK
3791





Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCYPK-
3792


amide





GVIINVKCKISRQCLKPCKKAGMRFGKCANGKCHCTPK-amide
3793





Ac-GVIINVKCKISRQCLKPCKKAGMRFGKCANGKCHCTPK
3794





Ac-GVIINVKCKISRQCLKPCKKAGMRFGKCANGKCHCTPK-
3795


amide





Ac-GVIINVKCKISRQCLEPCKDAGMRFGKCANGKCHCTPK
3796





GVIINVKCKISRQCLEPCKDAGMRFGKCANGKCHCTPK-amide
3797





Ac-GVIINVKCKISRQCLEPCKDAGMRFGKCANGKCHCTPK-
3798


amide





GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK-amide
3799





Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK
3800





Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK-
3801


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide
3802





Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3803





Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-
3804


amide





VIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK
3805





Ac-VIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK
3806





VIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK-amide
3807





Ac-VIINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK-
3808


amide





GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK
3809





Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK
3810





GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK-amide
3811





Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK-
3812


amide





VIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3813





Ac-VIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3814





VIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-
3815


amide





Ac-VIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-
3816


amide





NVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3817





Ac-NVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3818





NVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide
3819





Ac-NVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide
3820





KCKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3821





Ac-KCKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3822





KCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide
3823





Ac-KCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide
3824





CKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3825





Ac-CKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3826





CKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide
3827





Ac-CKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide
3828





GVIINVKCKISRQCLKPCKDAGMRNGKCANGKCHCTPK
3829





GVIINVKCKISRQCLKPCKDAGMRNGKCANGKCHCTPK-amide
3830





Ac-GVIINVKCKISRQCLKPCKDAGMRNGKCANGKCHCTPK
3831





Ac-GVIINVKCKISRQCLKPCKDAGMRNGKCANGKCHCTPK-
3832


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK
3833





GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-amide
3834





Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK
3835





Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-
3836


amide





GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK
3837





Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK
3838





GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK-amide
3839





Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCANGKCHCTPK-
3840


amide





TIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3841





Ac-TIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3842





TIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide
3843





Ac-TIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-
3844


amide





GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK
3845





Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK
3846





GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK-amide
3847





Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCHCTPK-
3848


amide





GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK
3849





GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3850





GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCACTPK
3851





Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK
3852





GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK-amide
3853





Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK-
3854


amide





Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCACTPK
3855





GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCACTPK-amide
3856





Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCACTPK-
3857


amide





Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK
3858





GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-amide
3859





Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCANGKCHCTPK-
3860


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCT
3861





GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCTPK
3862





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCTPK
3863





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCTPK
3864





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCTPK
3865





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCTPK
3866





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCTPK
3867





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCHCTPK
3868





GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCYPK
3869





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCYPK
3870





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCYPK
3871





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCYPK
3872





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCYPK
3873





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCYPK
3874





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCHCYPK
3875





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYPK
3876





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCGCYPK
3877





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACFPK
3878





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACWPK
3879





GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCACYPK
3880





GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACTPK
3881





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCACTPK
3882





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCACTPK
3883





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCACTPK
3884





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCTPK
3885





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCACTPK
3886





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCACTPK
3887





GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHC
3888





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHC
3889





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHC
3890





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHC
3891





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHC
3892





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHC
3893





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCHC
3894





GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCAC
3895





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCAC
3896





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCAC
3897





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCAC
3898





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHC
3899





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCAC
3900





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCAC
3901





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCGCYGG
3902





GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCYGG
3903





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCYGG
3904





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCYGG
3905





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCYGG
3906





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCYGG
3907





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCYGG
3908





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYGG
3909





GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACYGG
3910





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCACYGG
3911





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCACYGG
3912





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCACYGG
3913





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCYGG
3914





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCACYGG
3915





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCACYGG
3916





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYG
3917





GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCGGG
3918





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCGGG
3919





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCGGG
3920





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCGGG
3921





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCGGG
3922





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCGGG
3923





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACFGG
3924





GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACGGG
3925





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCACGGG
3926





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCACGGG
3927





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCACGGG
3928





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCACGGG
3929





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCACGGG
3930





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCACGGG
3931





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACGG
3932





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYG
3933





GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACGG
3934





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCTPK
3935





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCTPK
3936





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCTPK
3937





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCTPK
3938





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCTPK
3939





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCHCTPK
3940





GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCYPK
3941





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCYPK
3942





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCYPK
3943





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCYPK
3944





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCYPK
3945





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCYPK
3946





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCHCYPK
3947





GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCACTPK
3948





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCACTPK
3949





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCACTPK
3950





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCACTPK
3951





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCTPK
3952





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCACTPK
3953





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCACTPK
3954





GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHC
3955





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHC
3956





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHC
3957





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHC
3958





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHC
3959





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHC
3960





GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCAC
3961





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCAC
3962





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCAC
3963





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCAC
3964





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHC
3965





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCAC
3966





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCAC
3967





GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCHCYGG
3968





GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCYGG
3969





GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCHCYG
3970





GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCACYG
3971





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCYGG
3972





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCYCG
3973





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCYGG
3974





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCYGG
3975





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCYGG
3976





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCHCYGG
3977





GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCACYG
3978





GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCACYGG
3979





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCACYGG
3980





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCACYGG
3981





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCACYGG
3982





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCYGG
3983





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCACYGG
3984





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCACYGG
3985





GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCACFGG
3986





GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCGGG
3987





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCGGG
3988





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCGGG
3989





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCGGG
3990





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCGGG
3991





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCGGG
3992





GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCACGGG
3993





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCACGGG
3994





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCACGGG
3995





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCACGGG
3996





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCACTP
3997





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCACTP
3998





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCACTP
3999





GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCTPK-amide
4000





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCTPK-
4001


amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCTPK-
4002


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCTPK-
4003


amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCTPK-
4004


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCTPK-
4005


amide





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCHCTPK-
4006


amide





GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCYPK-amide
4007





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCYPK-
4008


amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCYPK-
4009


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCYPK-
4010


amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCYPK-
4011


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCYPK-
4012


amide





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCHCYPK-
4013


amide





GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACTPK-amide
4014





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCACTPK-
4015


amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCACTPK-
4016


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCACTPK-
4017


amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCACTPK-
4018


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCACTPK-
4019


amide





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCACTPK-
4020


amide





GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHC-amide
4021





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHC-
4022


amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHC-
4023


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHC-amide
4024





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHC-
4025


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHC-amide
4026





GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCAC-amide
4027





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCAC-
4028


amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCAC-
4029


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCAC-amide
4030





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHC-
4031


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCAC-amide
4032





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCAC-amide
4033





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCYGG-amide
4034





GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCYGG-amide
4035





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCYGG-
4036


amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCYGG-
4037


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCYGG-
4038


amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCYGG-
4039


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCYGG-
4040


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCFGG-amide
4041





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCYG-amide
4042





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYG-amide
4043





GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACYGG-amide
4044





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCACYGG-
4045


amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCACYGG-
4046


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCACYGG-
4047


amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCACYGG-
4048


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCACYGG-
4049


amide





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCACYGG-
4050


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYGG-amide
4051





GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCHCGGG-amide
4052





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCHCGGG-
4053


amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCHCGGG-
4054


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCHCGGG-
4055


amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCHCGGG-
4056


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCHCGGG-
4057


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACGGG-amide
4058





GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACFGG-amide
4059





GVIINVKCKISRQCLOPCKDAGMRFGKCANGKCACGGG-amide
4060





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCANGKCACGGG-
4061


amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCANGKCACGGG-
4062


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCANGKCACGGG-
4063


amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCANGKCACGGG-
4064


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCANGKCACGGG-
4065


amide





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCANGKCACGGG-
4066


amide





GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCTPK-amide
4067





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCTPK-
4068


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCTPK-
4069


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCTPK-
4070


amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCTPK-
4071


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCTPK-
4072


amide





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCHCTPK-
4073


amide





GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCYPK-amide
4074





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCYPK-
4075


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCYPK-
4076


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCYPK-
4077


amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCYPK-
4078


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCYPK-
4079


amide





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCHCYPK-
4080


amide





GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCACTPK-amide
4081





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCACTPK-
4082


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCACTPK-
4083


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCACTPK-
4084


amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCACTPK-
4085


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCACTPK-
4086


amide





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCACTPK-
4087


amide





GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHC-amide
4088





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHC-
4089


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHC-
4090


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHC-amide
4091





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHC-
4092


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHC-amide
4093





GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCAC-amide
4094





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCAC-
4095


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCAC-
4096


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCAC-amide
4097





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHC-
4098


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCAC-amide
4099





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCAC-amide
4100





GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCHCWGG-amide
4101





GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCYGG-amide
4102





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCYGG-
4103


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCYGG-
4104


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCYGG-
4105


amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCYGG-
4106


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCYGG-
4107


amide





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCHCYGG-
4108


amide





GVIINVKCKISRQCLKPCKEAGMRFGKCANGKCACYGG-amide
4109





GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCACYGG-amide
4110





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCACYGG-
4111


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCACYGG-
4112


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCACYGG-
4113


amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCYGG-
4114


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCACYGG-
4115


amide





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCACYGG-
4116


amide





GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCHCGGG-amide
4117





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCHCGGG-
4118


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCHCGGG-
4119


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCHCGGG-
4120


amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCHCGGG-
4121


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCHCGGG-
4122


amide





GVIINVKCKISRQCLOPCKEAGMRFGKCANGKCACGGG-amide
4123





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCANGKCACGGG-
4124


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCANGKCACGGG-
4125


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCANGKCACGGG-
4126


amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCANGKCACTP-
4127


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCANGKCACGGG-
4128


amide





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCANGKCACGGG-
4129


amide





GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCANGKCACYGG-
4130


amide





GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCANGKCACGGG-
4131


amide





GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCANGKCACY-
4132


amide





Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCACYGG-
4133


amide





GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCANGKCACYGG-
4134


amide





GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCANGKCHCYGG-
4135


amide





GVIINVKCKISRQCLKPCK[Aad]ACMRFGKCANGKCACYGG
4136





GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCACYGG-amide
4137





GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCACYGG
4138





GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCACY-amide
4139





GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCHCYGG-amide
4140





GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCHCYGG
4141





GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCHCYPK
4142





GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCAC
4143





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[1Nal]GG-
4144


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[1Nal]PK-
4145


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[2Nal]GG-
4146


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[Cha]GG-
4147


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[MePhe]GG-
4148


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[BiPhA]GG-
4149


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKC[Aib]CYGG-
4150


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKC[Abu]CYGG-
4151


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[1Nal]
4152





GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCAC[1Nal]GG-
4153


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCAC[4Bip]-
4154


amide





GVIINVKCKISRQCLHPCKDAGMRFGKCANGKCAC[4Bip]GG-
4155


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCANGKCHCGGG
4156
















TABLE 7H







Additional useful OSK1 peptide


analogs: Ala 30 Substituted Series









SEQ



ID


Sequence/structure
NO:





GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK
4157





GVIINVSCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK
4158





GVIINVKCKISRQCLKPCKKAGMRFGKCMAGKCHCTPK
4159





GVIINVKCKISRQCLEPCKDAGMRFGKCMAGKCHCTPK
4160





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4161





GVIINVSCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4162





GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK
4163





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCYPK
4164





Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK
4165





GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
4166





Ac-GVIINVKCKISPQCLKPCKDAGMRFGKCMAGKCHCTPK-
4167


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCYPK-amide
4168





Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCYPK
4169





Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCYPK-
4170


amide





GVIINVKCKISRQCLKPCKKAGMRFGKCMAGKCHCTPK-amide
4171





Ac-GVIINVKCKISRQCLKPCKKAGMRFGKCMAGKCHCTPK
4172





Ac-GVIINVKCKISRQCLKPCKKAGMRFGKCMAGKCHCTPK-
4173


amide





Ac-GVIINVKCKISRQCLEPCKDAGMRFGKCMAGKCHCTPK
4174





GVIINVKCKISRQCLEPCKDAGMRFGKCMAGKCHCTPK-amide
4175





Ac-GVIINVKCKISRQCLEPCKDAGMRFGKCMAGKCHCTPK-
4176


amide





GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK-amide
4177





Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK
4178





Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK-
4179


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
4180





Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4181





Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-
4182


amide





VIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK
4183





Ac-VIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK
4184





VIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK-amide
4185





Ac-VIINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK-
4186


amide





GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK
4187





Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK
4188





GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK-amide
4189





Ac-GVIINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK-
4190


amide





VIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4191





Ac-VIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4192





VIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
4193





Ac-VIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-
4194


amide





NVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4195





Ac-NVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4196





NVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
4197





Ac-NVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
4198





KCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4199





Ac-KCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4200





KCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
4201





Ac-KCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
4202





CKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4203





Ac-CKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4204





CKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
4205





Ac-CKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
4206





GVIINVKCKISRQCLKPCKDAGMRNGKCMAGKCHCTPK
4207





GVIINVKCKISRQCLKPCKDAGMRNGKCMAGKCHCTPK-amide
4208





Ac-GVIINVKCKISRQCLKPCKDAGMRNGKCMAGKCHCTPK
4209





Ac-GVIINVKCKISRQCLKPCKDAGMRNGKCMAGKCHCTPK-
4210


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK
4211





GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-amide
4212





Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK
4213





Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMNRKCHCTPK-
4214


amide





GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK
4215





Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK
4216





GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK-amide
4217





Ac-GVIINVKCKISKQCLKPCRDAGMRFGKCMAGKCHCTPK-
4218


amide





TIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4219





Ac-TIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4220





TIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
4221





Ac-TIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-
4222


amide





GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK
4223





Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK
4224





GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK-amide
4225





Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCHCTPK-
4226


amide





GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK
4227





GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4228





GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCACTPK
4229





Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK
4230





GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK-amide
4231





Ac-GVKINVKCKISRQCLEPCKKAGMRFGKCMAGKCACTPK-
4232


amide





Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCACTPK
4233





GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCACTPK-amide
4234





Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCACTPK-
4235


amide





Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK
4236





GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-amide
4237





Ac-GVKINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCTPK-
4238


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCT
4239





GVIINVKCKISRQCLOPCKDAGMRFGKCMACKCHCTPK
4240





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCTPK
4241





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCTPK
4242





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCTPK
4243





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCTPK
4244





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCTPK
4245





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCHCTPK
4246





GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCYPK
4247





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCYPK
4248





GVIINVKCKISRQCL[hArg]PCKDACMRFGKCMACKCHCYPK
4249





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCYPK
4250





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCYPK
4251





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCYPK
4252





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCHCYPK
4253





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYPK
4254





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCGCYPK
4255





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACEPK
4256





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACWPK
4257





GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCACYPK
4258





GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACTPK
4259





GVIINVKCKISRQCL[hLys]PCKDAGMREGKCMAGKCACTPK
4260





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCACTPK
4261





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCACTPK
4262





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCTPK
4263





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCACTPK
4264





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCACTPK
4265





GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHC
4266





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHC
4267





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHC
4268





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHC
4269





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHC
4270





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHC
4271





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCHC
4272





GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCAC
4273





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCAC
4274





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCAC
4275





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCAC
4276





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHC
4277





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCAC
4278





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCAC
4279





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCGCYGG
4280





GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCYGG
4281





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCYGG
4282





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCYGG
4283





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCYGG
4284





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCYGG
4285





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCYGG
4286





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYGG
4287





GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACYGG
4288





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCACYGG
4289





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCACYGG
4290





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCACYGG
4291





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCYGG
4292





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCACYGG
4293





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCACYGG
4294





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYG
4295





GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCGGG
4296





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCGGG
4297





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCGGG
4298





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCGGG
4299





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCGGG
4300





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCGGG
4301





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACFGG
4302





GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACGGG
4303





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCACGGG
4304





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCACGGG
4305





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCACGGG
4306





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCACGGG
4307





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCACGGG
4308





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCACGGG
4309





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACGG
4310





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYG
4311





GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACGG
4312





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCTPK
4313





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCTPK
4314





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCTPK
4315





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCTPK
4316





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCTPK
4317





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCHCTPK
4318





GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCYPK
4319





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCYPK
4320





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCYPK
4321





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCYPK
4322





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCYPK
4323





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCYPK
4324





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCHCYPK
4325





GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCACTPK
4326





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCACTPK
4327





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCACTPK
4328





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCACTPK
4329





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCTPK
4330





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCACTPK
4331





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCACTPK
4332





GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHC
4333





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHC
4334





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHC
4335





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHC
4336





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHC
4337





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHC
4338





GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCAC
4339





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCAC
4340





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCAC
4341





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCAC
4342





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHC
4343





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCAC
4344





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCAC
4345





GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCHCYGG
4346





GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCYGG
4347





GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCHCYG
4348





GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCACYG
4349





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCYGG
4350





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCYGG
4351





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCYGG
4352





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG
4353





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCYGG
4354





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCHCYGG
4355





GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCACYG
4356





GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCACYGG
4357





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCACYGG
4358





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCACYGG
4359





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCACYGG
4360





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG
4361





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCACYGG
4362





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCACYGG
4363





GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCACFGG
4364





GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCGGG
4365





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCGGG
4366





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCGGG
4367





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCGGG
4368





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCGGG
4369





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCGGG
4370





GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCACGGG
4371





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCACGGG
4372





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCACGGG
4373





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCACGGG
4374





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCACTP
4375





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCACTP
4376





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCACTP
4377





GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCTPK-amide
4378





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCTPK-
4379


amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCTPK-
4380


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCTPK-
4381


amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCTPK-
4382


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCTPK-
4383


amide





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCHCTPK-
4384


amide





GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCYPK-amide
4385





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCYPK-
4386


amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCYPK-
4387


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCYPK-
4388


amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCYPK-
4389


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCYPK-
4390


amide





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCHCYPK-
4391


amide





GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACTPK-amide
4392





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCACTPK-
4393


amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCACTPK-
4394


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCACTPK-
4395


amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCACTPK-
4396


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCACTPK-
4397


amide





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCACTPK-
4398


amide





GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHC-amide
4399





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHC-
4400


amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHC-
4401


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHC-amide
4402





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHC-
4403


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHC-amide
4404





GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCAC-amide
4405





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCAC-
4406


amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCAC-
4407


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCAC-amide
4408





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHC-
4409


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCAC-amide
4410





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCAC-amide
4411





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCYGG-amide
4412





GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCYGG-amide
4413





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCYGG-
4414


amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCYGG-
4415


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCYGG-
4416


amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCYGG-
4417


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCYGG-
4418


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCFGG-amide
4419





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCYG-amide
4420





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYG-amide
4421





GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACYGG-amide
4422





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCACYGG-
4423


amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCACYGG-
4424


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCACYGG-
4425


amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCACYGG-
4426


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCACYGG-
4427


amide





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCACYGG-
4428


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYGG-amide
4429





GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCHCGGG-amide
4430





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCHCGGG-
4431


amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCHCGGG-
4432


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCHCGGG-
4433


amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCHCGGG-
4434


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCHCGGG-
4435


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACGGG-amide
4436





GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACFGG-amide
4437





GVIINVKCKISRQCLOPCKDAGMRFGKCMAGKCACGGG-amide
4438





GVIINVKCKISRQCL[hLys]PCKDAGMRFGKCMAGKCACGGG-
4439


amide





GVIINVKCKISRQCL[hArg]PCKDAGMRFGKCMAGKCACGGG-
4440


amide





GVIINVKCKISRQCL[Cit]PCKDAGMRFGKCMAGKCACGGG-
4441


amide





GVIINVKCKISRQCL[hCit]PCKDAGMRFGKCMAGKCACGGG-
4442


amide





GVIINVKCKISRQCL[Dpr]PCKDAGMRFGKCMAGKCACGGG-
4443


amide





GVIINVKCKISRQCL[Dab]PCKDAGMRFGKCMAGKCACGGG-
4444


amide





GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCTPK-amide
4445





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCTPK-
4446


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCTPK-
4447


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCTPK-
4448


amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCTPK-
4449


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCTPK-
4450


amide





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCHCTPK-
4451


amide





GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCYPK-amide
4452





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCYPK-
4453


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCYPK-
4454


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCYPK-
4455


amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCYPK-
4456


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCYPK-
4457


amide





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCHCYPK-
4458


amide





GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCACTPK-amide
4459





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCACTPK-
4460


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCACTPK-
4461


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCACTPK-
4462


amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCACTPK-
4463


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCACTPK-
4464


amide





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCACTPK-
4465


amide





GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHC-amide
4466





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHC-
4467


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHC-
4468


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHC-amide
4469





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHC-
4470


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHC-amide
4471





GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCAC-amide
4472





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCAC-
4473


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCAC-
4474


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCAC-amide
4475





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHC-
4476


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCAC-amide
4477





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCAC-amide
4478





GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCHCWGG-amide
4479





GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCYGG-amide
4480





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCYGG-
4481


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCYGG-
4482


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCYGG-
4483


amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG-
4484


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCYGG-
4485


amide





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCHCYGG-
4486


amide





GVIINVKCKISRQCLKPCKEAGMRFGKCMAGKCACYGG-amide
4487





GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCACYGG-amide
4488





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCACYGG-
4489


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCACYGG-
4490


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCACYGG-
4491


amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCYGG-
4492


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCACYGG-
4493


amide





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCACYGG-
4494


amide





GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCHCGGG-amide
4495





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCHCGGG-
4496


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCHCGGG-
4497


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCHCGGG-
4498


amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCHCGGG-
4499


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCHCGGG-
4500


amide





GVIINVKCKISRQCLOPCKEAGMRFGKCMAGKCACGGG-amide
4501





GVIINVKCKISRQCL[hLys]PCKEAGMRFGKCMAGKCACGGG-
4502


amide





GVIINVKCKISRQCL[hArg]PCKEAGMRFGKCMAGKCACGGG-
4503


amide





GVIINVKCKISRQCL[Cit]PCKEAGMRFGKCMAGKCACGGG-
4504


amide





GVIINVKCKISRQCL[hCit]PCKEAGMRFGKCMAGKCACTP-
4505


amide





GVIINVKCKISRQCL[Dpr]PCKEAGMRFGKCMAGKCACGGG-
4506


amide





GVIINVKCKISRQCL[Dab]PCKEAGMRFGKCMAGKCACGGG-
4507


amide





GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMAGKCACYGG-
4508


amide





GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMAGKCACGGG-
4509


amide





GVIINVKCKISRQCLKPCK[Cpa]AGMRFGKCMAGKCACY-
4510


amide





Ac-GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCACYGG-
4511


amide





GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMAGKCACYGG-
4512


amide





GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMAGKCHCYGG-
4513


amide





GVIINVKCKISRQCLKPCK[Aad]AGMRFGKCMAGKCACYGG
4514





GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCACYGG-amide
4515





GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCACYGG
4516





GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCACY-amide
4517





GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCHCYGG-amide
4518





GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCHCYGG
4519





GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCHCYPK
4520





GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCAC
4521





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[1Nal]GG-
4522


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[1Nal]PK-
4523


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[2Nal]GG-
4524


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[Cha]GG-
4525


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[MePhe]GG-
4526


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[BiPhA]GG-
4527


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKC[Aib]CYGG-
4528


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKC[Abu]CYGG-
4529


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[1Nal]
4530





GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCAC[1Nal]GG-
4531


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCAC[4Bip]-
4532


amide





GVIINVKCKISRQCLHPCKDAGMRFGKCMAGKCAC[4Bip]GG-
4533


amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMAGKCHCGGG
4534

















TABLE 71







Additional useful OSK1 peptide analogs:



Combined Ala-11, 12, 27, 29, 30 Substituted


Series










SEQ




ID


Sequence/structure
NO:





GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK
4535






GVIINVSCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK
4536





GVIINVKCKIAAQCLKPCKKAGMRFGACAAGKCHCTPK
4537





GVIINVKCKIAAQCLEPCKDAGMRFGACAAGKCHCTPK
4538





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4539





GVIINVSCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4540





GVIINVKCKISPQCLKPCKDAGMRFGACAAGKCHCTPK
4541





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCYPK
4542





Ac-GVIINVKCKISPQCLKPCKDAGMRFGACAAGKCHCTPK
4543





GVIINVKCKISPQCLKPCKDAGMRFGACAAGKCHCTPK-amide
4544





Ac-GVIINVKCKISPQCLKPCKDAGMRFGACAAGKCHCTPK-
4545


amide





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCYPK-amide
4546





Ac-GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCYPK
4547





Ac-GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCYPK-
4548


amide





GVIINVKCKIAAQCLKPCKKAGMRFGACAAGKCHCTPK-amide
4549





Ac-GVIINVKCKIAAQCLKPCKKAGMRFGACAAGKCHCTPK
4550





Ac-GVIINVKCKIAAQCLKPCKKAGMRFGACAAGKCHCTPK-
4551


amide





Ac-GVIINVKCKIAAQCLEPCKDAGMRFGACAAGKCHCTPK
4552





GVIINVKCKIAAQCLEPCKDAGMRFGACAAGKCHCTPK-amide
4553





Ac-GVIINVKCKIAAQCLEPCKDAGMRFGACAAGKCHCTPK-
4554


amide





GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK-amide
4555





Ac-GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK
4556





Ac-GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK-
4557


amide





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide
4558





Ac-GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4559





Ac-GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-
4560


amide





VIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK
4561





Ac-VIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK
4562





VIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK-amide
4563





Ac-VIINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK-
4564


amide





GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK
4565





Ac-GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK
4566





GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK-amide
4567





Ac-GVIINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK-
4568


amide





VIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4569





Ac-VIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4570





VIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide
4571





Ac-VIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-
4572


amide





NVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4573





Ac-NVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4574





NVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide
4575





Ac-NVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide
4576





KCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4577





Ac-KCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4578





KCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide
4579





Ac-KCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide
4580





CKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4581





Ac-CKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4582





CKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide
4583





Ac-CKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide
4584





GVIINVKCKIAAQCLKPCKDAGMRNGACAAGKCHCTPK
4585





GVIINVKCKIAAQCLKPCKDAGMRNGACAAGKCHCTPK-amide
4586





Ac-GVIINVKCKIAAQCLKPCKDAGMRNGACAAGKCHCTPK
4587





Ac-GVIINVKCKIAAQCLKPCKDAGMRNGACAAGKCHCTPK-
4588


amide





GVIINVKCKIAAQCLKPCKDAGMRFGKCMNRKCHCTPK
4589





GVIINVKCKIAAQCLKPCKDAGMRFGKCMNRKCHCTPK-amide
4590





Ac-GVIINVKCKIAAQCLKPCKDAGMRFGKCMNRKCHCTPK
4591





Ac-GVIINVKCKIAAQCLKPCKDAGMRFGKCMNRKCHCTPK-
4592


amide





GVIINVKCKISKQCLKPCRDAGMRFGACAAGKCHCTPK
4593





Ac-GVIINVKCKISKQCLKPCRDAGMRFGACAAGKCHCTPK
4594





GVIINVKCKISKQCLKPCRDAGMRFGACAAGKCHCTPK-amide
4595





Ac-GVIINVKCKISKQCLKPCRDAGMRFGACAAGKCHCTPK-
4596


amide





TIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4597





Ac-TIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4598





TIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide
4599





Ac-TIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-
4600


amide





GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK
4601





Ac-GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK
4602





GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK-amide
4603





Ac-GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCHCTPK-
4604


amide





GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK
4605





GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4606





GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCACTPK
4607





Ac-GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK
4608





GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK-amide
4609





Ac-GVKINVKCKIAAQCLEPCKKAGMRFGACAAGKCACTPK-
4610


amide





Ac-GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCACTPK
4611





GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCACTPK-amide
4612





Ac-GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCACTPK-
4613


amide





Ac-GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK
4614





GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-amide
4615





Ac-GVKINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCTPK-
4616


amide





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCT
4617





GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCTPK
4618





GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCTPK
4619





GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCTPK
4620





GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCTPK
4621





GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCTPK
4622





GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCTPK
4623





GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCHCTPK
4624





GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCYPK
4625





GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCYPK
4626





GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCYPK
4627





GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCYPK
4628





GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCYPK
4629





GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCYPK
4630





GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCHCYPK
4631





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYPK
4632





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCGCYPK
4633





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACFPK
4634





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACWPK
4635





GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCACYPK
4636





GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACTPK
4637





GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCACTPK
4638





GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCACTPK
4639





GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCACTPK
4640





GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCTPK
4641





GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCACTPK
4642





GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCACTPK
4643





GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHC
4644





GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHC
4645





GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHC
4646





GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHC
4647





GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHC
4648





GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHC
4649





GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCHC
4650





GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCAC
4651





GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCAC
4652





GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCAC
4653





GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCAC
4654





GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHC
4655





GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCAC
4656





GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCAC
4657





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCGCYGG
4658





GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCYGG
4659





GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCYGG
4660





GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCYGG
4661





GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCYGG
4662





GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCYGG
4663





GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCYGG
4664





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYGG
4665





GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACYGG
4666





GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCACYGG
4667





GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCACYGG
4668





GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCACYGG
4669





GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCYGG
4670





GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCACYGG
4671





GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCACYGG
4672





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYG
4673





GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCGGG
4674





GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCGGG
4675





GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCGGG
4676





GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCGGG
4677





GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCGGG
4678





GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCGGG
4679





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACFGG
4680





GVIINVKCKIAAQCLOPCKDAGMRPGACAAGKCACGGG
4681





GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCACGGG
4682





GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCACGGG
4683





GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCACGGG
4684





GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCACGGG
4685





GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCACGGG
4686





GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCACGGG
4687





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACGG
4688





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYG
4689





GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACGG
4690





GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCTPK
4691





GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCTPK
4692





GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCTPK
4693





GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCTPK
4694





GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCTPK
4695





GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCHCTPK
4696





GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCYPK
4697





GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCYPK
4698





GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCYPK
4699





GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCYPK
4700





GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCYPK
4701





GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCYPK
4702





GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCHCYPK
4703





GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCACTPK
4704





GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCACTPK
4705





GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCACTPK
4706





GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCACTPK
4707





GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCTPK
4708





GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCACTPK
4709





GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCACTPK
4710





GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHC
4711





GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHC
4712





GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHC
4713





GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHC
4714





GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHC
4715





GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHC
4716





GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCAC
4717





GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCAC
4718





GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCAC
4719





GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCAC
4720





GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHC
4721





GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCAC
4722





GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCAC
4723





GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCHCYGG
4724





GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCYGG
4725





GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCHCYG
4726





GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCACYG
4727





GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCYGG
4728





GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCYGG
4729





GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCYGG
4730





GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCYGG
4731





GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCYGG
4732





GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCHCYGG
4733





GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCACYG
4734





GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCACYGG
4735





GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCACYGG
4736





GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCACYGG
4737





GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCACYGG
4738





GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCYGG
4739





GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCACYGG
4740





GVIINVKCKIAAQCL[Dab]PCKEAGMRPGACAAGKCACYGG
4741





GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCACFGG
4742





GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCGGG
4743





GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCGGG
4744





GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCGGG
4745





GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCGGG
4746





GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCGGG
4747





GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCGGG
4748





GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCACGGG
4749





GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCACGGG
4750





GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCACGGG
4751





GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCACGGG
4752





GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCACTP
4753





GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCACTP
4754





GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCACTP
4755





GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCTPK-amide
4756





GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCTPK-
4757


amide





GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCTPK-
4758


amide





GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCTPK-
4759


amide





GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCTPK-
4760


amide





GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCTPK-
4761


amide





GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCHCTPK-
4762


amide





GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCYPK-amide
4763





GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCYPK-
4764


amide





GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCYPK-
4765


amide





GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCYPK-
4766


amide





GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCYPK-
4767


amide





GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCYPK-
4768


amide





GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCHCYPK-
4769


amide





GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACTPK-amide
4770





GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCACTPK-
4771


amide





GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCACTPK-
4772


amide





GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCACTPK-
4773


amide





GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCACTPK-
4774


amide





GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCACTPK-
4775


amide





GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCACTPK-
4776


amide





GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHC-amide
4777





GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHC-
4778


amide





GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHC-
4779


amide





GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHC-amide
4780





GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHC-
4781


amide





GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHC-amide
4782





GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCAC-amide
4783





GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCAC-
4784


amide





GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCAC-
4785


amide





GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCAC-amide
4786





GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHC-
4787


amide





GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCAC-amide
4788





GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCAC-amide
4789





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCYGG-amide
4790





GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCYCG-amide
4791





GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCYGG-
4792


amide





GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCYGG-
4793


amide





GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCYGG-
4794


amide





GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCYGG-
4795


amide





GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCYGG-
4796


amide





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCFGG-amide
4797





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCYG-amide
4798





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYG-amide
4799





GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACYGG-amide
4800





GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCACYGG-
4801


amide





GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCACYGG-
4802


amide





GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCACYGG-
4803


amide





GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCACYGG-
4804


amide





GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCACYGG-
4805


amide





GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCACYGG-
4806


amide





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYGG-amide
4807





GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCHCGGG-amide
4808





GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCHCGGG-
4809


amide





GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCHCGGG-
4810


amide





GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCHCGGG-
4811


amide





GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCHCGGG-
4812


amide





GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCHCGGG-
4813


amide





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACGGG-amide
4814





GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACGGG-amide
4815





GVIINVKCKIAAQCLOPCKDAGMRFGACAAGKCACGGG-amide
4816





GVIINVKCKIAAQCL[hLys]PCKDAGMRFGACAAGKCACGGG-
4817


amide





GVIINVKCKIAAQCL[hArg]PCKDAGMRFGACAAGKCACGGG-
4818


amide





GVIINVKCKIAAQCL[Cit]PCKDAGMRFGACAAGKCACGGG-
4819


amide





GVIINVKCKIAAQCL[hCit]PCKDAGMRFGACAAGKCACGGG-
4820


amide





GVIINVKCKIAAQCL[Dpr]PCKDAGMRFGACAAGKCACGGG-
4821


amide





GVIINVKCKIAAQCL[Dab]PCKDAGMRFGACAAGKCACGGG-
4822


amide





GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCTPK-amide
4823





GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCTPK-
4824


amide





GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCTPK-
4825


amide





GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCTPK-
4826


amide





GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCTPK-
4827


amide





GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCTPK-
4828


amide





GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCHCTPK-
4829


amide





GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCYPK-amide
4830





GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCYPK-
4831


amide





GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCYPK-
4832


amide





GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCYPK-
4833


amide





GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCYPK-
4834


amide





GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCYPK-
4835


amide





GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCHCYPK-
4836


amide





GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCACTPK-amide
4837





GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCACTPK-
4838


amide





GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCACTPK-
4839


amide





GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCACTPK-
4840


amide





GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCACTPK-
4841


amide





GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCACTPK-
4842


amide





GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCACTPK-
4843


amide





GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHC-amide
4844





GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHC-
4845


amide





GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHC-
4846


amide





GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHC-amide
4847





GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHC-
4848


amide





GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHC-amide
4849





GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCAC-amide
4850





GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCAC-
4851


amide





GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCAC-
4852


amide





GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCAC-amide
4853





GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHC-
4854


amide





GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCAC-amide
4855





GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCAC-amide
4856





GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCHCWGG-amide
4857





GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCYGG-amide
4858





GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCYGG-
4859


amide





GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCYGG-
4860


amide





GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCYGG-
4861


amide





GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCYGG-
4862


amide





GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCYGG-
4863


amide





GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCHCYGG-
4864


amide





GVIINVKCKIAAQCLKPCKEAGMRFGACAAGKCACYGG-amide
4865





GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCACYGG-amide
4866





GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCACYGG-
4867


amide





GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCACYGG-
4868


amide





GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCACYGG-
4869


amide





GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCYGG-
4870


amide





GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCACYGG-
4871


amide





GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCACYGG-
4872


amide





GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCHCGGG-amide
4873





GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCHCGGG-
4874


amide





GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCHCGGG-
4875


amide





GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCHCGGG-
4876


amide





GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCHCGGG-
4877


amide





GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCHCGGG-
4878


amide





GVIINVKCKIAAQCLOPCKEAGMRFGACAAGKCACGGG-amide
4879





GVIINVKCKIAAQCL[hLys]PCKEAGMRFGACAAGKCACGGG-
4880


amide





GVIINVKCKIAAQCL[hArg]PCKEAGMRFGACAAGKCACGGG-
4881


amide





GVIINVKCKIAAQCL[Cit]PCKEAGMRFGACAAGKCACGGG-
4882


amide





GVIINVKCKIAAQCL[hCit]PCKEAGMRFGACAAGKCACTP-
4883


amide





GVIINVKCKIAAQCL[Dpr]PCKEAGMRFGACAAGKCACGGG-
4884


amide





GVIINVKCKIAAQCL[Dab]PCKEAGMRFGACAAGKCACGGG-
4885


amide





GVIINVKCKIAAQCLKPCK[Cpa]AGMRFGACAAGKCACYGG-
4886


amide





GVIINVKCKIAAQCLKPCK[Cpa]AGMRFGACAAGKCACGGG-
4887


amide





GVIINVKCKIAAQCLKPCK[Cpa]AGMRFGACAAGKCACY-
4888


amide





Ac-GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCACYGG-
4889


amide





GVIINVKCKIAAQCLKPCK[Aad]AGMRFGACAAGKCACYGG-
4890


amide





GVIINVKCKIAAQCLKPCK[Aad]AGMRFGACAAGKCHCYGG-
4891


amide





GVIINVKCKIAAQCLKPCK[Aad]AGMRFGACAAGKCACYGG
4892





GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCACYGG-amide
4893





GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCACYGG
4894





GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCACY-amide
4895





GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCHCYGG-amide
4896





GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCHCYGG
4897





GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCHCYPK
4898





GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCAC
4899





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[1Nal]GG-
4900


amide





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[1Nal]PK-
4901


amide





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[2Nal]GG-
4902


amide





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[Cha]GG-
4903


amide





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[MePhe]GG-
4904


amide





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[BiPhA]GG-
4905


amide





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKC[Aib]CYGG-
4906


amide





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKC[Abu]CYGG-
4907


amide





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[1Nal]
4908





GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCAC[1Nal]GG-
4909


amide





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCAC[4Bip]-
4910


amide





GVIINVKCKIAAQCLHPCKDAGMRFGACAAGKCAC[4Bip]GG-
4911


amide





GVIINVKCKIAAQCLKPCKDAGMRFGACAAGKCHCGGG
4912





GIINVKCKISAQCLKPCRDAGMRFGKCMNGKCACTPK
4916
















TABLE 7J







Additional useful OSK1 peptide analogs












SEQ




Short-hand
ID


Sequence/Structure
designation
NO:





GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCG
[Gly34]
4930



CTPK
OSK1





GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCS
[Ser34]
4931


CTPK
OSK1





GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCT
[Thr34]
4932


CTPK
OSK1





GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCN
[Asn34]
4933


CTPK
OSK1





GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCV
[Val34]
4934


CTPK
OSK1





GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCL
[Leu34]
4935


CTPK
OSK1





GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCI
[Ile34]
4936


CTPK
OSK1





GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCP
[Pro34]
4937


CTPK
OSK1





GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCM
[Met34]
4938


CTPK
OSK1





GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCQ
[Gln34]
4939


CTPK
OSK1





GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCK
[Lys34]
4940


CTPK
OSK1





GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCD
[Asp34]
4941


CTPK
OSK1






WVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH

[Trp1]
4942


CWPK
OSK1





GVWINVKCKISRQCLEPCKKAGMRFGKCMNGKCH
[Trp3]
4943


CTPK
OSK1





GVIIWVKCKISRQCLEPCKKAGMRFGKCMNGKCH
[Trp5]
4944


CTPK
OSK1






[1Nal]VIINVKCKISRQCLEPCKKAGMRFGKCM

[1Nal1]
4945


NGKCHCWPK
OSk1





GV[1Nal]INVKCKISRQCLEPCKKAGMRFGKCM
[1Nal3]
4946


NGKCHCTPK
OSK1





GVII[1Nal]VKCKISRQCLEPCKKAGMRFGKCM
[1Na15]
4947


NGKCHCTPK
OSK1





GVIKNVKCKISRQCLEPCKKAGMRFGKCMNGKCH
[Lys4]
4948


CTPK
OSK1





GVIKNVKCKISRQCLEPCKKAGMRFGKCMNGKCA
[Lys4,
4949


CTPK
Ala34]



OSK1






[1Nal]VIINVKCKISRQCLEPCKKAGMRFGKCM

[1Nal1;
4950


NGKCACWPK
Ala34]



OSK1





GV[1Nal]INVKCKISRQCLEPCKKAGMRFGKCM
[1Nal3;
4951


NGKCACTPK
Ala34]



OSK1





GVII[1Nal]VKCKISRQCLEPCKKAGMRFGKCM
[1Nal5;
4952


NGKCACTPK
Ala34]



OSK1






WVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCA

[Trp1;
4953


CWPK
Ala34]



OSK1





GVWINVKCKISRQCLEPCKKAGMRFGKCMNGKCA
[Trp3;
4954


CTPK
Ala34]



OSK1





GVIIWVKCKISRQCLEPCKKAGMRFGKCMNGKCA
[Trp5;
4955


CTPK
Ala34]



OSK1






WVWIWVKCKISRQCLEPCKKAGMRFGKCMNGKCA

[Trp1,3,
4956


CTPK
5;Ala34]



OSK1






[1Nal]V[1Nal]I[1Nal]VKCKISRQCLEPCK

[1Nal1,3,
4957


KAGMRFGKCMNGKCACTPK
5;Ala34]



OSK1





CKISRQCLEPCKKAGMRFGKCMNGKCACTPK
Δ1-7,
4958



[Ala34]



OSK1





KCKISRQCLEPCKKAGMRFGKCMNGKCACTPK
Δ1-6,
4959



[Ala34]



OSK1





GVIINVKCKI[1Nal]RQCLEPCKKAGMRFGKCA
[1Nal11;
4960


NGKCACWPK
Ala29,34]



Osk-1





GVIINVKCKIRRQCLEPCKKAGMRFGKCANGKCA
[Arg11;
4961


CWPK
Ala29,34]



Osk-1





GVIINVKCKISRQCEEPCKKAGMRFGKCANGKCA
[Glu15;
4962


CWPK
Ala29,34]



Osk-1





GVIINVKCKIRRQCLEPCKKAGMRFGKCMNGKCA
[Arg11;
4963


CWPK
Ala34]



Osk-1





GVIINVKCKISRQCEEPCKKAGMRFGKCMNGKCA
[Glu15;
4964


CWPK
Ala34]



Osk-1





CKIRRQCEEPCKKAGMRFGKCANGKCACTPK
Δ1-7,
4965



[Arg11;



Glu45;



Ala29,34]



OSK1





GVIINVKCKIRRQCEEPCKKAGMRFGKCANGKCA
[Arg11;
4966


CTPK
Glu15;



Ala29,34]



OSK1






CVIINVKCKIRRQCEEPCKKAGMRFGKCANGKCA

[Cys1,37;
4967


CTCK
Arg11;



Glu15;



Ala29,34]



OSK1





GVIINVKCKIRAQCEEPCKKAGMRFGKCANGKCA
[Arg11;
4968


CTPK
Ala12,29,



34;Glu15]



OSK1





GVIINVKCKIRAQCEEPCKKAGMRFGKCANGKCA
[Arg11;
4969


CTPK-NH2
Glu15;



Ala12,29,



34]OSK1-



amide





Ac-
Ac-
4970


GVIINVKCKIRAQCEEPCKKAGMRFGKCANGKCA
[Arg11;


CTPK-NH2
Glu15;



Ala12,29,



34]OSK1-



amide





GVIINVKCKI[1Nal]AQCEEPCKKAGMRFGKCA
[1Nal11;
4971


NGKCACTPK
Glu15;



Ala12,29,



34]OSK1





GVIINVKCKISRQCLEPCKKAGMRFGKCANGKC
[Ala29;
4972



[1Nal]CWPK

1Nal34]



Osk-1





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCH
[Ala12;
4973


CTPK
Lys16;



Asp20]



Osk-1





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCA
[Ala12,
4974


CTPK
34;Lys16;



Asp20]



Osk-1





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCA
[Ala12,
4975


CTPK
29,34;



Lys16;



Asp20]



Osk-1





GVIINVKCKIRAQCLKPCKDAGMRFGKCANGKCA
[Arg11;
4976


CTPK
Ala12,29,



34;Lys16;



Asp20]



Osk-1





GVIINVKCKISAQCEKPCKDAGMRFGKCANGKCA
[Ala12,
4977


CTPK
29,34;



Glu15,



Lys16;



Asp20]



Osk-1





GVIINVKCKI[1Nal]AQCLKPCKDAGMRFGKCA
[1Nal11;
4978


NGKCACTPK
Ala12,29,



34;Lys16;



Asp20]



Osk-1





GVIINVKCKIRAQCEKPCKDAGMRFGKCANGKCA
[Arg11;
4979


CTPK
Ala12,29,



34;Glu15;



Lys16;



Asp20]



Osk-1





GVIINVKCKIRAQCEKPCKDAGMRFGKCMNGKCA
[Arg11;
4980


CTPK
Ala12,34;



Glu15;



Lys16;



Asp20]



Osk-1





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKC
[A12,
4981


[1Nal]CTPK
K16, D20,



Nal34]-



OSK1





GVIINVKCKISAQCLKPCKDAGMRFGKCMNGKCA
[A12,
4982


CTPK
K16, D20,



A34]-



OSK1





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKC
[A12,
4983


[1Nal]CTPK
K16, D20,



A29,



Nal34]-



OSK1





GVIINVKCKISAQCLKPCKDAGMRFGKCANGKCA
[A12,
4984


CTPK
K16, D20,



A29, A34]-



OSK1





{Acetyl}GVIINVKCKISRQCLEPCK
Ac-
4985


(Glycyl)KAGMRFGKCMNGKCACTPK
[K(Gly)



19,Ala



34]-Osk1





{Acetyl}GVIK(Glycyl)NVKCKISRQCLEPC
Ac-
4986


KKAGMRFGKCMNGKCACTPK
[K(Gly)4,



Ala34]-



Osk1





{Acetyl}GVIINVKCKISRQCLEPCKKAGMRFG
Ac-
4987


KCMNGKCACTPK(Glycyl)
[Ala34,K



(Gly)38]-



Osk1





GVIINVKCKISRQCLEPCKKAGMRFGKCANGKC
[A29,
4988


[1Nal]CTPK
Nal34]-



OSK1





CKISRQCLKPCKDAGMRFGKCMNGKCHC
OSK1
4989


{Amide}
[des1-7,



E16K,



K20D,



des36-



38]-amide





GVIINVKCKISRQCLKPCKDAGMRFGKCMNGKCA
OSK1-
4990


CTPK
K16, D20,



A34





GVIINVKCKI[1Nal]AQCLEPCKKAGMRFGKCA
Osk-1
4991


NGKC[1Nal]CTPK
[1Nal11,



A12,A29,1-



Na134]





GVIINVKCKI[1Nal]AQCLEPCKKAGMRFGKCA
[1Nal11,
4992


NGKC[1Nal]CTEK
A12,A29,



1Nal34,



E37]Osk-1





GVIINVKCKI[1Nal]AQCEEPCKKAGMRFGKCA
Osk-1[1-
4993


NGKC[1Nal]CEEK
Nal11,



A12,E15



A29,



1Nal34,



E36,E37]





GVIINVKCKI[1Nal]AQCLEPCKKAGFRFGKCA
[1Nal11,
4994


NGKC[1Nal]CTPK
A12,F23,



A29,



1Nal34]



Osk-1





GVIINVKCKI[1Nal]AQCLEPCKKAG[Nle]RF
[1Nal11,
4995


GKCANGKC[1Nal]CTEK
A12,



Nle23,



A29,



1Nal34,



E37]Osk-1





GVIINVKCKISPQCLKPCKDAGMRFGKCMNGKCA
[Pro12,
4996


CTY[Nle]
Lys16,



Asp20,



Ala34,



Tyr37,



Nle38]



Osk-1-



amide





GVIINVKCKISPQCLOPCKEAGMRFGKCMNGKCA
[P12,
4997


CTY[Nle]
Orn16,



E20,A34,



Y37,



Nle38]



Osk-1-



amide





NVKCKISRQCLEPCKKAGMRFGKCANGKC
des1-4,
4998


[1Nal]CTPK
[A29,



Nal34]-



OSK1





NVKCKISRQCLEPCKKAGMRFGKCANGKCACTPK
des1-4,
4999



[A29,



A34]-



OSK1





GVIINVKCKIRRQCLEPCKKAGMRFGKCANGKCA
[R11,
5000


CTPK
A29,



A34]-



OSK1





GVIINVKCKIRAQCLEPCKKAGMRFGKCANGKCA
[R11,
5001


CTPK
A12,



A29,



A34]-



OSK1





CKISRQCLEPCKKAGMRFGKCMNGKCACTPK
[Ala34]
5002



OSK1



(8-35)





CKISRQCLEPCKKAGMRFGKCMNGKCAC
[Ala34]
5003



OSK1



(8-35)





CKIRRQCLEPCKKAGMRFGKCANGKCAC
[Arg11;
5004



Ala29,34]



Osk-1



(8-35)





CKISAQCLEPCKKAGMRFGKCANGKCAC
[Ala12;
5005



Ala29,34]



Osk-1



(8-35)





CKISAQCLEPCKKAGMRFGKCMNGKCAC
[Ala12;
5006



Ala34]



Osk-1



(8-35)





GVI[Dpr(AOA)]NVKCKISRQCLEPCKKAGMRF
[Dpr(AoA)4]
5009


GKCMNGKCHCTPK
Osk1





GVI[Dpr(AOA-PEG)]NVKCKISRQCLEPCKKA
[Dpr(AOA)-
5010


GMRFGKCMNGKCHCTPK

PEG)4]Osk1






GCIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH
[C2, C37]-
5012


TCK
OSK1






SCIINVKCKISRQCLEPCKKAGMRFGKCMNGKCH

[S1, C2,
5013


CTCK
C37]-OSK1






SCIINVKCKISRQCLEPCKKAGMRFGKCMNGKCA

[S1, C2,
5014


CTCK
A34, C37]-



OSK1





SCVIINVKCKISRQCLEPCKKAGMRFGKCMNGKC
Ser-[C1,
5015


HCTCK
C37]-OSK1









Any OSK1 peptide analog that comprises an amino acid sequence selected from SEQ ID NOS: 1391 through 4912, 4916, 4920 through 5006, 5009, 5010, and 5012 through 5015 as set forth in Tables 7, 7A, 7B, 7C, 7D, 7E, 7F, 7G, 7H or 7I, is useful in accordance with the present invention. Any of these can also be derivatized at either its N-terminal or C-terminal, e.g., with a fatty acid having from 4 to 10 carbon atoms and from 0 to 2 carbon-carbon double bonds, or a derivative thereof such as an ω-amino-fatty acid. (E.g., Mouhat et al., WO 2006/002850 A2, which is incorporated by reference in its entirety). Examples of such fatty acids include valeric acid or (for the C-terminal) co-amino-valeric acid.


Among useful OSK1 peptide analog sequences of the present invention are analog sequences that introduce amino acid residues that can form an intramolecular covalent bridge (e.g., a disulfide bridge) or non-covalent interactions (e.g. hydrophobic, ionic, stacking) between the first and last beta strand, which may enhance the stability of the structure of the unconjugated or conjugated (e.g., PEGylated) OSK1 peptide analog molecule. Examples of such sequences include SEQ ID NOS: 4985-4987 and 5012-5015.


In some embodiments of the composition of matter, the C-terminal carboxylic acid moiety of the OSK1 peptide analog is replaced with a moiety selected from:


(A) —COOR, where R is independently (C1-C8)alkyl, haloalkyl, aryl or heteroaryl;


(B) —C(═O)NRR, where R is independently hydrogen, (C1-C8)alkyl, haloalkyl, aryl or heteroaryl; and


(C) —CH2OR where R is hydrogen, (C1-C8) alkyl, aryl or heteroaryl.


“Aryl” is phenyl or phenyl vicinally-fused with a saturated, partially-saturated, or unsaturated 3-, 4-, or 5 membered carbon bridge, the phenyl or bridge being substituted by 0, 1, 2 or 3 substituents selected from C18 alkyl, C14 haloalkyl or halo.


“Heteroaryl” is an unsaturated 5, 6 or 7 membered monocyclic or partially-saturated or unsaturated 6-, 7-, 8-, 9-, 10- or 11 membered bicyclic ring, wherein at least one ring is unsaturated, the monocyclic and the bicyclic rings containing 1, 2, 3 or 4 atoms selected from N, O and S, wherein the ring is substituted by 0, 1, 2 or 3 substituents selected from C18 alkyl, C14 haloalkyl and halo.


In other embodiments of the composition of matter comprising a half-life extending moiety, the OSK1 peptide analog comprises an amino acid sequence of the formula:










SEQ ID NO: 5011









G1V2I3I4N5V6K7C8K9I10Xaa11Xaa12Q13C14Xaa15Xaa16P17






C18Xaa19Xaa20A21G22M23R24F25G26Xaa27C28Xaa29Xaa30





G31Xaa32C33Xaa34C35Xaa35Xaa37Xaa38






wherein:


amino acid residues 1 through 7 are optional (Thus, the OSK1 peptide analog optionally includes residues 1-7 as indicated above in SEQ ID NO:5011, or a N-terminal truncation leaving present residues 2-7, 3-7, 4-7, 5-7, 6-7, or 7, or alternatively, a N-terminal truncation wherein all of residues 1-7 are entirely absent.);

    • Xaa11 is a neutral, basic, or acidic amino acid residue (e.g., Ser, Thr, Ala, Gly, Leu, Ile, Val, Met, Cit, Homocitrulline, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Guf, and 4-Amino-Phe, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Lys, His, Trp, Arg, Nα Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
    • Xaa12 is a neutral or acidic amino acid residue (e.g., Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
    • Xaa15 is a neutral or acidic amino acid residue (e.g., Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
    • Xaa16 is a neutral or basic amino acid residue (e.g., Lys, His, Arg, Trp, Arg, Nα Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn, Cit, Nα-Methyl-Cit, Homocitrulline, His, Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Ser, Thr, Guf, and 4-Amino-Phe);
    • Xaa19 is a neutral or basic amino acid residue (e.g., Lys, His, Arg, Trp, Arg, Nα Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn, Cit, Nα-Methyl-Cit, Homocitrulline, His, Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Ser, Thr, Guf, and 4-Amino-Phe);
    • Xaa20 is a neutral or basic amino acid residue (e.g., Lys, His, Arg, Trp, Arg, Nα Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn, Cit, Nα-Methyl-Cit, Homocitruiline, His, Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Ser, Thr, Guf, and 4-Amino-Phe);
    • Xaa27 is a neutral, basic, or acidic amino acid residue (e.g., Ser, Thr, Ala, Gly, Leu, Ile, Val, Met, Cit, Homocitrulline, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Guf, and 4-Amino-Phe, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Lys, His, Trp, Arg, Nα Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
    • Xaa29 is a neutral or acidic amino acid residue (e.g., Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
    • Xaa30 is a neutral or acidic amino acid residue (e.g., Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
    • Xaa32 is a neutral, basic, or acidic amino acid residue (e.g., Ser, Thr, Ala, Gly, Leu, Ile, Val, Met, Cit, Homocitrulline, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Guf, and 4-Amino-Phe, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Lys, His, Trp, Arg, Nα Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
    • Xaa34 is a neutral or acidic amino acid residue (e.g., Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
    • Xaa36 is optional, and if present, is a neutral amino acid residue (e.g., Pro, Ala, Gly, Leu, Ile, Val, Met, Oic, Hyp, Tic, D-Tic, D-Pro, Thz, Nα-Methyl-Cit, Homocitrulline, Aib, Sar, Pip, Tyr, Thr, Ser, Phe, Trp, 1-Nal, 2-Nal, and Bip;
    • Xaa37 is optional, and if present, is a neutral amino acid residue (e.g., Pro, Ala, Gly, Leu, Ile, Val, Met, Oic, Hyp, Tic, D-Tic, D-Pro, Thz, Nα-Methyl-Cit, Homocitrulline, Aib, Sar, Pip, Tyr, Thr, Ser, Phe, Trp, 1-Nal, 2-Nal, and Bip); and
    • Xaa38 is optional, and if present, is a basic amino acid residue (e.g., Lys, His, Orn, Trp, D-Orn, Arg, Nα Methyl-Arg; homoarginine, Cit, Nα-Methyl-Cit, Homocitrulline, His, Guf, and 4-Amino-Phe).


In some other embodiments of the composition of matter comprising a half-life extending moiety, the OSK1 peptide analog comprises an amino acid sequence of the formula:










SEQ ID NO: 4913









G1V2I3I4N5V6K7C8K9I10Xaa11Xaa12Q13C14L15Xaa16P17






C18K19Xaa20A21G22M23R24F25G26Xaa27C28Xaa29Xaa30G31





K32C33Xaa34C35Xaa36Xaa37Xaa38






wherein:

    • amino acid residues 1 to 7 are optional (Thus, the OSK1 peptide analog optionally includes residues 1-7 as indicated above in SEQ ID NO:4913, or a N-terminal truncation leaving present residues 2-7, 3-7, 4-7, 5-7, 6-7, or 7, or alternatively, a N-terminal truncation wherein all of residues 1-7 are entirely absent.);
    • Xaa11 is a neutral, basic or acidic amino acid residue (e.g., Ser, Thr, Ala, Gly, Leu, Ile, Val, Met, Cit, Homocitrulline, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Guf, and 4-Amino-Phe, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Lys, His, Trp, Arg, Nα Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
    • Xaa12 is a neutral or acidic amino acid residue (e.g., Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
    • Xaa16 is a neutral or basic amino acid residue (e.g., Lys, His, Arg, Trp, Arg, Nα Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn, Cit, Nα-Methyl-Cit, Homocitrulline, His, Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Ser, Thr, Guf, and 4-Amino-Phe);
    • Xaa20 is a neutral or basic amino acid residue (e.g., Lys, His, Arg, Trp, Arg, Nα Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn, Cit, Nα-Methyl-Cit, Homocitrulline, His, Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Ser, Thr, Guf, and 4-Amino-Phe);
    • Xaa27 is a neutral, basic, or acidic amino acid residue (e.g., Ser, Thr, Ala, Gly, Leu, Ile, Val, Met, Cit, Homocitrulline, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Guf, and 4-Amino-Phe, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Lys, His, Trp, Arg, Nα Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
    • Xaa29 is a neutral or acidic amino acid residue (e.g., Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
    • Xaa30 is a neutral or acidic amino acid residue (e.g., Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
    • Xaa35 is a neutral or acidic amino acid residue (e.g., Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine);
    • Xaa36 is optional, and if present, is a neutral amino acid residue (e.g., Pro, Ala, Gly, Leu, Ile, Val, Met, Oic, Hyp, Tic, D-Tic, D-Pro, Thz, Nα-Methyl-Cit, Homocitrulline, Aib, Sar, Pip, Tyr, Thr, Ser, Phe, Trp, 1-Nal, 2-Nal, and Bip;
    • Xaa37 is optional, and if present, is a neutral amino acid residue (e.g., Pro, Ala, Gly, Leu, Ile, Val, Met, Oic, Hyp, Tic, D-Tic, D-Pro, Thz, Nα-Methyl-Cit, Homocitrulline, Aib, Sar, Pip, Tyr, Thr, Ser, Phe, Trp, 1-Nal, 2-Nal, and Bip); and
    • Xaa38 is optional, and if present, is a basic amino acid residue (e.g., Lys, His, Orn, Trp, D-Orn, Arg, Nα Methyl-Arg; homoarginine, Cit, Nα-Methyl-Cit, Homocitruiline, His, Guf, and 4-Amino-Phe).









TABLE 8







Pi2 peptide and PiP2 s peptide analog equences












SEQ




Short-hand
ID


Sequence/structure
designation
NO:





TISCTNPKQCYPHCKKETGYPNAKCMNRKCKCFGR
Pi2
 17






TISCTNPXQCYPHCKKETGYPNAKCMNRKCKCFGR
Pi2-X8
299





TISCTNPAQCYPHCKKETGYPNAKCMNRKCKCFGR
Pi2-A8
300





TISCTNPKQCYPHCXKETGYPNAKCMNRKCKCFGR
Pi2-X15
301





TISCTNPKQCYPHCAKETGYPNAKCMNRKCKCFGR
Pi2-A15
302





TISCTNPKQCYPHCKXETGYPNAKCMNRKCKCFGR
Pi2-X16
303





TISCTNPKQCYPHCKAETGYPNAKCMNRKCKCFGR
Pi2-A16
304





TISCTNPKQCYPHCKKETGYPNAXCMNRKCKCFGR
Pi2-X24
305





TISCTNPKQCYPHCKKETGYPNAACMNRKCKCFGR
Pi2-A24
306





TISCTNPKQCYPHCKKETGYPNAKCMNXKCKCFGR
Pi2-X28
307





TISCTNPKQCYPHCKKETGYPNAKCMNAKCKCFGR
Pi2-A28
308





TISCTNPKQCYPHCKKETGYPNAKCMNRXCKCFGR
Pi2-X29
309





TISCTNPKQCYPHCKKETGYPNAKCMNRACKCFGR
Pi2-A29
310





TISCTNPKQCYPHCKKETGYPNAKCMNRKCXCFGR
Pi2-X31
311





TISCTNPKQCYPHCKKETGYPNAKCMNRKCACFGR
Pi2-A31
312





TISCTNPKQCYPHCKKETGYPNAKCMNRKCKCFGX
Pi2-X35
313





TISCTNPKQCYPHCKKETGYPNAKCMNRKCKCFGA
Pi2-A35
314





TISCTNPKQCYPHCKKETGYPNAKCMNRKCKCFG
Pi2-d35
315

















TABLE 9







Anuroctoxin (AnTx) peptide and peptide analog



sequences












SEQ




Short-hand
ID


Sequence/structure
designation
NO:





ZKECTGPQHCTNFCRKNKCTHGKCMNRKCKCFNCK
Anuroctoxin
 62




(AnTx)





 KECTGPQHCTNFCRKNKCTHGKCMNRKCKCFNCK
AnTx-d1
316





 XECTGPQHCTNFCRKNKCTHGKCMNRKCKCFNCK
AnTx-d1,X2
317





 AECTGPQHCTNFCRKNKCTHGKCMNRKCKCFNCK
AnTx-d1,A2
318

















TABLE 10







Noxiustoxin (NTX) peptide and NTX peptide



analog sequences












SEQ




Short-hand
ID


Sequence/structure
designation
NO:





TIINVKCTSPKQCSKPCKELYGSSAGAKCMNGKCK
NTX
 30



CYNN





TIINVACTSPKQCSKPCKELYGSSAGAKCMNGKCK
NTX-A6
319


CYNN





TIINVKCTSPKQCSKPCKELYGSSRGAKCMNGKCK
NTX-R25
320


CYNN





TIINVKCTSSKQCSKPCKELYGSSAGAKCMNGKCK
NTX-S10
321


CYNN





TIINVKCTSPKQCWKPCKELYGSSAGAKCMNGKCK
NTX-W14
322


CYNN





TIINVKCTSPKQCSKPCKELYGSSGAKCMNGKCKC
NTX-A25d
323


YNN





TIINVKCTSPKQCSKPCKELFGVDRGKCMNGKCKC
NTX-IbTx1
324


YNN





TIINVKCTSPKQCWKPCKELFGVDRGKCMNGKCKC
NTX-IBTX2
325


YN

















TABLE 11







Kaliotoxin1 (KTX1) peptide and KTX1 peptide



analog sequences












SEQ




Short-hand
ID


Sequence/structure
designation
NO:





GVEINVKCSGSPQCLKPCKDAGMRFGKCMNRKCHC
KTX1
 24



TPK





 VRIPVSCKHSGQCLKPCKDAGMRFGKCMNGKCDC
KTX2
326


TPK





GVEINVSCSGSPQCLKPCKDAGMRFGKCMNRKCHC
KTX1-S7
327


TPK





GVEINVACSGSPQCLKPCKDAGMRFGKCMNRKCHC
KTX1-A7
328


TPK
















TABLE 12







IKCa1 inhibitor peptide sequences












SEQ




Short-hand
ID


Sequence/structure
designation
NO:





VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX
 20






QFTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRC
ChTx
 36


YS





QFTQESCTASNQCWSICKRLHNTNRGKCMNKKCRC
ChTx-Lq2
329


YS

















TABLE 13







Maurotoxin (MTx) peptide amd MTx peptide analog



sequences












SEQ




Short-hand
ID


Sequence/structure
designation
NO:





VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX
 20






VSCAGSKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-A4
330





VSCTGAKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-A6
331





VSCTGSADCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-A7
332





VSCTGSKDCAAPCRKQTGCPNAKCINKSCKCYGC
MTX-A10
333





VSCTGSKDCYAPCQKQTGCPNAKCINKSCKCYGC
MTX-Q14
334





VSCTGSKDCYAPCRQQTGCPNAKCINKSCKCYGC
MTX-Q15
335





VSCTGSKDCYAPCQQQTGCPNAKCINKSCKCYGC
MTX-Q14,15
336





VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYAC
MTX-A33
337





VSCTGSKDCYAPCRKQTGCPYGKCMNRKCKCNRC
MTX-HsTx1
338





VSCTGSKDCYAACRKQTGCANAKCINKSCKCYGC
MTX-A12,20
339





VSCTGSKDCYAPCRKQTGXM19PNAKCINKSCKCY
MTX-X19,34
340


GXM34





VSCTGSKDCYAPCRKQTGSPNAKCINKSCKCYGS
MTX-S19,34
341





VSCTGSADCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-A7
342





VVIGQRCTGSKDCYAPCRKQTGCPNAKCINKSCKC
TsK-MTX
343


YGC





VSCRGSKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-R4
1301





VSCGGSKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-G4
1302





VSCTTSKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-T5
1304





VSCTASKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-A5
1305





VSCTGTKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-T6
1306





VSCTGPKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-P6
1307





VSCTGSKDCGAPCRKQTGCPNAKCINKSCKCYGC
MTX-G10
1309





VSCTGSKDCYRPCRKQTGCPNAKCINKSCKCYGC
MTX-R11
1311





VSCTGSKDCYDPCRKQTGCPNAKCINKSCKCYGC
MTX-D11
1312





VSCTGSKDCYAPCRKRTGCPNAKCINKSCKCYGC
MTX-R16
1315





VSCTGSKDCYAPCRKETGCPNAKCINKSCKCYGC
MTX-E16
1316





VSCTGSKDCYAPCRKQTGCPYAKCINKSCKCYGC
MTX-Y21
1317





VSCTGSKDCYAPCRKQTGCPNSKCINKSCKCYGC
MTX-S22
1318





VSCTGSKDCYAPCRKQTGCPNGKCINKSCKCYGC
MTX-G22
1319





VSCTGSKDCYAPCRKQTGCPNAKCINRSCKCYGC
MTX-R27
1320





VSCTGSKDCYAPCRKQTGCPNAKCINKTCKCYGC
MTX-T28
1321





VSCTGSKDCYAPCRKQTGCPNAKCINKMCKCYGC
MTX-M28
1322





VSCTGSKDCYAPCRKQTGCPNAKCINKKCKCYGC
MTX-K28
1323





VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCNGC
MTX-N32
1324





VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYRC
MTX-R33
1325





VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGCS
MTX-S35
1326





 SCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-d1
1327





 SCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGCS
MTX-S35d1
1328





VSCTGSKDCYAPCAKQTGCPNAKCINKSCKCYGC
MTX-A14
1329





VSCTGSKDCYAPCRAQTGCPNAKCINKSCKCYGC
MTX-A15
1330





VSCTGSKDCYAPCRKQTGCPNAACINKSCKCYGC
MTX-A23
1331





VSCTGSKDCYAPCRKQTGCPNAKCINASCKCYGC
MTX-A27
1332





VSCTGSKDCYAPCRKQTGCPNAKCINKSCACYGC
MTX-A30
1333





VSCTGSKDCYAPCRKQTGCPNAKCINKSCKCAGC
MTX-A32
1334





ASCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-A1
1335





MSCTGSKDCYAPCRKQTGCPNAKCINKSCKCYGC
MTX-M1
1336









In Table 13 and throughout this specification, Xm19 and Xm34 are each independently nonfunctional residues.









TABLE 14







Charybdotoxin(ChTx) peptide and ChTx peptide analog sequences











Short-hand




Sequence/structure
designation
SEQ ID NO:













QFTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS
ChTx
36






QFTNVSCTTSKECWSVCQRLHNTSRGKCMNKECRCYS
ChTx-E32
59





QFTNVSCTTSKECWSVCQRLHNTSRGKCMNKDCRCYS
ChTx-D32
344





CTTSKECWSVCQRLHNTSRGKCMNKKCRCYS
ChTx-d1-d6
345





QFTNVSCTTSKECWSVCQRLFGVDRGKCMGKKCRCYQ
ChTx-IbTx
346





QFTNVSCTTSKECWSVCQRLHNTSRGKCMNGKCRCYS
ChTx-G31
1369





QFTNVSCTTSKECLSVCQRLHNTSRGKCMNKKCRCYS
ChTx-L14
1370





QFTNVSCTTSKECASVCQRLHNTSRGKCMNKKCRCYS
ChTx-A14
1371





QFTNVSCTTSKECWAVCQRLHNTSRGKCMNKKCRCYS
ChTx-A15
1372





QFTNVSCTTSKECWPVCQRLHNTSRGKCMNKKCRCYS
ChTx-P15
1373





QFTNVSCTTSKECWSACQRLHNTSRGKCMNKKCRCYS
ChTx-A16
1374





QFTNVSCTTSKECWSPCQRLHNTSRGKCMNKKCRCYS
ChTx-P16
1375





QFTNVSCTTSKECWSVCQRLHNTSAGKCMNKKCRCYS
ChTx-A25
1376





QFTNVACTTSKECWSVCQRLHNTSRGKCMNKKCRCYS
ChTx-A6
1377





QFTNVKCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS
ChTx-K6
1378





QFTNVSCTTAKECWSVCQRLHNTSRGKCMNKKCRCYS
ChTx-A10
1379





QFTNVSCTTPKECWSVCQRLHNTSRGKCMNKKCRCYS
ChTx-P10
1380





QFTNVSCTTSKACWSVCQRLHNTSRGKCMNKKCRCYS
ChTx-A12
1381





QFTNVSCTTSKQCWSVCQRLHNTSRGKCMNKKCRCYS
ChTx-Q12
1382





AFTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS
ChTx-A1
1383





TFTNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS
ChTx-T1
1384





QATNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS
ChTx-A2
1385





QITNVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS
ChTx-I2
1386





QFANVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS
ChTx-A3
1387





QFINVSCTTSKECWSVCQRLHNTSRGKCMNKKCRCYS
ChTx-I3
1388





TIINVKCTSPKQCLPPCKAQFGTSRGKCMNKKCRCYSP
ChTx-MgTx
1389





TIINVSCTSPKQCLPPCKAQFGTSRGKCMNKKCRCYSP
ChTx-MgTx-b
1390
















TABLE 15







SKCa inhibitor peptide sequences










Short-hand
SEQ ID


Sequence/structure
designation
NO:





CNCKAPETALCARRCQQHG
Apamin
68





AFCNLRMCQLSCRSLGLLGKCIGDKCECVKH
ScyTx
51





AVCNLKRCQLSCRSLGLLGKCIGDKCECVKHG
BmP05
50





TVCNLRRCQLSCRSLGLLGKCIGVKCECVKH
P05
52





AFCNLRRCELSCRSLGLLGKCIGEECKCVPY
Tamapin
53





VSCEDCPEHCSTQKAQAKCDNDKCVCEPI
P01
16





VVIGQRCYRSPDCYSACKKLVGKATGKCT
TsK
47


NGRCDC
















TABLE 16







Apamin peptide and peptide analog


inhibitor sequences












Short-hand




Sequence/structure
designation
SEQ ID NO:















CNCKAPETALCARRCQQHG
Apamin (Ap)
68







CNCXAPETALCARRCQQHG
Ap-X4
348







CNCAAPETALCARRCQQHG
Ap-A4
349







CNCKAPETALCAXRCQQHG
Ap-X13
350







CNCKAPETALCAARCQQHG
Ap-A13
351







CNCKAPETALCARXCQQHG
Ap-X14
352







CNCKAPETALCARACQQHG
Ap-A14
353

















TABLE 17







Scyllatoxin (ScyTx), BmP05, P05, Tamapin,


P01 peptide and peptide analog inhibitor


sequences










Short-hand
SEQ ID


Sequence/structure
designation
NO:












AFCNLRMCQLSCRSLGLLGKCIGDKCECVKH
ScyTx
51





AFCNLRRCQLSCRSLGLLGKCIGDKCECVKH
ScyTx-R7
354





AFCNLRMCQLSCRSLGLLGKCMGKKCRCVKH
ScyTx-IbTx
355





AFSNLRMCQLSCRSLGLLGKSIGDKCECVKH
ScyTx-C/S
356





AFCNLRRCELSCRSLGLLGKCIGEECKCVPY
Tamapin
53
















TABLE 18







BKCa inhibitor peptide sequences










Short-hand
SEQ ID


Sequence/structure
designation
NO:





QFTDVDCSVSKECWSVCKDLFGVDRGKCMGK
IbTx
38


KCRCYQ





TFIDVDCTVSKECWAPCKAAFGVDRGKCMGKK
Slotoxin
39


CKCYV
(SloTx)





QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNG
BmTx1
40


KCRCYS





WCSTCLDLACGASRECYDPCFKAFGRAHGKCM
BuTx
41


NNKCRCYTN





FGLIDVKCFASSECWTACKKVTGSGQGKCQNN
MartenTx
35


QCRCY





ITINVKCTSPQQCLRPCKDRFGQHAGGKCING
CllTx1
29


KCKCYP

















TABLE 19







IbTx, Slotoxin, BmTx1, & BuTX (Slotoxin family) peptide



and peptide analog inhibitor sequences











Short-hand




Sequence/structure
designation
SEQ ID NO:













QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKKCRCYQ
IbTx
38






QFTDVDCSVSXECWSVCKDLFGVDRGKCMGKKCRCYQ
IbTx-X11
357





QFTDVDCSVSAECWSVCKDLFGVDRGKCMGKKCRCYQ
IbTx-A11
358





QFTDVDCSVSKECWSVCXDLFGVDRGKCMGKKCRCYQ
IbTx-X18
359





QFTDVDCSVSKECWSVCADLFGVDRGKCMGKKCRCYQ
IbTx-A18
360





QFTDVDCSVSKECWSVCKDLFGVDXGKCMGKKCRCYQ
IbTx-X25
361





QFTDVDCSVSKECWSVCKDLFGVDAGKCMGKKCRCYQ
IbTx-A25
362





QFTDVDCSVSKECWSVCKDLFGVDRGXCMGKKCRCYQ
IbTx-X27
363





QFTDVDCSVSKECWSVCKDLFGVDRGACMGKKCRCYQ
IbTx-A27
364





QFTDVDCSVSKECWSVCKDLFGVDRGKCMGXKCRCYQ
IbTx-X31
365





QFTDVDCSVSKECWSVCKDLFGVDRGKCMGAKCRCYQ
IbTx-A31
366





QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKXCRCYQ
IbTx-X32
367





QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKACRCYQ
IbTx-A32
368





QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKKCXCYQ
IbTx-X34
369





QFTDVDCSVSKECWSVCKDLFGVDRGKCMGKKCACYQ
IbTx-A34
370





QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGKCRCYS
BmTx1
371





QFTDVXCTGSKQCWPVCKQMFGKPNGKCMNGKCRCYS
BmTx1-X6
372





QFTDVACTGSKQCWPVCKQMFGKPNGKCMNGKCRCYS
BmTx1-A6
373





QFTDVKCTGSXQCWPVCKQMFGKPNGKCMNGKCRCYS
BmTx1-X11
374





QFTDVKCTGSAQCWPVCKQMFGKPNGKCMNGKCRCYS
BmTx1-A11
375





QFTDVKCTGSKQCWPVCXQMFGKPNGKCMNGKCRCYS
BmTx1-X18
376





QFTDVKCTGSKQCWPVCAQMFGKPNGKCMNGKCRCYS
BmTx1-A18
377





QFTDVKCTGSKQCWPVCKQMFGXPNGKCMNGKCRCYS
BmTx1-X23
378





QFTDVKCTGSKQCWPVCKQMFGAPNGKCMNGKCRCYS
BmTx1-A23
379





QFTDVKCTGSKQCWPVCKQMFGKPNGXCMNGKCRCYS
BmTx1-X27
380





QFTDVKCTGSKQCWPVCKQMFGKPNGACMNGKCRCYS
BmTx1-A27
381





QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGXCRCYS
BmTx1-X32
382





QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGARCYS
BmTx1-A32
383





QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGKCXYS
BmTx1-X34
384





QFTDVKCTGSKQCWPVCKQMFGKPNGKCMNGKCAYS
BmTx1-A34
385





WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNKCRCYTN
BuTx
386





WCSTCLDLACGASXCYDPCFKAFGRAHGKCMNNKCRCYTN
BuTx-X14
387





WCSTCLDLACGASACYDPCFKAFGRAHGKCMNNKCRCYTN
BuTx-A14
388





WCSTCLDLACGASRECYDPCFXFGRAHGKCMNNKCRCYTN
BuTx-X22
389





WCSTCLDLACGASRECYDPCFAGRAHGKCMNNKCRCYTN
BuTx-A22
390





WCSTCLDLACGASRECYDPCFKAFGXHGKCMNNKCRCYTN
BuTx-X26
391





WCSTCLDLACGASRECYDPCFKAFGAHGKCMNNKCRCYTN
BuTx-A26
392





WCSTCLDLACGASRECYDPCFKAFGRAHGXMNNKCRCYTN
BuTx-X30
393





WCSTCLDLACGASRECYDPCFKAFGRAHGAMNNKCRCYTN
BuTx-A30
394





WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNXRCYTN
BuTx-X35
395





WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNARCYTN
BuTx-A35
396





WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNKCXYTN
BuTx-X37
397





WCSTCLDLACGASRECYDPCFKAFGRAHGKCMNNKCAYTN
BuTx-A37
398
















TABLE 20







Martentoxin peptide and peptide analog inhibitor sequences











Short-hand




Sequence/structure
designation
SEQ ID NO:













FGLIDVKCFASSECWTACKKVTGSGQGKCQNNQCRCY
MartenTx
35






FGLIDVXCFASSECWTACKKVTGSGQGKCQNNQCRCY
MartenTx-X7
399





FGLIDVACFASSECWTACKKVTGSGQGKCQNNQCRCY
MartenTx-A7
400





FGLIDVKCFASSECWTACXKVTGSGQGKCQNNQCRCY
MartenTx-X19
401





FGLIDVKCFASSECWTACAKVTGSGQGKCQNNQCRCY
MartenTx-A19
402





FGLIDVKCFASSECWTACKXVTGSGQGKCQNNQCRCY
MartenTx-X20
403





FGLIDVKCFASSECWTACKAVTGSGQGKCQNNQCRCY
MartenTx-A20
404





FGLIDVKCFASSECWTACKKVTGSGQGXCQNNQCRCY
MartenTx-X28
405





FGLIDVKCFASSECWTACKKVTGSGQGACQNNQCRCY
MartenTx-A28
406





FGLIDVKCFASSECWTACKKVTGSGQGKCQNNQCXCY
MartenTx-X35
407





FGLIDVKCFASSECWTACKKVTGSGQGKCQNNQCACY
MartenTx-A35
408
















TABLE 21







N type Ca2+ channel inhibitor peptide sequences










Short-hand
SEQ ID


Sequence/structure
designation
NO:












CKGKGAKCSRLMYDCCTGSCRSGKC
MVIIA
65





CKSPGSSCSPTSYNCCRSCNPYTKRCY
GVIA
64





CKSKGAKCSKLMYDCCTGSCSGTVGRC
CVIA
409





CKLKGQSCRKTSYDCCSGSCGRSGKC
SVIB
347





AEKDCIAPGAPCFGTDKPCCNPRAWCSSY
Ptu1
66


ANKCL





CKGKGASCRKTMYDCCRGSCRSGRC
CVIB
1364





CKGKGQSCSKLMYDCCTGSCSRRGKC
CVIC
1365





CKSKGAKCSKLMYDCCSGSCSGTVGRC
CVID
1366





CLSXGSSCSXTSYNCCRSCNXYSRKCY
TVIA
1367
















TABLE 22







ωMVIIA peptide and peptide analog inhibitor


sequences










Short-hand



Sequence/structure
designation
SEQ ID NO:












CKGKGAKCSRLMYDCCTGSCRSGKC
MVIIA
65





CXGKGAKCSRLMYDCCTGSCRSGKC
MVIIA-X2
410





CAGKGAKCSRLMYDCCTGSCRSGKC
MVIIA-A2
411





CKGXGAKCSRLMYDCCTGSCRSGKC
MVIIA-X4
412





CKGAGAKCSRLMYDCCTGSCRSGKC
MVIIA-A4
413





CKGKGAXCSRLMYDCCTGSCRSGKC
MVIIA-X7
414





CKGKGAACSRLMYDCCTGSCRSGKC
MVIIA-A7
415





CKGKGAKCSXLMYDCCTGSCRSGKC
MVIIA-X10
416





CKGKGAKCSALMYDCCTGSCRSGKC
MVIIA-A10
417





CKGKGAKCSRLMYDCCTGSCXSGKC
MVIIA-X21
418





CKGKGAKCSRLMYDCCTGSCASGKC
MVIIA-A21
419





CKGKGAKCSRLMYDCCTGSCRSGXC
MVIIA-X24
420





CKGKGAKCSRLMYDCCTGSCRSGAC
MVIIA-A24
421
















TABLE 23







□GVIA peptide and peptide analog inhibitor


sequences










Short-hand



Sequence/structure
designation
SEQ ID NO:












CKSPGSSCSPTSYNCCRSCNPYTKRCY
GVIA
64





CXSPGSSCSPTSYNCCRSCNPYTKRCY
GVIA-X2
422





CASPGSSCSPTSYNCCRSCNPYTKRCY
GVIA-A2
423





CKSPGSSCSPTSYNCCXSCNPYTKRCY
GVIA-X17
424





CKSPGSSCSPTSYNCCASCNPYTKRCY
GVIA-A17
425





CKSPGSSCSPTSYNCCRSCNPYTXRCY
GVIA-X24
426





CKSPGSSCSPTSYNCCRSCNPYTARCY
GVIA-A24
427





CKSPGSSCSPTSYNCCRSCNPYTKXCY
GVIA-X25
428





CKSPGSSCSPTSYNCCRSCNPYTKACY
GVIA-A25
429
















TABLE 24







Ptu1 peptide and peptide analog inhibitor


sequences











SEQ



Short-hand
ID


Sequence/structure
designation
NO:












AEKDCIAPGAPCFGTDKPCCNPRAWCSSYANKCL
Ptu1
66





AEXDCIAPGAPCFGTDKPCCNPRAWCSSYANKCL
Ptu1-X3
430





AEADCIAPGAPCFGTDKPCCNPRAWCSSYANKCL
Ptu1-A3
431





AEKDCIAPGAPCFGTDXPCCNPRAWCSSYANKCL
Ptu1-X17
432





AEKDCIAPGAPCFGTDAPCCNPRAWCSSYANKCL
Ptu1-A17
433





AEKDCIAPGAPCFGTDKPCCNPXAWCSSYANKCL
Ptu1-X23
434





AEKDCIAPGAPCFGTDKPCCNPAAWCSSYANKCL
Ptu1-A23
435





AEKDCIAPGAPCFGTDKPCCNPRAWCSSYANXCL
Ptu1-X32
436





AEKDCIAPGAPCFGTDKPCCNPRAWCSSYANACL
Ptu1-A32
437
















TABLE 25







Thrixopelma pruriens (ProTx1) and ProTx1 peptide


analogs and other T type Ca2+ 


channel inhibitor peptide sequences











SEQ



Short-hand
ID


Sequence/structure
designation
NO:












ECRYWLGGCSAGQTCCKHLVCSRRHGWCVWD
ProTx1
56


GTFS





ECXYWLGGCSAGQTCCKHLVCSRRHGWCVWD
ProTx1-X3
438


GTFS





ECAYWLGGCSAGQTCCKHLVCSRRHGWCVWD
ProTx1-A3
439


GTFS





ECRYWLGGCSAGQTCCXHLVCSRRHGWCVWD
ProTx1-X17
440


GTFS





ECRYWLGGCSAGQTCCAHLVCSRRHGWCVWD
ProTx1-A17
441


GTFS





ECRYWLGGCSAGQTCCKHLVCSXRHGWCVWD
ProTx1-X23
442


GTFS





ECRYWLGGCSAGQTCCKHLVCSARHGWCVWD
ProTx1-A23
443


GTFS





ECRYWLGGCSAGQTCCKHLVCSRXHGWCVWD
ProTx1-X24
444


GTFS





ECRYWLGGCSAGQTCCKHLVCSRAHGWCVWD
ProTx1-A24
445


GTFS





KIDGYPVDYW NCKRICWYNN KYCNDLCKGL
Kurtoxin
1276


KADSGYCWGW TLSCYCQGLP DNARIKRSGR


CRA
















TABLE 26







BeKM1 M current inhibitor peptide and BeKM1


peptide analog sequences











SEQ



Short-hand
ID


Sequence/structure
designation
NO:












RPTDIKCSESYQCFPVCKSRFGKTNGRCVNGFCDCF
BeKM1
63





PTDIKCSESYQCFPVCKSRFGKTNGRCVNGFCDCF
BeKM1-d1
446





XPTDIKCSESYQCFPVCKSRFGKTNGRCVNGFCDCF
BeKM1-X1
447





APTDIKCSESYQCFPVCKSRFGKTNGRCVNGFCDCF
BeKM1-A1
448





RPTDIXCSESYQCFPVCKSRFGKTNGRCVNGFCDCF
BeKM1-X6
449





RPTDIACSESYQCFPVCKSRFGKTNGRCVNGFCDCF
BeKM1-A6
450





RPTDIKCSESYQCFPVCXSRFGKTNGRCVNGFCDCF
BeKM1-X18
451





RPTDIKCSESYQCFPVCASRFGKTNGRCVNGFCDCF
BeKM1-A18
452





RPTDIKCSESYQCFPVCKSXFGKTNGRCVNGFCDCF
BeKM1-X20
453





RPTDIKCSESYQCFPVCKSAFGKTNGRCVNGFCDCF
BeKM1-A20
454





RPTDIKCSESYQCFPVCKSRFGXTNGRCVNGFCDCF
BeKM1-X23
455





RPTDIKCSESYQCFPVCKSRFGATNGRCVNGFCDCF
BeKM1-A23
456





RPTDIKCSESYQCFPVCKSRFGKTNGXCVNGFCDCF
BeKM1-X27
457





RPTDIKCSESYQCFPVCKSRFGKTNGACVNGFCDCF
BeKM1-A27
458
















TABLE 27







Na+ channel inhibitor peptide sequences










Short-hand
SEQ ID


Sequence/structure
designation
NO:





QRCCNGRRGCSSRWCRDHSRCC
SmIIIa
459





RDCCTOOKKCKDRQCKOQRCCA
μ-GIIIA
460





RDCCTOORKCKDRRCKOMRCCA
μ-GIIIB
461





ZRLCCGFOKSCRSRQCKOHRCC
μ-PIIIA
462





ZRCCNGRRGCSSRWCRDHSRCC
μ-SmIIIA
463





ACRKKWEYCIVPIIGFIYCCPGLICGPFVCV
μO-MrVIA
464





ACSKKWEYCIVPIIGFIYCCPGLICGPFVCV
μO-MrVIB
465





EACYAOGTFCGIKOGLCCSEFCLPGVCFG
δ-PVIA
466





DGCSSGGTFCGIHOGLCCSEFCFLWCITFID
δ-SVIE
467





WCKQSGEMCNLLDQNCCDGYCIVLVCT
δ-TxVIA
468





VKPCRKEGQLCDPIFQNCCRGWNCVLFCV
δ-GmVIA
469
















TABLE 28







Cl− channel inhibitor peptide sequences











Short-hand




Sequence/structure
designation
SEQ ID NO:













MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCR
CTX
67






MCMPCFTTDHQMAXKCDDCCGGKGRGKCYGPQCLCR
CTX-X14
470





MCMPCFTTDHQMAAKCDDCCGGKGRGKCYGPQCLCR
CTX-A14
471





MCMPCFTTDHQMARXCDDCCGGKGRGKCYGPQCLCR
CTX-X15
472





MCMPCFTTDHQMARACDDCCGGKGRGKCYGPQCLCR
CTX-A15
473





MCMPCFTTDHQMARKCDDCCGGXGRGKCYGPQCLCR
CTX-X23
474





MCMPCFTTDHQMARKCDDCCGGAGRGKCYGPQCLCR
CTX-A23
475





MCMPCFTTDHQMARKCDDCCGGKGXGKCYGPQCLCR
CTX-X25
476





MCMPCFTTDHQMARKCDDCCGGKGAGKCYGPQCLCR
CTX-A25
477





MCMPCFTTDHQMARKCDDCCGGKGRGXCYGPQCLCR
CTX-X27
478





MCMPCFTTDHQMARKCDDCCGGKGRGACYGPQCLCR
CTX-A27
479





MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCX
CTX-X36
480





MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLCA
CTX-A36
481





MCMPCFTTDHQMARKCDDCCGGKGRGKCYGPQCLC
CTX-d36
482





QTDGCGPCFTTDANMARKCRECCGGNGKCFGPQCLCNRE
Bm-12b
483





QTDGCGPCFTTDANMAXKCRECCGGNGKCFGPQCLCNRE
Bm-12b-X17
484





QTDGCGPCFTTDANMAAKCRECCGGNGKCFGPQCLCNRE
Bm-12b-A17
485





QTDGCGPCFTTDANMARXCRECCGGNGKCFGPQCLCNRE
Bm-12b-X18
486





QTDGCGPCFTTDANMARACRECCGGNGKCFGPQCLCNRE
Bm-12b-A18
487





QTDGCGPCFTTDANMARKCXECCGGNGKCFGPQCLCNRE
Bm-12b-X20
488





QTDGCGPCFTTDANMARKCAECCGGNGKCFGPQCLCNRE
Bm-12b-A20
489





QTDGCGPCFTTDANMARKCRECCGGNGXCFGPQCLCNRE
Bm-12b-X28
490





QTDGCGPCFTTDANMARKCRECCGGNGACFGPQCLCNRE
Bm-12b-A28
491





QTDGCGPCFTTDANMARKCRECCGGNGKCFGPQCLCNXE
Bm-12b-X38
492





QTDGCGPCFTTDANMARKCRECCGGNGKCFGPQCLCNAE
Bm-12b-A38
493
















TABLE 29







Kv2.1 inhibitor peptide sequences











SEQ



Short-hand
ID


Sequence/structure
designation
NO:





ECRYLFGGCKTTSDCCKHLGCKFRDKYCAWDFTFS
HaTx1
494





ECXYLFGGCKTTSDCCKHLGCKFRDKYCAWDFTFS
HaTx1-X3
495





ECAYLFGGCKTTSDCCKHLGCKFRDKYCAWDFTFS
HaTx1-A3
496





ECRYLFGGCXTTSDCCKHLGCKFRDKYCAWDFTFS
HaTx1-X10
497





ECRYLFGGCATTSDCCKHLGCKFRDKYCAWDFTFS
HaTx1-A10
498





ECRYLFGGCKTTSDCCXHLGCKFRDKYCAWDFTFS
HaTx1-X17
499





ECRYLFGGCKTTSDCCAHLGCKFRDKYCAWDFTFS
HaTx1-A17
500





ECRYLFGGCKTTSDCCKHLGCXFRDKYCAWDFTFS
HaTx1-X22
501





ECRYLFGGCKTTSDCCKHLGCAFRDKYCAWDFTFS
HaTx1-A22
502





ECRYLFGGCKTTSDCCKHLGCKFXDKYCAWDFTFS
HaTx1-X24
503





ECRYLFGGCKTTSDCCKHLGCKFADKYCAWDFTFS
HaTx1-A24
504





ECRYLFGGCKTTSDCCKHLGCKFRDXYCAWDFTFS
HaTx1-X26
505





ECRYLFGGCKTTSDCCKHLGCKFRDAYCAWDFTFS
HaTx1-A26
506
















TABLE 30







Kv4.3 & Kv4.2 inhibitor peptide sequences










Short-hand
SEQ


Sequence/structure
designation
ID NO:












YCQKWMWTCDEERKCCEGLVCRLWCKRIINM
PaTx2
57





YCQXWMWTCDEERKCCEGLVCRLWCKRIINM
PaTx2-X4
507





YCQAWMWTCDEERKCCEGLVCRLWCKRIINM
PaTx2-A4
508





YCQKWMWTCDEEXKCCEGLVCRLWCKRIINM
PaTx2-X13
509





YCQKWMWTCDEEAKCCEGLVCRLWCKRIINM
PaTx2-A13
510





YCQKWMWTCDEERXCCEGLVCRLWCKRIINM
PaTx2-X14
511





YCQKWMWTCDEERACCEGLVCRLWCKRIINM
PaTx2-A14
512





YCQKWMWTCDEERKCCEGLVCXLWCKRIINM
PaTx2-X22
513





YCQKWMWTCDEERKCCEGLVCALWCKRIINM
PaTx2-A22
514





YCQKWMWTCDEERKCCEGLVCRLWCXRIINM
PaTx2-X26
515





YCQKWMWTCDEERKCCEGLVCRLWCARIINM
PaTx2-A26
516





YCQKWMWTCDEERKCCEGLVCRLWCKXIINM
PaTx2-X27
517





YCQKWMWTCDEERKCCEGLVCRLWCKAIINM
PaTx2-A27
518
















TABLE 31







nACHR channel inhibitor peptide sequences











SEQ



Short-hand
ID


Sequence/structure
designation
NO:





GCCSLPPCAANNPDYC
PnIA
519





GCCSLPPCALNNPDYC
PnIA-L10
520





GCCSLPPCAASNPDYC
PnIA-S11
521





GCCSLPPCALSNPDYC
PnIB
522





GCCSLPPCAASNPDYC
PnIB-A10
523





GCCSLPPCALNNPDYC
PnIB-N11
524





GCCSNPVCHLEHSNLC
MII
525





GRCCHPACGKNYSC
α-MI
526





RD(hydroxypro)CCYHPTCNMSNPQIC
α-EI
527





GCCSYPPCFATNPDC
α-AuIB
528





RDPCCSNPVCTVHNPQIC
α-PIA
529





GCCSDPRCAWRC
α-ImI
530





ACCSDRRCRWRC
α-ImII
531





ECCNPACGRHYSC
α-GI
532





GCCGSY(hydroxypro)NAACH
αA-PIVA
533


CSCKDR(hydroxypro)SYCGQ





GCCPY(hydroxypro)NAACH(hydroxypro)
αA-EIVA
534


CGCKVGR(hydroxypro)(hydroxypro)


YCDR(hydroxypro)S


GG





H(hydroxypro)(hydroxypro)CCLYGKCRRY
ψ-PIIIE
535


(hydroxypro)GCSSASCCQR





GCCSDPRCNMNNPDYC
EpI
536





GCCSHPACAGNNQHIC
GIC
537





IRD(γ-carboxyglu) CCSNPACRVNN
GID
538


(hydroxypro)HVC





GGCCSHPACAANNQDYC
AnIB
539





GCCSYPPCFATNSDYC
AuIA
540





GCCSYPPCFATNSGYC
AuIC
541

















TABLE 32







Agelenopsis aperta (Agatoxin) toxin peptides and peptide



analogs and other Ca2+ channel inhibiter peptides











Short-hand




Sequence/structure
designation
SEQ ID NO:













KKKCIAKDYG RCKWGGTPCC RGRGCICSIM
ω-Aga-IVA
959



GTNCECKPRL IMEGLGLA





EDNCIAEDYG KCTWGGTKCC RGRPCRCSMI
ω-Aga-IVB
960


GTNCECTPRL IMEGLSFA





SCIDIGGDCD GEKDDCQCCR RNGYCSCYSL
ω-Aga-IIIA
961


FGYLKSGCKC VVGTSAEFQG ICRRKARQCY


NSDPDKCESH NKPKRR





SCIDIGGDCD GEKDDCQCCR RNGYCSCYSL
ω-Aga-IIIA-
962


FGYLKSGCKC VVGTSAEFQG ICRRKARTCY
T58


NSDPDKCESH NKPKRR





SCIDFGGDCD GEKDDCQCCR SNGYCSCYSL
ω-Aga-IIIB
963


FGYLKSGCKC EVGTSAEFRR ICRRKAKQCY


NSDPDKCVSV YKPKRR





SCIDFGGDCD GEKDDCQCCR SNGYCSCYNL
ω-Aga-IIIB-
964


FGYLKSGCKC EVGTSAEFRR
N29


ICRRKAKQCYNSDPDKCVSV YKPKRR





SCIDFGGDCD GEKDDCQCCR SNGYCSCYNL
ω-Aga-IIIB-
965


FGYLRSGCKC EVGTSAEFRR ICRRKAKQCY
N29/R35


NSDPDKCVSV YKPKRR





NCIDFGGDCD GEKDDCQCCX RNGYCSCYNL
ω-Aga-IIIC
966


FGYLKRGCKX EVG





SCIKIGEDCD GDKDDCQCCR TNGYCSXYXL FGYLKSG
ω-Aga-IIID
967





GCIEIGGDCD GYQEKSYCQC CRNNGFCS
ω-Aga-IIA
968





AKAL PPGSVCDGNE SDCKCYGKWH KCRCPWKWHF
ω-Aga-IA
969


TGEGPCTCEK GMKHTCITKL HCPNKAEWGL DW
(major chain)





ECVPENGHCR DWYDECCEGF YCSCRQPPKC ICRNNNX
μ-Aga
970





DCVGESQQCA DWAGPHCCDG YYCTCRYFPK CICVNNN
μ-Aga-6
971





ACVGENKQCA DWAGPHCCDG YYCTCRYFPK CICRNNN
μ-Aga-5
972





ACVGENQQCA DWAGPHCCDG YYCTCRYFPK CICRNNN
μ-Aga-4
973





ADCVGDGQRC ADWAGPYCCS GYYCSCRSMP
μ-Aga-3
1275


YCRCRSDS





ECATKNKRCA DWAGPWCCDG LYCSCRSYPG CMCRPSS
μ-Aga-2
974





ECVPENGHCR DWYDECCEGF YCSCRQPPKC ICRNNN
μ-Aga-1
975





AELTSCFPVGHECDGDASNCNCCGDDVYCGCGWGRWNCKC
Tx-1
1277


KVADQSYAYGICKDKVNCPNRHLWPAKVCKKPCRREC





GCANAYKSCNGPHTCCWGYNGYKKACICSGXNWK
Tx3-3
1278





SCINVGDFCDGKKDCCQCDRDNAFCSCSVIFGYKTNCRCE
Tx3-4
1279





SCINVGDFCDGKKDDCQCCRDNAFCSCSVIFGYKTNCRCE
ω-PtXIIA
1280


VGTTATSYGICMAKHKCGRQTTCTKPCLSKRCKKNH





AECLMTIGDTSCVPRLGRRCCYGAWCYCDQQLSCRRVGRKR
Dw13.3
1281


ECGWVEVNCKCGWSWSQRIDDWRADYSCKCPEDQ





GGCLPHNRFCNALSGPRCCSGLKCKELSIWDSRCL
Agelenin
1282





DCVRFWGKCSQTSDCCPHLACKSKWPRNICVWDGSV
ω-GTx-SIA
1283





GCLEVDYFCG IPFANNGLCC SGNCVFVCTP Q
ω-conotoxin
1284



PnVIA





DDDCEPPGNF CGMIKIGPPC CSGWCFFACA
ω-conotoxin
1285



PnVIB





VCCGYKLCHP C
Lambda-
1286



conotoxin



CMrVIA





MRCLPVLIIL LLLTASAPGV VVLPKTEDDV
Lambda-
1287


PMSSVYGNGK SILRGILRNG VCCGYKLCHP C
conotoxin



CMrVIB





KIDGYPVDYW NCKRICWYNN KYCNDLCKGL
Kurtoxin
1276


KADSGYCWGW TLSCYCQGLP DNARIKRSGR CRA





CKGKGAPCRKTMYDCCSGSCGRRGKC
MVIIC
1368









In accordance with this invention are molecules in which at least one of the toxin peptide (P) portions of the molecule comprises a Kv1.3 antagonist peptide. Amino acid sequences selected from ShK, HmK, MgTx, AgTx1, AgTx2, Heterometrus spinnifer (HsTx1), OSK1, Anuroctoxin (AnTx), Noxiustoxin (NTX), KTX1, Hongotoxin, ChTx, Titystoxin, BgK, BmKTX, BmTx, AeK, AsKS Tc30, Tc32, Pi1, Pi2, and/or Pi3 toxin peptides and peptide analogs of any of these are preferred. Examples of useful Kv1.3 antagonist peptide sequences include those having any amino acid sequence set forth in Table 1, Table 2, Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, and/or Table 11 herein above;


Other embodiments of the inventive composition include at least one toxin peptide (P) that is an IKCa1 antagonist peptide. Useful IKCa1 antagonist peptides include Maurotoxin (MTx), ChTx, peptides and peptide analogs of either of these, examples of which include those having any amino acid sequence set forth in Table 12, Table 13, and/or Table 14;


Other embodiments of the inventive composition include at least one toxin peptide (P) that is a SKCa inhibitor peptide. Useful SKCa inhibitor peptides include, Apamin, ScyTx, BmP05, P01, P05, Tamapin, TsK, and peptide analogs of any of these, examples of which include those having any amino acid sequence set forth in Table 15;


Other embodiments of the inventive composition include at least one toxin peptide (P) that is an apamin peptide, and peptide analogs of apamin, examples of which include those having any amino acid sequence set forth in Table 16;


Other embodiments of the inventive composition include at least one toxin peptide (P) that is a Scyllotoxin family peptide, and peptide analogs of any of these, examples of which include those having any amino acid sequence set forth in Table 17;


Other embodiments of the inventive composition include at least one toxin peptide (P) that is a BKCa inhibitor peptide, examples of which include those having any amino acid sequence set forth in Table 18;


Other embodiments of the inventive composition include at least one toxin peptide (P) that is a Slotoxin family peptide, and peptide analogs of any of these, examples of which include those having any amino acid sequence set forth in Table 19;


Other embodiments of the inventive composition include at least one toxin peptide (P) that is a Martentoxin peptide, and peptide analogs thereof, examples of which include those having any amino acid sequence set forth in Table 20;


Other embodiments of the inventive composition include at least one toxin peptide (P) that is a N-type Ca2+ channel inhibitor peptide, examples of which include those having any amino acid sequence set forth in Table 21;


Other embodiments of the inventive composition include at least one toxin peptide (P) that is a ωMVIIA peptide, and peptide analogs thereof, examples of which include those having any amino acid sequence set forth in Table 22;


Other embodiments of the inventive composition include at least one toxin peptide (P) that is a ωGVIA peptide, and peptide analogs thereof, examples of which include those having any amino acid sequence set forth in Table 23;


Other embodiments of the inventive composition include at least one toxin peptide (P) that is a Ptu1 peptide, and peptide analogs thereof, examples of which include those having any amino acid sequence set forth in Table 24;


Other embodiments of the inventive composition include at least one toxin peptide (P) that is a ProTx1 peptide, and peptide analogs thereof, examples of which include those having any amino acid sequence set forth in Table 25;


Other embodiments of the inventive composition include at least one toxin peptide (P) that is a BeKM1 peptide, and peptide analogs thereof, examples of which include those having any amino acid sequence set forth in Table 26;


Other embodiments of the inventive composition include at least one toxin peptide (P) that is a Na+ channel inhibitor peptide, examples of which include those having any amino acid sequence set forth in Table 27;


Other embodiments of the inventive composition include at least one toxin peptide (P) that is a Cl channel inhibitor peptide, examples of which include those having any amino acid sequence set forth in Table 28;


Other embodiments of the inventive composition include at least one toxin peptide (P) that is a Kv2.1 inhibitor peptide, examples of which include those having any amino acid sequence set forth in Table 29;


Other embodiments of the inventive composition include at least one toxin peptide (P) that is a Kv4.2/Kv4.3 inhibitor peptide, examples of which include those having any amino acid sequence set forth in Table 30;


Other embodiments of the inventive composition include at least one toxin peptide (P) that is a nACHR inhibitor peptide, examples of which include those having any amino acid sequence set forth in Table 31; and


Other embodiments of the inventive composition include at least one toxin peptide (P) that is an Agatoxin peptide, a peptide analog thereof or other calcium channel inhibitor peptide, examples of which include those having any amino acid sequence set forth in Table 32.


Half-life extending moieties. This invention involves the presence of at least one half-life extending moiety (F1 and/or F2 in Formula I) attached to a peptide through the N-terminus, C-terminus or a sidechain of one of the intracalary amino acid residues. Multiple half-life extending moieties can also be used; e.g., Fc's at each terminus or an Fc at a terminus and a PEG group at the other terminus or at a sidechain. In other embodiments the Fc domain can be PEGylated (e.g., in accordance with the formulae F1—F2-(L)f-P; P-(L)g-F1—F2; or P-(L)9-F1—F2-(L)f-P).


The half-life extending moiety can be selected such that the inventive composition achieves a sufficient hydrodynamic size to prevent clearance by renal filtration in vivo. For example, a half-life extending moiety can be selected that is a polymeric macromolecule, which is substantially straight chain, branched-chain, or dendritic in form. Alternatively, a half-life extending moiety can be selected such that, in vivo, the inventive composition of matter will bind to a serum protein to form a complex, such that the complex thus formed avoids substantial renal clearance. The half-life extending moiety can be, for example, a lipid; a cholesterol group (such as a steroid); a carbohydrate or oligosaccharide; or any natural or synthetic protein, polypeptide or peptide that binds to a salvage receptor.


Exemplary half-life extending moieties that can be used, in accordance with the present invention, include an immunoglobulin Fc domain, or a portion thereof, or a biologically suitable polymer or copolymer, for example, a polyalkylene glycol compound, such as a polyethylene glycol or a polypropylene glycol. Other appropriate polyalkylene glycol compounds include, but are not limited to, charged or neutral polymers of the following types: dextran, polylysine, colominic acids or other carbohydrate based polymers, polymers of amino acids, and biotin derivatives. In some monomeric fusion protein embodiments an immunoglobulin (including light and heavy chains) or a portion thereof, can be used as a half-life-extending moiety, preferably an immunoglobulin of human origin, and including any of the immunoglobulins, such as, but not limited to, IgG1, IgG2, IgG3 or IgG4.


Other examples of the half-life extending moiety, in accordance with the invention, include a copolymer of ethylene glycol, a copolymer of propylene glycol, a carboxymethylcellulose, a polyvinyl pyrrolidone, a poly-1,3-dioxolane, a poly-1,3,6-trioxane, an ethylene/maleic anhydride copolymer, a polyaminoacid (e.g., polylysine), a dextran n-vinyl pyrrolidone, a poly n-vinyl pyrrolidone, a propylene glycol homopolymer, a propylene oxide polymer, an ethylene oxide polymer, a polyoxyethylated polyol, a polyvinyl alcohol, a linear or branched glycosylated chain, a polyacetal, a long chain fatty acid, a long chain hydrophobic aliphatic group, an immunoglobulin light chain and heavy chain, an immunoglobulin Fc domain or a portion thereof (see, e.g., Feige et al., Modified peptides as therapeutic agents, U.S. Pat. No. 6,660,843), a CH2 domain of Fc, an albumin (e.g., human serum albumin (HSA)); see, e.g., Rosen et al., Albumin fusion proteins, U.S. Pat. No. 6,926,898 and US 2005/0054051; Bridon et al., Protection of endogenous therapeutic peptides from peptidase activity through conjugation to blood components, U.S. Pat. No. 6,887,470), a transthyretin (TTR; see, e.g., Walker et al., Use of transthyretin peptide/protein fusions to increase the serum half-life of pharmacologically active peptides/proteins, US 2003/0195154 A1; 2003/0191056 A1), or a thyroxine-binding globulin (TBG). Thus, exemplary embodiments of the inventive compositions also include HSA fusion constructs such as but not limited to: HSA fusions with ShK, OSK1, or modified analogs of those toxin peptides. Examples include HSA-L10-ShK(2-35); HSA-L10-OsK1(1-38); HSA-L10-ShK(2-35); and HSA-L10-OsK1(1-38).


Other embodiments of the half-life extending moiety, in accordance with the invention, include peptide ligands or small (organic) molecule ligands that have binding affinity for a long half-life serum protein under physiological conditions of temperature, pH, and ionic strength. Examples include an albumin-binding peptide or small molecule ligand, a transthyretin-binding peptide or small molecule ligand, a thyroxine-binding globulin-binding peptide or small molecule ligand, an antibody-binding peptide or small molecule ligand, or another peptide or small molecule that has an affinity for a long half-life serum protein. (See, e.g., Blaney et al., Method and compositions for increasing the serum half-life of pharmacologically active agents by binding to transthyretin-selective ligands, U.S. Pat. No. 5,714,142; Sato et al., Serum albumin binding moieties, US 2003/0069395 A1; Jones et al., Pharmaceutical active conjugates, U.S. Pat. No. 6,342,225). A “long half-life serum protein” is one of the hundreds of different proteins dissolved in mammalian blood plasma, including so-called “carrier proteins” (such as albumin, transferrin and haptoglobin), fibrinogen and other blood coagulation factors, complement components, immunoglobulins, enzyme inhibitors, precursors of substances such as angiotensin and bradykinin and many other types of proteins. The invention encompasses the use of any single species of pharmaceutically acceptable half-life extending moiety, such as, but not limited to, those described herein, or the use of a combination of two or more different half-life extending moieties, such as PEG and immunoglobulin Fc domain or a CH2 domain of Fc, albumin (e.g., HSA), an albumin-binding protein, transthyretin or TBG, or a combination such as immunoglobulin (light chain+heavy chain) and Fc domain (the combination so-called “hemibody”).


In some embodiments of the invention an Fc domain or portion thereof, such as a CH2 domain of Fc, is used as a half-life extending moiety. The Fc domain can be fused to the N-terminal (e.g., in accordance with the formula F1-(L)f-P) or C-terminal (e.g., in accordance with the formula P-(L)g-F1) of the toxin peptides or at both the N and C termini (e.g., in accordance with the formulae F1-(L)f-P-(L)g-F2 or P-(L)g-F1-(L)f-P). A peptide linker sequence can be optionally included between the Fc domain and the toxin peptide, as described herein. Examples of the formula F1-(L)f-P include: Fc-L10-ShK(K22A)[2-35]; Fc-L10-ShK(R1K/K22A)[1-35]; Fc-L10-ShK(R1H/K22A)[1-35]; Fc-L110-ShK(R1Q/K22A)[1-35]; Fc-L110-ShK(R1Y/K22A)[1-35]; Fc-L10-PP-ShK(K22A) [1-35]; and any other working examples described herein. Examples of the formula P-(L)g-F1 include: ShK(1-35)-L10-Fc; OsK1(1-38)-L10-Fc; Met-ShK(1-35)-L10-Fc; ShK(2-35)-L10-Fc; Gly-ShK(1-35)-L10-Fc; Osk1(1-38)-L10-Fc; and any other working examples described herein.


Fc variants are suitable half-life extending moieties within the scope of this invention. A native Fc can be extensively modified to form an Fc variant in accordance with this invention, provided binding to the salvage receptor is maintained; see, for example WO 97/34631, WO 96/32478, and WO 04/110 472. In such Fc variants, one can remove one or more sites of a native Fc that provide structural features or functional activity not required by the fusion molecules of this invention. One can remove these sites by, for example, substituting or deleting residues, inserting residues into the site, or truncating portions containing the site. The inserted or substituted residues can also be altered amino acids, such as peptidomimetics or D-amino acids. Fc variants can be desirable for a number of reasons, several of which are described below. Exemplary Fc variants include molecules and sequences in which:

    • 1. Sites involved in disulfide bond formation are removed. Such removal can avoid reaction with other cysteine-containing proteins present in the host cell used to produce the molecules of the invention. For this purpose, the cysteine-containing segment at the N-terminus can be truncated or cysteine residues can be deleted or substituted with other amino acids (e.g., alanyl, seryl). In particular, one can truncate the N-terminal 20-amino acid segment of SEQ ID NO: 2 or delete or substitute the cysteine residues at positions 7 and 10 of SEQ ID NO: 2. Even when cysteine residues are removed, the single chain Fc domains can still form a dimeric Fc domain that is held together non-covalently.
    • 2. A native Fc is modified to make it more compatible with a selected host cell. For example, one can remove the PA sequence near the N-terminus of a typical native Fc, which can be recognized by a digestive enzyme in E. coli such as proline iminopeptidase. One can also add an N-terminal methionine residue, especially when the molecule is expressed recombinantly in a bacterial cell such as E. coli. The Fc domain of SEQ ID NO: 2 (FIG. 4A-4B) is one such Fc variant.
    • 3. A portion of the N-terminus of a native Fc is removed to prevent N-terminal heterogeneity when expressed in a selected host cell. For this purpose, one can delete any of the first 20 amino acid residues at the N-terminus, particularly those at positions 1, 2, 3, 4 and 5.
    • 4. One or more glycosylation sites are removed. Residues that are typically glycosylated (e.g., asparagine) can confer cytolytic response. Such residues can be deleted or substituted with unglycosylated residues (e.g., alanine).
    • 5. Sites involved in interaction with complement, such as the C1q binding site, are removed. For example, one can delete or substitute the EKK sequence of human IgG1. Complement recruitment may not be advantageous for the molecules of this invention and so can be avoided with such an Fc variant.
    • 6. Sites are removed that affect binding to Fc receptors other than a salvage receptor. A native Fc can have sites for interaction with certain white blood cells that are not required for the fusion molecules of the present invention and so can be removed.
    • 7. The ADCC site is removed. ADCC sites are known in the art; see, for example, Molec. Immunol. 29 (5): 633-9 (1992) with regard to ADCC sites in IgG1. These sites, as well, are not required for the fusion molecules of the present invention and so can be removed.
    • 8. When the native Fc is derived from a non-human antibody, the native Fc can be humanized. Typically, to humanize a native Fc, one will substitute selected residues in the non-human native Fc with residues that are normally found in human native Fc. Techniques for antibody humanization are well known in the art.


Preferred Fc variants include the following. In SEQ ID NO: 2, the leucine at position 15 can be substituted with glutamate; the glutamate at position 99, with alanine; and the lysines at positions 101 and 103, with alanines. In addition, phenyalanine residues can replace one or more tyrosine residues.


An alternative half-life extending moiety would be a protein, polypeptide, peptide, antibody, antibody fragment, or small molecule (e.g., a peptidomimetic compound) capable of binding to a salvage receptor. For example, one could use as a half-life extending moiety a polypeptide as described in U.S. Pat. No. 5,739,277, issued Apr. 14, 1998 to Presta et al. Peptides could also be selected by phage display for binding to the FcRn salvage receptor. Such salvage receptor-binding compounds are also included within the meaning of “half-life extending moiety” and are within the scope of this invention. Such half-life extending moieties should be selected for increased half-life (e.g., by avoiding sequences recognized by proteases) and decreased immunogenicity (e.g., by favoring non-immunogenic sequences, as discovered in antibody humanization).


As noted above, polymer half-life extending moieties can also be used for F1 and F2. Various means for attaching chemical moieties useful as half-life extending moieties are currently available, see, e.g., Patent Cooperation Treaty (“PCT”) International Publication No. WO 96/11953, entitled “N-Terminally Chemically Modified Protein Compositions and Methods,” herein incorporated by reference in its entirety. This PCT publication discloses, among other things, the selective attachment of water-soluble polymers to the N-terminus of proteins.


In some embodiments of the inventive compositions, the polymer half-life extending moiety is polyethylene glycol (PEG), as F1 and/or F2, but it should be understood that the inventive composition of matter, beyond positions F1 and/or F2, can also include one or more PEGs conjugated at other sites in the molecule, such as at one or more sites on the toxin peptide. Accordingly, some embodiments of the inventive composition of matter further include one or more PEG moieties conjugated to a non-PEG half-life extending moiety, which is F1 and/or F2, or to the toxin peptide(s) (P), or to any combination of any of these. For example, an Fc domain or portion thereof (as F1 and/or F2) in the inventive composition can be made mono-PEGylated, di-PEGylated, or otherwise multi-PEGylated, by the process of reductive alkylation.


Covalent conjugation of proteins and peptides with poly(ethylene glycol) (PEG) has been widely recognized as an approach to significantly extend the in vivo circulating half-lives of therapeutic proteins. PEGylation achieves this effect predominately by retarding renal clearance, since the PEG moiety adds considerable hydrodynamic radius to the protein. (Zalipsky, S., et al., Use of functionalized poly(ethylene glycol)s for modification of polypeptides., in poly(ethylene glycol) chemistry: Biotechnical and biomedical applications., J. M. Harris, Ed., Plenum Press: New York., 347-370 (1992)). Additional benefits often conferred by PEGylation of proteins and peptides include increased solubility, resistance to proteolytic degradation, and reduced immunogenicity of the therapeutic polypeptide. The merits of protein PEGylation are evidenced by the commercialization of several PEGylated proteins including PEG-Adenosine deaminase (Adagen™/Enzon Corp.), PEG-L-asparaginase (Oncaspar™/Enzon Corp.), PEG-Interferon α-2b (PEG-Intron™/Schering/Enzon), PEG-Interferon α-2a (PEGASYS™/Roche) and PEG-G-CSF (Neulasta™/Amgen) as well as many others in clinical trials.


Briefly, the PEG groups are generally attached to the peptide portion of the composition of the invention via acylation or reductive alkylation (or reductive amination) through a reactive group on the PEG moiety (e.g., an aldehyde, amino, thiol, or ester group) to a reactive group on the inventive compound (e.g., an aldehyde, amino, or ester group).


A useful strategy for the PEGylation of synthetic peptides consists of combining, through forming a conjugate linkage in solution, a peptide and a PEG moiety, each bearing a special functionality that is mutually reactive toward the other. The peptides can be easily prepared with conventional solid phase synthesis (see, for example, FIGS. 5 and 6 and the accompanying text herein). The peptides are “preactivated” with an appropriate functional group at a specific site. The precursors are purified and fully characterized prior to reacting with the PEG moiety. Ligation of the peptide with PEG usually takes place in aqueous phase and can be easily monitored by reverse phase analytical HPLC. The PEGylated peptides can be easily purified by preparative HPLC and characterized by analytical HPLC, amino acid analysis and laser desorption mass spectrometry.


PEG is a well-known, water soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). In the present application, the term “PEG” is used broadly to encompass any polyethylene glycol molecule, in mono-, bi-, or poly-functional form, without regard to size or to modification at an end of the PEG, and can be represented by the formula:





X—O(CH2CH2O)n-1CH2CH2OH,  (X)


where n is 20 to 2300 and X is H or a terminal modification, e.g., a C1-4 alkyl.


In some useful embodiments, a PEG used in the invention terminates on one end with hydroxy or methoxy, i.e., X is H or CH3 (“methoxy PEG”). It is noted that the other end of the PEG, which is shown in formula (II) terminating in OH, covalently attaches to an activating moiety via an ether oxygen bond, an amine linkage, or amide linkage. When used in a chemical structure, the term “PEG” includes the formula (II) above without the hydrogen of the hydroxyl group shown, leaving the oxygen available to react with a free carbon atom of a linker to form an ether bond. More specifically, in order to conjugate PEG to a peptide, the peptide must be reacted with PEG in an “activated” form. Activated PEG can be represented by the formula:





(PEG)-(A)  (XI)


where PEG (defined supra) covalently attaches to a carbon atom of the activation moiety (A) to form an ether bond, an amine linkage, or amide linkage, and (A) contains a reactive group which can react with an amino, azido, alkyne, imino, maleimido, N-succinimidyl, carboxyl, aminooxy, seleno, or thiol group on an amino acid residue of a peptide or a linker moiety covalently attached to the peptide, e.g., the OSK1 peptide analog. Residues baring chemoselective reactive groups can be introduced into the toxin peptide, e.g., an OSK1 peptide analog during assembly of the peptide sequence solid-phase synthesis as protected derivatives. Alternatively, chemoselective reactive groups can be introduced in the toxin peptide after assembly of the peptide sequence by solid-phase synthesis via the use of orthogonal protecting groups at specific sites. Examples of amino acid residues useful for chemoselective reactions include, but are not limited to, (amino-oxyacetyl)-L-diaminopropionic acid, p-azido-phenylalanine, azidohomolalanine, para-propargyloxy-phenylalanine, selenocysteine, para-acetylphenylalanine, (Nε-levulinyl)-Lysine, (Nε-pyruvyl)-Lysine, selenocysteine, and orthogonally protected cysteine and homocysteine.


Accordingly, in some embodiments of the composition of matter, the toxin peptide, e.g., the OSK1 peptide analog, is conjugated to a polyethylene glycol (PEG) at:


(a) 1, 2, 3 or 4 amino functionalized sites in the toxin peptide;


(b) 1, 2, 3 or 4 thiol functionalized sites in the toxin peptide; (c) 1 or 2 ketone functionalized sites in the toxin peptide; (d) 1 or 2 azido functionalized sites of the toxin peptide; (e) 1 or 2 carboxyl functionalized sites in the toxin peptide; (f) 1 or 2 aminooxy functionalized sites in the toxin peptide; or (g) 1 or 2 seleno functionalized sites in the toxin peptide.


In other embodiments of the composition of matter, the toxin peptide, e.g., the OSK1 peptide analog, is conjugated to a polyethylene glycol (PEG) at:


(a) 1, 2, 3 or 4 amino functionalized sites of the PEG;


(b) 1, 2, 3 or 4 thiol functionalized sites of the PEG;


(c) 1, 2, 3 or 4 maleimido functionalized sites of the PEG;


(d) 1, 2, 3 or 4 N-succinimidyl functionalized sites of the PEG;


(e) 1, 2, 3 or 4 carboxyl functionalized sites of the PEG; or


(f) 1, 2, 3 or 4 p-nitrophenyloxycarbonyl functionalized sites of the PEG.


Techniques for the preparation of activated PEG and its conjugation to biologically active peptides are well known in the art. (E.g., see U.S. Pat. Nos. 5,643,575, 5,919,455, 5,932,462, and 5,990,237; Thompson et al., PEGylation of polypeptides, EP 0575545 B1; Petit, Site specific protein modification, U.S. Pat. Nos. 6,451,986, and 6,548,644; S. Herman et al., Poly(ethylene glycol) with reactive endgroups: I. Modification of proteins, J. Bioactive Compatible Polymers, 10:145-187 (1995); Y. Lu et al., Pegylated peptides III: Solid-phase synthesis with PEGylating reagents of varying molecular weight: synthesis of multiply PEGylated peptides, Reactive Polymers, 22:221-229 (1994); A. M. Felix et al., PEGylated Peptides IV: Enhanced biological activity of site-directed PEGylated GRF analogs, Int. J. Peptide Protein Res., 46:253-264 (1995); A. M. Felix, Site-specific poly(ethylene glycol)ylation of peptides, ACS Symposium Series 680(poly(ethylene glycol)): 218-238 (1997); Y. Ikeda et al., Polyethylene glycol derivatives, their modified peptides, methods for producing them and use of the modified peptides, EP 0473084 B1; G. E. Means et al., Selected techniques for the modification of protein side chains, in: Chemical modification of proteins, Holden Day, Inc., 219 (1971)).


Activated PEG, such as PEG-aldehydes or PEG-aldehyde hydrates, can be chemically synthesized by known means or obtained from commercial sources, e.g., Shearwater Polymers, (Huntsville, Ala.) or Enzon, Inc. (Piscataway, N.J.).


An example of a useful activated PEG for purposes of the present invention is a PEG-aldehyde compound (e.g., a methoxy PEG-aldehyde), such as PEG-propionaldehyde, which is commercially available from Shearwater Polymers (Huntsville, Ala.). PEG-propionaldehyde is represented by the formula PEG-CH2CH2CHO. (See, e.g., U.S. Pat. No. 5,252,714). Other examples of useful activated PEG are PEG acetaldehyde hydrate and PEG bis aldehyde hydrate, which latter yields a bifunctionally activated structure. (See., e.g., Bentley et al., Poly(ethylene glycol) aldehyde hydrates and related polymers and applications in modifying amines, U.S. Pat. No. 5,990,237).


Another useful activated PEG for generating the PEG-conjugated peptides of the present invention is a PEG-maleimide compound, such as, but not limited to, a methoxy PEG-maleimide, such as maleimido monomethoxy PEG, are particularly useful for generating the PEG-conjugated peptides of the invention. (E.g., Shen, N-maleimidyl polymer derivatives, U.S. Pat. No. 6,602,498; C. Delgado et al., The uses and properties of PEG-linked proteins., Crit. Rev. Therap. Drug Carrier Systems, 9:249-304 (1992); S. Zalipsky et al., Use of functionalized poly(ethylene glycol)s for modification of polypeptides, in: Poly(ethylene glycol) chemistry: Biotechnical and biomedical applications (J. M. Harris, Editor, Plenum Press: New York, 347-370 (1992); S. Herman et al., Poly(ethylene glycol) with reactive endgroups: I. Modification of proteins, J. Bioactive Compatible Polymers, 10:145-187 (1995); P. J. Shadle et al., Conjugation of polymer to colony stimulating factor-1, U.S. Pat. No. 4,847,325; G. Shaw et al., Cysteine added variants IL-3 and chemical modifications thereof, U.S. Pat. No. 5,166,322 and EP 0469074 B1; G. Shaw et al., Cysteine added variants of EPO and chemical modifications thereof, EP 0668353 A1; G. Shaw et al., Cysteine added variants G-CSF and chemical modifications thereof, EP 0668354 A1; N. V. Katre et al., Interleukin-2 muteins and polymer conjugation thereof, U.S. Pat. No. 5,206,344; R. J. Goodson and N. V. Katre, Site-directed pegylation of recombinant interleukin-2 at its glycosylation site, Biotechnology, 8:343-346 (1990)).


A poly(ethylene glycol) vinyl sulfone is another useful activated PEG for generating the PEG-conjugated peptides of the present invention by conjugation at thiolated amino acid residues, e.g., at C residues. (E.g., M. Morpurgo et al., Preparation and characterization of poly(ethylene glycol) vinyl sulfone, Bioconj. Chem., 7:363-368 (1996); see also Harris, Functionalization of polyethylene glycol for formation of active sulfone-terminated PEG derivatives for binding to proteins and biologically compatible materials, U.S. Pat. Nos. 5,446,090; 5,739,208; 5,900,461; 6,610,281 and 6,894,025; and Harris, Water soluble active sulfones of poly(ethylene glycol), WO 95/13312 A1).


Another activated form of PEG that is useful in accordance with the present invention, is a PEG-N-hydroxysuccinimide ester compound, for example, methoxy PEG-N-hydroxysuccinimidyl (NHS) ester.


Heterobifunctionally activated forms of PEG are also useful. (See, e.g., Thompson et al., PEGylation reagents and biologically active compounds formed therewith, U.S. Pat. No. 6,552,170).


Typically, a toxin peptide or, a fusion protein comprising the toxin peptide, is reacted by known chemical techniques with an activated PEG compound, such as but not limited to, a thiol-activated PEG compound, a diol-activated PEG compound, a PEG-hydrazide compound, a PEG-oxyamine compound, or a PEG-bromoacetyl compound. (See, e.g., S. Herman, Poly(ethylene glycol) with Reactive Endgroups: I. Modification of Proteins, J. Bioactive and Compatible Polymers, 10:145-187 (1995); S. Zalipsky, Chemistry of Polyethylene Glycol Conjugates with Biologically Active Molecules, Advanced Drug Delivery Reviews, 16:157-182 (1995); R. Greenwald et al., Poly(ethylene glycol) conjugated drugs and prodrugs: a comprehensive review, Critical Reviews in Therapeutic Drug Carrier Systems, 17:101-161 (2000)).


Methods for N-terminal PEGylation are exemplified herein in Examples 31-34, 45 and 47-48, but these are in no way limiting of the PEGylation methods that can be employed by one skilled in the art.


Any molecular mass for a PEG can be used as practically desired, e.g., from about 1,000 or 2,000 Daltons (Da) to about 100,000 Da (n is 20 to 2300). Preferably, the combined or total molecular mass of PEG used in a PEG-conjugated peptide of the present invention is from about 3,000 Da or 5,000 Da, to about 50,000 Da or 60,000 Da (total n is from 70 to 1,400), more preferably from about 10,000 Da to about 40,000 Da (total n is about 230 to about 910). The most preferred combined mass for PEG is from about 20,000 Da to about 30,000 Da (total n is about 450 to about 680). The number of repeating units “n” in the PEG is approximated for the molecular mass described in Daltons. It is preferred that the combined molecular mass of PEG on an activated linker is suitable for pharmaceutical use. Thus, the combined molecular mass of the PEG molecule should not exceed about 100,000 Da.


Polysaccharide polymers are another type of water-soluble polymer that can be used for protein modification. Dextrans are polysaccharide polymers comprised of individual subunits of glucose predominantly linked by α1-6 linkages. The dextran itself is available in many molecular weight ranges, and is readily available in molecular weights from about 1 kDa to about 70 kDa. Dextran is a suitable water-soluble polymer for use in the present invention as a half-life extending moiety by itself or in combination with another half-life extending moiety (e.g., Fc). See, for example, WO 96/11953 and WO 96/05309. The use of dextran conjugated to therapeutic or diagnostic immunoglobulins has been reported; see, for example, European Patent Publication No. 0 315 456, which is hereby incorporated by reference in its entirety. Dextran of about 1 kDa to about 20 kDa is preferred when dextran is used as a half-life extending moiety in accordance with the present invention.


Linkers. Any “linker” group or moiety (i.e., “-(L)g-” or “-(L)g-” in Formulae I-IX) is optional. When present, its chemical structure is not critical, since it serves primarily as a spacer. As stated herein above, the linker moiety (-(L)f- and/or -(L)g-), if present, can be independently the same or different from any other linker, or linkers, that may be present in the inventive composition. For example, an “(L)f” can represent the same moiety as, or a different moiety from, any other “(L)f” or any “(L)g” in accordance with the invention. The linker is preferably made up of amino acids linked together by peptide bonds. Some of these amino acids can be glycosylated, as is well understood by those in the art. For example, a useful linker sequence constituting a sialylation site is X1X2NX4X5G (SEQ ID NO: 637), wherein X1, X2, X4 and X5 are each independently any amino acid residue.


As stated above, in some embodiments, a peptidyl linker is present (i.e., made up of amino acids linked together by peptide bonds) that is made in length, preferably, of from 1 up to about 40 amino acid residues, more preferably, of from 1 up to about 20 amino acid residues, and most preferably of from 1 to about 10 amino acid residues. Preferably, but not necessarily, the amino acid residues in the linker are from among the twenty canonical amino acids, more preferably, cysteine, glycine, alanine, proline, asparagine, glutamine, and/or serine. Even more preferably, a peptidyl linker is made up of a majority of amino acids that are sterically unhindered, such as glycine, serine, and alanine linked by a peptide bond. It is also desirable that, if present, a peptidyl linker be selected that avoids rapid proteolytic turnover in circulation in vivo. Thus, preferred linkers include polyglycines (particularly (Gly)4 (SEQ ID NO: 4918), (Gly)5) (SEQ ID NO: 4919), poly(Gly-Ala), and polyalanines. Other preferred linkers are those identified herein as “L5” (GGGGS; SEQ ID NO: 638), “L10” (GGGGSGGGGS; SEQ ID NO:79), “L25” GGGGSGGGGSGGGGSGGGGSGGGGS; SEQ ID NO:84) and any linkers used in the working examples hereinafter. The linkers described herein, however, are exemplary; linkers within the scope of this invention can be much longer and can include other residues.


In some embodiments of the compositions of this invention, which comprise a peptide linker moiety (L), acidic residues, for example, glutamate or aspartate residues, are placed in the amino acid sequence of the linker moiety (L). Examples include the following peptide linker sequences:












GGEGGG;
(SEQ ID NO: 639)







GGEEEGGG;
(SEQ ID NO: 640)







GEEEG;
(SEQ ID NO: 641)







GEEE;
(SEQ ID NO: 642)







GGDGGG;
(SEQ ID NO: 643)







GGDDDGG;
(SEQ ID NO: 644)







GDDDG;
(SEQ ID NO: 645)







GDDD;
(SEQ ID NO: 646)







GGGGSDDSDEGSDGEDGGGGS;
(SEQ ID NO: 647)







WEWEW;
(SEQ ID NO: 648)







FEFEF;
(SEQ ID NO: 649)







EEEWWW;
(SEQ ID NO: 650)







EEEFFF;
(SEQ ID NO: 651)







WWEEEWW;
(SEQ ID NO: 652)



or







FFEEEFF.
(SEQ ID NO: 653)






In other embodiments, the linker constitutes a phosphorylation site, e.g., X1X2YX3X4G (SEQ ID NO: 654), wherein X1, X2, X3 and X4 are each independently any amino acid residue; X1X2SX3X4G (SEQ ID NO: 655), wherein X1, X2, X3 and X4 are each independently any amino acid residue; or X1X2TX3X4G (SEQ ID NO: 656), wherein X1, X2, X3 and X4 are each independently any amino acid residue.


Non-peptide linkers are also possible. For example, alkyl linkers such as —NH—(CH2)s—C(O)—, wherein s=2-20 could be used. These alkyl linkers can further be substituted by any non-sterically hindering group such as lower alkyl (e.g., C1-C6) lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc. An exemplary non-peptide linker is a PEG linker,







wherein n is such that the linker has a molecular weight of 100 to 5000 kDa, preferably 100 to 500 kDa. The peptide linkers can be altered to form derivatives in the same manner as described above.


Useful linker embodiments also include aminoethyloxyethyloxy-acetyl linkers as disclosed by Chandy et al. (Chandy et al., WO 2006/042151 A2, incorporated herein by reference in its entirety).


Derivatives. The inventors also contemplate derivatizing the peptide and/or half-life extending moiety portion of the compounds. Such derivatives can improve the solubility, absorption, biological half-life, and the like of the compounds. The moieties can alternatively eliminate or attenuate any undesirable side-effect of the compounds and the like. Exemplary derivatives include compounds in which:

  • 1. The compound or some portion thereof is cyclic. For example, the peptide portion can be modified to contain two or more Cys residues (e.g., in the linker), which could cyclize by disulfide bond formation.
  • 2. The compound is cross-linked or is rendered capable of cross-linking between molecules. For example, the peptide portion can be modified to contain one Cys residue and thereby be able to form an intermolecular disulfide bond with a like molecule. The compound can also be cross-linked through its C-terminus, as in the molecule shown below.







  • 3. Non-peptidyl linkages (bonds) replace one or more peptidyl [—C(O)NR—] linkages. Exemplary non-peptidyl linkages are —CH2-carbamate [—CH2—OC(O)NR—], phosphonate, —CH2-sulfonamide [—CH2—S(O)2NR—], urea [—NHC(O)NH—], —CH2-secondary amine, and alkylated peptide [—C(O)NR6— wherein R6 is lower alkyl].

  • 4. The N-terminus is chemically derivatized. Typically, the N-terminus can be acylated or modified to a substituted amine. Exemplary N-terminal derivative groups include —NRR1 (other than —NH2), —NRC(O)R1, —NRC(O)OR1, —NRS(O)2R1, —NHC(O)NHR1, succinimide, or benzyloxycarbonyl-NH— (CBZ-NH—), wherein R and R1 are each independently hydrogen or lower alkyl and wherein the phenyl ring can be substituted with 1 to 3 substituents selected from the group consisting of C1-C4 alkyl, C1-C4 alkoxy, chloro, and bromo.

  • 5. The free C-terminus is derivatized. Typically, the C-terminus is esterified or amidated. For example, one can use methods described in the art to add (NH—CH2—CH2—NH2)2 to compounds of this invention having any of SEQ ID NOS: 504 to 508 at the C-terminus. Likewise, one can use methods described in the art to add —NH2 to compounds of this invention having any of SEQ ID NOS: 924 to 955, 963 to 972, 1005 to 1013, or 1018 to 1023 at the C-terminus. Exemplary C-terminal derivative groups include, for example, —C(O)R2 wherein R2 is lower alkoxy or —NR3R4 wherein R3 and R4 are independently hydrogen or C1-C8 alkyl (preferably C1-C4 alkyl).

  • 6. A disulfide bond is replaced with another, preferably more stable, cross-linking moiety (e.g., an alkylene). See, e.g., Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9; Alberts et al. (1993) Thirteenth Am. Pep. Symp., 357-9.

  • 7. One or more individual amino acid residues are modified. Various derivatizing agents are known to react specifically with selected sidechains or terminal residues, as described in detail below.



Lysinyl residues and amino terminal residues can be reacted with succinic or other carboxylic acid anhydrides, which reverse the charge of the lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.


Arginyl residues can be modified by reaction with any one or combination of several conventional reagents, including phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginyl residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents can react with the groups of lysine as well as the arginine epsilon-amino group.


Specific modification of tyrosyl residues has been studied extensively, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.


Carboxyl sidechain groups (aspartyl or glutamyl) can be selectively modified by reaction with carbodiimides (R′—N═C═N—R′) such as 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues can be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.


Glutaminyl and asparaginyl residues can be deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.


Cysteinyl residues can be replaced by amino acid residues or other moieties either to eliminate disulfide bonding or, conversely, to stabilize cross-linking. See, e.g., Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9.


Derivatization with bifunctional agents is useful for cross-linking the peptides or their functional derivatives to a water-insoluble support matrix or to other macromolecular half-life extending moieties. Commonly used cross-linking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.


Carbohydrate (oligosaccharide) groups can conveniently be attached to sites that are known to be glycosylation sites in proteins. Generally, O-linked oligosaccharides are attached to serine (Ser) or threonine (Thr) residues while N-linked oligosaccharides are attached to asparagine (Asn) residues when they are part of the sequence Asn-X-Ser/Thr, where X can be any amino acid except proline. X is preferably one of the 19 naturally occurring amino acids other than proline. The structures of N-linked and O-linked oligosaccharides and the sugar residues found in each type are different. One type of sugar that is commonly found on both is N-acetylneuraminic acid (referred to as sialic acid). Sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides and, by virtue of its negative charge, can confer acidic properties to the glycosylated compound. Such site(s) can be incorporated in the linker of the compounds of this invention and are preferably glycosylated by a cell during recombinant production of the polypeptide compounds (e.g., in mammalian cells such as CHO, BHK, COS). However, such sites can further be glycosylated by synthetic or semi-synthetic procedures known in the art.


Other possible modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, oxidation of the sulfur atom in Cys, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains. Creighton, Proteins: Structure and Molecule Properties (W. H. Freeman and Co., San Francisco), pp. 79-86 (1983).


Compounds of the present invention can be changed at the DNA level, as well. The DNA sequence of any portion of the compound can be changed to codons more compatible with the chosen host cell. For E. coli, which is the preferred host cell, optimized codons are known in the art. Codons can be substituted to eliminate restriction sites or to include silent restriction sites, which can aid in processing of the DNA in the selected host cell. The half-life extending moiety, linker and peptide DNA sequences can be modified to include any of the foregoing sequence changes.


A process for preparing conjugation derivatives is also contemplated. Tumor cells, for example, exhibit epitopes not found on their normal counterparts. Such epitopes include, for example, different post-translational modifications resulting from their rapid proliferation. Thus, one aspect of this invention is a process comprising:

    • a) selecting at least one randomized peptide that specifically binds to a target epitope; and
    • b) preparing a pharmacologic agent comprising (i) at least one half-life extending moiety (Fc domain preferred), (ii) at least one amino acid sequence of the selected peptide or peptides, and (iii) an effector molecule. The target epitope is preferably a tumor-specific epitope or an epitope specific to a pathogenic organism. The effector molecule can be any of the above-noted conjugation partners and is preferably a radioisotope.


Methods of Making


The present invention also relates to nucleic acids, expression vectors and host cells useful in producing the polypeptides of the present invention. Host cells can be eukaryotic cells, with mammalian cells preferred and CHO cells most preferred. Host cells can also be prokaryotic cells, with E. coli cells most preferred.


The compounds of this invention largely can be made in transformed host cells using recombinant DNA techniques. To do so, a recombinant DNA molecule coding for the peptide is prepared. Methods of preparing such DNA molecules are well known in the art. For instance, sequences coding for the peptides could be excised from DNA using suitable restriction enzymes. Alternatively, the DNA molecule could be synthesized using chemical synthesis techniques, such as the phosphoramidate method. Also, a combination of these techniques could be used.


The invention also includes a vector capable of expressing the peptides in an appropriate host. The vector comprises the DNA molecule that codes for the peptides operatively linked to appropriate expression control sequences. Methods of effecting this operative linking, either before or after the DNA molecule is inserted into the vector, are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosomal binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved with the control of transcription or translation.


The resulting vector having the DNA molecule thereon is used to transform an appropriate host. This transformation can be performed using methods well known in the art.


Any of a large number of available and well-known host cells can be used in the practice of this invention. The selection of a particular host is dependent upon a number of factors recognized by the art. These include, for example, compatibility with the chosen expression vector, toxicity of the peptides encoded by the DNA molecule, rate of transformation, ease of recovery of the peptides, expression characteristics, bio-safety and costs. A balance of these factors must be struck with the understanding that not all hosts can be equally effective for the expression of a particular DNA sequence. Within these general guidelines, useful microbial hosts include bacteria (such as E. coli sp.), yeast (such as Saccharomyces sp.) and other fungi, insects, plants, mammalian (including human) cells in culture, or other hosts known in the art.


Next, the transformed host is cultured and purified. Host cells can be cultured under conventional fermentation conditions so that the desired compounds are expressed. Such fermentation conditions are well known in the art. Finally, the peptides are purified from culture by methods well known in the art.


In some embodiments of the inventive DNA, the DNA encodes a recombinant fusion protein composition of the invention, preferably, but not necessarily, monovalent with respect to the toxin peptide, for expression in a mammalian cell, such as, but not limited to, CHO or HEK293. The encoded fusion protein includes (a)-(c) immediately below, in the N-terminal to C-terminal direction:


(a) an immunoglobulin, which includes the constant and variable regions of the immunoglobulin light and heavy chains, or a portion of an immunoglobulin (e.g., an Fc domain, or the variable regions of the light and heavy chains); if both immunoglobulin light chain and heavy chain components are to be included in the construct, then a peptidyl linker, as further described in (b) immediately below, is also included to separate the immunoglobulin components (See, e.g., FIG. 92A-C); useful coding sequences for immunoglobulin light and heavy chains are well known in the art;


(b) a peptidyl linker, which is at least 4 (or 5) amino acid residues long and comprises at least one protease cleavage site (e.g., a furin cleavage site, which is particularly useful for intracellular cleavage of the expressed fusion protein); typically, the peptidyl linker sequence can be up to about 35 to 45 amino acid residues long (e.g., a 7×L5 linker modified to include the desired protease cleavage site(s)), but linkers up to about 100 to about 300 amino acid residues long are also useful; and


(c) an immunoglobulin Fc domain or a portion thereof. The Fc domain of (c) can be from the same type of immunoglobulin in (a), or different. In such embodiments, the DNA encodes a toxin peptide covalently linked to the N-terminal or C-terminal end of (a) or (c) above, either directly or indirectly via a peptidyl linker (a linker minus a protease cleavage site). Any toxin peptide or peptide analog thereof as described herein can be encoded by the DNA (e.g., but not limited to, ShK, HmK, MgTx, AgTx1, AgTx2, HsTx1, OSK1, Anuroctoxin, Noxiustoxin, Hongotoxin, HsTx1, ChTx, MTx, Titystoxin, BgK, BmKTX, BmTx, Tc30, Tc32, Pi1, Pi2, Pi3 toxin peptide, or a peptide analog of any of these). For example, an OSK1 peptide analog comprising an amino acid sequence selected from SEQ ID NOS: 25, 294 through 298, 562 through 636, 980 through 1274, 1303, 1308, 1391 through 4912, 4916, 4920 through 5006, 5009, 5010, and 5012 through 5015, as set forth in Tables 7 and Tables 7A-J, can be employed. Alternatively, an ShK peptide analog comprising an amino acid sequence selected from SEQ ID NOS: 5, 88 through 200, 548 through 561, 884 through 950, and 1295 through 1300 as set forth in Table 2, can be employed. Any other toxin peptide sequence described herein that can alternatively be expressed recombinantly using recombinant and protein engineering techniques known in the art can also be used. The immunoglobulin of (a) and (c) above can be in each instance independently selected from any desired type, such as but not limited to, IgG1, IgG2, IgG3, and IgG4. The variable regions can be non-functional in vivo (e.g., CDRs specifically binding KLH), or alternatively, if targeting enhancement function is also desired, the variable regions can be chosen to specifically bind (non-competitively) the ion channel target of the toxin peptide (e.g., Kv1.3) or specifically bind another antigen typically found associated with, or in the vicinity of, the target ion channel. In addition, the inventive DNA optionally further encodes, 5′ to the coding region of (a) above, a signal peptide sequence (e.g., a secretory signal peptide) operably linked to the expressed fusion protein. An example of the inventive DNA encoding a recombinant fusion protein for expression in a mammalian cell, described immediately above, is a DNA that encodes a fusion protein comprising, in the N-terminal to C-terminal direction:

    • (a) an immunoglobulin light chain;
    • (b) a first peptidyl linker at least 4 amino acid residues long comprising at least one protease cleavage site, as described above;
    • (c) an immunoglobulin heavy chain;
    • (d) a second peptidyl linker at least 4 amino acid residues long comprising at least one protease cleavage site, as described above; and
    • (e) an immunoglobulin Fc domain or a portion thereof. Here, the Fc domain of (e) can be from the same type of immunoglobulin as the heavy chain in (c), or different. The DNA encodes a toxin peptide covalently linked to the N-terminal or C-terminal end of (a), (c), or (e) of the expressed fusion protein, either directly or indirectly via a peptidyl linker (a linker minus a protease cleavage site). FIG. 92A-C illustrates schematically an embodiment, in which the toxin peptide (e.g., an OSK1, ShK, or a peptide analog of either of these) is covalently linked to the C-terminal end of the Fc domain of (e). In FIG. 92A-C, a linker is shown covalently linking the toxin peptide to the rest of the molecule, but as previously described, this linker is optional.


In some embodiments particularly suited for the recombinant expression of monovalent dimeric Fc-toxin peptide fusions or “peptibodies” (see, FIG. 2B and Example 56) by mammalian cells, such as, but not limited to, CHO or HEK293, the inventive DNA encodes a recombinant expressed fusion protein that comprises, in the N-terminal to C-terminal direction:

    • (a) a first immunoglobulin Fc domain or portion thereof;
    • (b) a peptidyl linker at least 4 (or 5) amino acid residues long comprising at least one protease cleavage site (e.g., a furin cleavage site, which is particularly useful for intracellular cleavage of the expressed fusion protein); typically, the peptidyl linker sequence can be up to about 35 to 45 amino acid residues long (e.g., a 7×L5 linker modified to include the desired protease cleavage site(s)), but linkers up to about 100 to about 300 amino acid residues long are also useful; and
    • (c) a second immunoglobulin Fc domain or portion thereof (which may be the same or different from the first Fc domain, but should be expressed in the same orientation as the first Fc domain).


      For such embodiments, the DNA encodes a toxin peptide covalently linked to the N-terminal or C-terminal end of (a) or (c) of the expressed fusion protein, either directly or indirectly via a peptidyl linker (a linker minus a protease cleavage site); Example 56 describes an embodiment in which the toxin peptide is conjugated to the C-terminal end of the second immunoglobulin Fc domain (c). Any toxin peptide or peptide analog thereof as described herein can be encoded by the DNA (e.g., but not limited to, ShK, HmK, MgTx, AgTx1, AgTx2, HsTx1, OSK1, Anuroctoxin, Noxiustoxin, Hongotoxin, HsTx1, ChTx, MTx, Titystoxin, BgK, BmKTX, BmTx, Tc30, Tc32, Pi1, Pi2, Pi3 toxin peptide, or a peptide analog of any of these). For example, an OSK1 peptide analog comprising an amino acid sequence selected from SEQ ID NOS: 25, 294 through 298, 562 through 636, 980 through 1274, 1303, 1308, 1391 through 4912, 4916, 4920 through 5006, 5009, 5010, and 5012 through 5015, as set forth in Tables 7 and Tables 7A-J, can be employed. Alternatively, an ShK peptide analog comprising an amino acid sequence selected from SEQ ID NOS: 5, 88 through 200, 548 through 561, 884 through 950, and 1295 through 1300 as set forth in Table 2, can be employed. Any other toxin peptide sequence described herein that can alternatively be expressed using recombinant and protein engineering techniques known in the art can also be used. In addition, the inventive DNA optionally further encodes, 5′ to the coding region of (a) above, a signal peptide sequence (e.g., a secretory signal peptide) operably linked to the expressed fusion protein.


DNA constructs similar to those described above are also useful for recombinant expression by mammalian cells of other dimeric Fc fusion proteins (“peptibodies”) or chimeric immunoglobulin (light chain+heavy chain)-Fc heterotrimers (“hemibodies”), conjugated to pharmacologically active peptides (e.g., agonist or antagonist peptides) other than toxin peptides.


Peptide compositions of the present invention can also be made by synthetic methods. Solid phase synthesis is the preferred technique of making individual peptides since it is the most cost-effective method of making small peptides. For example, well known solid phase synthesis techniques include the use of protecting groups, linkers, and solid phase supports, as well as specific protection and deprotection reaction conditions, linker cleavage conditions, use of scavengers, and other aspects of solid phase peptide synthesis. Suitable techniques are well known in the art. (E.g., Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.); Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985), Biochem. Intl. 10: 394-414; Stewart and Young (1969), Solid Phase Peptide Synthesis; U.S. Pat. No. 3,941,763; Finn et al. (1976), The Proteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins (3rd ed.) 2: 257-527; “Protecting Groups in Organic Synthesis,” 3rd Edition, T. W. Greene and P. G. M. Wuts, Eds., John Wiley & Sons, Inc., 1999; NovaBiochem Catalog, 2000; “Synthetic Peptides, A User's Guide,” G. A. Grant, Ed., W.H. Freeman & Company, New York, N.Y., 1992; “Advanced Chemtech Handbook of Combinatorial & Solid Phase Organic Chemistry,” W. D. Bennet, J. W. Christensen, L. K. Hamaker, M. L. Peterson, M. R. Rhodes, and H. H. Saneii, Eds., Advanced Chemtech, 1998; “Principles of Peptide Synthesis, 2nd ed.,” M. Bodanszky, Ed., Springer-Verlag, 1993; “The Practice of Peptide Synthesis, 2nd ed.,” M. Bodanszky and A. Bodanszky, Eds., Springer-Verlag, 1994; “Protecting Groups,” P. J. Kocienski, Ed., Georg Thieme Verlag, Stuttgart, Germany, 1994; “Fmoc Solid Phase Peptide Synthesis, A Practical Approach,” W. C. Chan and P. D. White, Eds., Oxford Press, 2000, G. B. Fields et al., Synthetic Peptides: A User's Guide, 1990, 77-183).


Whether the compositions of the present invention are prepared by synthetic or recombinant techniques, suitable protein purification techniques can also be involved, when applicable. In some embodiments of the compositions of the invention, the toxin peptide portion and/or the half-life extending portion, or any other portion, can be prepared to include a suitable isotopic label (e.g., 125I, 14C, 13C, 35S, 3H, 2H, 13N, 15N, 15O, 17O, etc.), for ease of quantification or detection.


Compounds that contain derivatized peptides or which contain non-peptide groups can be synthesized by well-known organic chemistry techniques.


Uses of the Compounds


In general. The compounds of this invention have pharmacologic activity resulting from their ability to bind to proteins of interest as agonists, mimetics or antagonists of the native ligands of such proteins of interest. Heritable diseases that have a known linkage to ion channels (“channelopathies”) cover various fields of medicine, some of which include neurology, nephrology, myology and cardiology. A list of inherited disorders attributed to ion channels includes:

    • cystic fibrosis (Cl channel; CFTR),
    • Dent's disease (proteinuria and hypercalciuria; Cl channel; CLCN5),
    • osteopetrosis (Cl channel; CLCN7),
    • familial hyperinsulinemia (SUR1; KCNJ11; K channel),
    • diabetes (KATP/SUR channel),
    • Andersen syndrome (KCNJ2, Kir2.1 K channel),
    • Bartter syndrome (KCNJ1; Kir1.1/ROMK; K channel),
    • hereditary hearing loss (KCNQ4; K channel),
    • hereditary hypertension (Liddle's syndrome; SCNN1; epithelial Na channel),
    • dilated cardiomyopathy (SUR2, K channel),
    • long-QT syndrome or cardiac arrhythmias (cardiac potassium and sodium channels),
    • Thymothy syndrome (CACNA1C, Cav1.2),
    • myasthenic syndromes (CHRNA, CHRNB, CNRNE; nAChR), and a variety of other myopathies,
    • hyperkalemic periodic paralysis (Na and K channels),
    • epilepsy (Na+ and K+ channels),
    • hemiplegic migraine (CACNA1A, Cav2.1 Ca2+ channel and ATP1A2),
    • central core disease (RYR1, RYR1; Ca2+ channel), and
    • paramyotonia and myotonia (Na+, Cl channels)


      See L. J. Ptacek and Y—H Fu (2004), Arch. Neurol. 61: 166-8; B. A. Niemeyer et al. (2001), EMBO reports 21: 568-73; F. Lehmann-Horn and K. Jurkat-Rott (1999), Physiol. Rev. 79: 1317-72. Although the foregoing list concerned disorders of inherited origin, molecules targeting the channels cited in these disorders can also be useful in treating related disorders of other, or indeterminate, origin.


In addition to the aforementioned disorders, evidence has also been provided supporting ion channels as targets for treatment of:

    • sickle cell anemia (IKCa1)—in sickle cell anemia, water loss from erythrocytes leads to hemoglobin polymerization and subsequent hemolysis and vascular obstruction. The water loss is consequent to potassium efflux through the so-called Gardos channel i.e., IKCa1. Therefore, block of IKCa1 is a potential therapeutic treatment for sickle cell anemia.
    • glaucoma (BKCa),—in glaucoma the intraocular pressure is too high leading to optic nerve damage, abnormal eye function and possibly blindness. Block of BKCa potassium channels can reduce intraocular fluid secretion and increase smooth muscle contraction, possibly leading to lower intraocular pressure and neuroprotection in the eye.
    • multiple sclerosis (Kv, KCa),
    • psoriasis (Kv, KCa),
    • arthritis (Kv, KCa),
    • asthma (KCa, Kv),
    • allergy (KCa, Kv),
    • COPD (KCa, Kv, Ca),
    • allergic rhinitis (KCa, Kv),
    • pulmonary fibrosis,
    • lupus (IKCa1, Kv),
    • transplantation, GvHD (KCa, Kv),
    • inflammatory bone resorption (KCa, Kv),
    • periodontal disease (KCa, Kv),
    • diabetes, type I (Kv),—type I diabetes is an autoimmune disease that is characterized by abnormal glucose, protein and lipid metabolism and is associated with insulin deficiency or resistance. In this disease, Kv1.3-expressing T-lymphocytes attack and destroy pancreatic islets leading to loss of beta-cells. Block of Kv1.3 decreases inflammatory cytokines. In addition block of Kv1.3 facilitates the translocation of GLUT4 to the plasma membrane, thereby increasing insulin sensitivity.
    • obesity (Kv),—Kv1.3 appears to play a critical role in controlling energy homeostasis and in protecting against diet-induced obesity. Consequently, Kv1.3 blockers could increase metabolic rate, leading to greater energy utilization and decreased body weight.
    • restenosis (KCa, Ca2+),—proliferation and migration of vascular smooth muscle cells can lead to neointimal thickening and vascular restenosis. Excessive neointimal vascular smooth muscle cell proliferation is associated with elevated expression of IKCa1. Therefore, block of IKCa1 could represent a therapeutic strategy to prevent restenosis after angioplasty.
    • ischaemia (KCa, Ca2+),—in neuronal or cardiac ischemia, depolarization of cell membranes leads to opening of voltage-gated sodium and calcium channels. In turn this can lead to calcium overload, which is cytotoxic. Block of voltage-gated sodium and/or calcium channels can reduce calcium overload and provide cytoprotective effects. In addition, due to their critical role in controlling and stabilizing cell membrane potential, modulators of voltage- and calcium-activated potassium channels can also act to reduce calcium overload and protect cells.
    • renal incontinence (KCa), renal incontinence is associated with overactive bladder smooth muscle cells. Calcium-activated potassium channels are expressed in bladder smooth muscle cells, where they control the membrane potential and indirectly control the force and frequency of cell contraction. Openers of calcium-activated potassium channels therefore provide a mechanism to dampen electrical and contractile activity in bladder, leading to reduced urge to urinate.
    • osteoporosis (Kv),
    • pain, including migraine (Nav, TRP [transient receptor potential channels], P2X, Ca2+), N-type voltage-gated calcium channels are key regulators of nociceptive neurotransmission in the spinal cord. Ziconotide, a peptide blocker of N-type calcium channels reduces nociceptive neurotransmission and is approved worldwide for the symptomatic alleviation of severe chronic pain in humans. Novel blockers of nociceptor-specific N-type calcium channels would be improved analgesics with reduced side-effect profiles.
    • hypertension (Ca2+),—L-type and T-type voltage-gated calcium channels are expressed in vascular smooth muscle cells where they control excitation-contraction coupling and cellular proliferation. In particular, T-type calcium channel activity has been linked to neointima formation during hypertension. Blockers of L-type and T-type calcium channels are useful for the clinical treatment of hypertension because they reduce calcium influx and inhibit smooth muscle cell contraction.
    • wound healing, cell migration serves a key role in wound healing. Intracellular calcium gradients have been implicated as important regulators of cellular migration machinery in keratinocytes and fibroblasts. In addition, ion flux across cell membranes is associated with cell volume changes. By controlling cell volume, ion channels contribute to the intracellular environment that is required for operation of the cellular migration machinery. In particular, IKCa1 appears to be required universally for cell migration. In addition, Kv1.3, Kv3.1, NMDA receptors and N-type calcium channels are associated with the migration of lymphocytes and neurons.
    • stroke,
    • Alzheimer's,
    • Parkenson's Disease (nACHR, Nav)
    • Bipolar Disorder (Nav, Cav)
    • cancer, many potassium channel genes are amplified and protein subunits are upregulated in many cancerous condition. Consistent with a pathophysiological role for potassium channel upregulation, potassium channel blockers have been shown to suppress proliferation of uterine cancer cells and hepatocarcinoma cells, presumably through inhibition of calcium influx and effects on calcium-dependent gene expression.
    • a variety of neurological, cardiovascular, metabolic and autoimmune diseases.


Both agonists and antagonists of ion channels can achieve therapeutic benefit. Therapeutic benefits can result, for example, from antagonizing Kv1.3, IKCa1, SKCa, BKCa, N-type or T-type Ca2+ channels and the like. Small molecule and peptide antagonists of these channels have been shown to possess utility in vitro and in vivo. Limitations in production efficiency and pharmacokinetics, however, have largely prevented clinical investigation of inhibitor peptides of ion channels.


Compositions of this invention incorporating peptide antagonists of the voltage-gated potassium channel Kv1.3, in particular OSK1 peptide analogs, whether or not conjugated to a half-life extending moiety, are useful as immunosuppressive agents with therapeutic value for autoimmune diseases. For example, such molecules are useful in treating multiple sclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease, and rheumatoid arthritis. (See, e.g., H. Wulff et al. (2003) J. Clin. Invest. 111, 1703-1713 and H. Rus et al. (2005) PNAS 102, 11094-11099; Beeton et al., Targeting effector memory T cells with a selective inhibitor peptide of Kv1.3 channels for therapy of autoimmune diseases, Molec. Pharmacol. 67(4):1369-81 (2005); 1 Beeton et al. (2006), Kv1.3: therapeutic target for cell-mediated autoimmune disease, electronic preprint at //webfiles.uci.edu/xythoswfs/webui/2670029.1). Inhibitors of the voltage-gated potassium channel Kv1.3 have been examined in a variety of preclinical animal models of inflammation. Small molecule and peptide inhibitors of Kv1.3 have been shown to block delayed type hypersensitivity responses to ovalbumin [C. Beeton et al. (2005) Mol. Pharmacol. 67, 1369] and tetanus toxoid [G. C. Koo et al. (1999) Clin. Immunol. 197, 99]. In addition to suppressing inflammation in the skin, inhibitors also reduced antibody production [G. C. Koo et al. (1997) J. Immunol. 158, 5120]. Kv1.3 antagonists have shown efficacy in a rat adoptive-transfer experimental autoimmune encephalomyelitis (AT-EAE) model of multiple sclerosis (MS). The Kv1.3 channel is overexpressed on myelin-specific T cells from MS patients, lending further support to the utility Kv1.3 inhibitors may provide in treating MS. Inflammatory bone resorption was also suppressed by Kv1.3 inhibitors in a preclinical adoptive-transfer model of periodontal disease [P. Valverde et al. (2004) J. Bone Mineral Res. 19, 155]. In this study, inhibitors additionally blocked antibody production to a bacterial outer membrane protein,—one component of the bacteria used to induce gingival inflammation. Recently in preclinical rat models, efficacy of Kv1.3 inhibitors was shown in treating pristane-induced arthritis and diabetes [C. Beeton et al. (2006) preprint available at //webfiles.uci.edu/xythoswfs/webui/_xy-26700291.]. The Kv1.3 channel is expressed on all subsets of T cells and B cells, but effector memory T cells and class-switched memory B cells are particularly dependent on Kv1.3 [H. Wulff et al. (2004) J. Immunol. 173, 776]. Gad5/insulin-specific T cells from patients with new onset type 1 diabetes, myelin-specific T cells from MS patients and T cells from the synovium of rheumatoid arthritis patients all overexpress Kv1.3 [C. Beeton et al. (2006) preprint at //webfiles.uci.edu/xythoswfs/webui/_xy-26700291.]. Because mice deficient in Kv1.3 gained less weight when placed on a high fat diet [J. Xu et al. (2003) Human Mol. Genet. 12, 551] and showed altered glucose utilization [J. Xu et al. (2004) Proc. Natl. Acad. Sci. 101, 3112], Kv1.3 is also being investigated for the treatment of obesity and diabetes. Breast cancer specimens [M. Abdul et al. (2003) Anticancer Res. 23, 3347] and prostate cancer cell lines [S. P. Fraser et al. (2003) Pflugers Arch. 446, 559] have also been shown to express Kv1.3, and Kv1.3 blockade may be of utility for treatment of cancer. Disorders that can be treated in accordance with the inventive method of treating an autoimmune disorder, involving Kv1.3 inhibitor toxin peptide(s), include multiple sclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease, contact-mediated dermatitis, rheumatoid arthritis, psoriatic arthritis, asthma, allergy, restinosis, systemic sclerosis, fibrosis, scleroderma, glomerulonephritis, Sjogren syndrome, inflammatory bone resorption, transplant rejection, graft-versus-host disease, and systemic lupus erythematosus (SLE) and other forms of lupus.


Some of the cells that express the calcium-activated potassium of intermediate conductance IKCa1 include T cells, B cells, mast cells and red blood cells (RBCs). T cells and RBCs from mice deficient in IKCa1 show defects in volume regulation [T. Begenisich et al. (2004) J. Biol. Chem. 279, 47681]. Preclinical and clinical studies have demonstrated IKCa1 inhibitors utility in treating sickle cell anemia [J. W. Stocker et al. (2003) Blood 101, 2412; www.icagen.com]. Blockers of the IKCa1 channel have also been shown to block EAE, indicating they may possess utility in treatment of MS [E. P. Reich et al. (2005) Eur. J. Immunol. 35, 1027]. IgE-mediated histamine production from mast cells is also blocked by IKCa1 inhibitors [S. Mark Duffy et al. (2004) J. Allergy Clin. Immunol. 114, 66], therefore they may also be of benefit in treating asthma. The IKCa1 channel is overexpressed on activated T and B lymphocytes [H. Wulff et al. (2004) J. Immunol. 173, 776] and thus may show utility in treatment of a wide variety of immune disorders. Outside of the immune system, IKCa1 inhibitors have also shown efficacy in a rat model of vascular restinosis and thus might represent a new therapeutic strategy to prevent restenosis after angioplasty [R. Kohler et al. (2003) Circulation 108, 1119]. It is also thought that IKCa1 antagonists are of utility in treatment of tumor angiogenesis since inhibitors suppressed endothelial cell proliferation and angionenesis in vivo [I. Grgic et al. (2005) Arterioscler. Thromb. Vasc. Biol. 25, 704]. The IKCa1 channel is upregulated in pancreatic tumors and inhibitors blocked proliferation of pancreatic tumor cell lines [H. Jager et al. (2004) Mol Pharmacol. 65, 630]. IKCa1 antagonists may also represent an approach to attenuate acute brain damage caused by traumatic brain injury [F. Mauler (2004) Eur. J. Neurosci. 20, 1761]. Disorders that can be treated with IKCa1 inhibitors include multiple sclerosis, asthma, psoriasis, contact-mediated dermatitis, rheumatoid & psoriatic arthritis, inflammatory bowel disease, transplant rejection, graft-versus-host disease, Lupus, restinosis, pancreatic cancer, tumor angiogenesis and traumatic brain injury.


Accordingly, molecules of this invention incorporating peptide antagonists of the calcium-activated potassium channel of intermediate conductance, IKCa can be used to treat immune dysfunction, multiple sclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease, contact-mediated dermatitis, rheumatoid arthritis, psoriatic arthritis, asthma, allergy, restinosis, systemic sclerosis, fibrosis, scleroderma, glomerulonephritis, Sjogren syndrome, inflammatory bone resorption, transplant rejection, graft-versus-host disease, and lupus.


Accordingly, the present invention includes a method of treating an autoimmune disorder, which involves administering to a patient who has been diagnosed with an autoimmune disorder, such as multiple sclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease, contact-mediated dermatitis, rheumatoid arthritis, psoriatic arthritis, asthma, allergy, restinosis, systemic sclerosis, fibrosis, scleroderma, glomerulonephritis, Sjogren syndrome, inflammatory bone resorption, transplant rejection, graft-versus-host disease, or lupus, a therapeutically effective amount of the inventive composition of matter, whereby at least one symptom of the disorder is alleviated in the patient. “Alleviated” means to be lessened, lightened, diminished, softened, mitigated (i.e., made more mild or gentle), quieted, assuaged, abated, relieved, nullified, or allayed, regardless of whether the symptom of interest is entirely erased, eradicated, eliminated, or prevented in a particular patient.


The present invention is further directed to a method of preventing or mitigating a relapse of a symptom of multiple sclerosis, which method involves administering to a patient, who has previously experienced at least one symptom of multiple sclerosis, a prophylactically effective amount of the inventive composition of matter, such that the at least one symptom of multiple sclerosis is prevented from recurring or is mitigated.


The inventive compositions of matter preferred for use in practicing the inventive method of treating an autoimmune disorder, e.g., inflammatory bowel disease (IBD, including Crohn's Disease and ulcerative colitis), and the method of preventing or mitigating a relapse of a symptom of multiple sclerosis include as P (conjugated as in Formula I), a Kv1.3 or IKCa1 antagonist peptide, such as a ShK peptide, an OSK1 peptide or an OSK1 peptide analog, a ChTx peptide and/or a Maurotoxin (MTx) peptide, or peptide analogs of any of these.


For example, the conjugated ShK peptide or ShK peptide analog can comprise an amino acid sequence selected from the following:

    • SEQ ID NOS: 5, 88 through 200, 548 through 561, 884 through 950, or 1295 through 1300 as set forth in Table 2.


The conjugated OSK1 peptide, or conjugated or unconjugated OSK1 peptide analog, can comprise an amino acid sequence selected from the following:

    • SEQ ID NOS: 25, 294 through 298, 562 through 636, 980 through 1274, GVIINVSCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK (OSK1-S7) (SEQ ID NO: 1303), or GVIINVSCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK (OSK1-S7,K16,D20) (SEQ ID NO: 1308) as set forth in Table 7, or any of SEQ ID NOS: 1391 through 4912, 4916, 4920 through 5006, 5009, 5010, and 5012 through 5015 as set forth in Table 7A, Table 7B, Table 7C, Table 7D, Table 7E, Table 7F, Table 7G, Table 7H, Table 7I, or Table 7J.


Also by way of example, a the conjugated MTX peptide, MTX peptide analog, ChTx peptide or ChTx peptide analog can comprise an amino acid sequence selected from:

    • SEQ ID NOS: 20, 330 through 343, 1301, 1302, 1304 through 1307, 1309, 1311, 1312, or 1315 through 1336 as set forth in Table 13; or SEQ ID NOS: 36, 59, 344 through 346, or 1369 through 1390 as set forth in Table 14.


Also useful in these methods conjugated, or unconjugated, are a Kv1.3 or IKCa1 inhibitor toxin peptide analog that comprises an amino acid sequence selected from:

    • SEQ ID NOS: 88, 89, 92, 148 through 200, 548 through 561, 884 through 949, or 1295 through 1300 as set forth in Table 2; or
    • SEQ ID NOS: 980 through 1274, GVIINVSCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK (OSK1-S7) (SEQ ID NO: 1303), or GVIINVSCKISRQCLKPCKDAGMRFGKCMNGKCHCTPK (OSK1-S7,K16,D20) (SEQ ID NO: 1308) as set forth in Table 7; or
    • SEQ ID NOS: 330 through 337, 341, 1301, 1302, 1304 through 1307, 1309, 1311, 1312, and 1315 through 1336 as set forth in Table 13.


In accordance with these inventive methods, a patient who has been diagnosed with an autoimmune disorder, such as, but not limited to multiple sclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease, contact-mediated dermatitis, rheumatoid arthritis, psoratic arthritis, asthma, allergy, restinosis, systemic sclerosis, fibrosis, scleroderma, glomerulonephritis, Sjogren syndrome, inflammatory bone resorption, transplant rejection, graft-versus-host disease, or lupus, or a patient who has previously experienced at least one symptom of multiple sclerosis, are well-recognizable and/or diagnosed by the skilled practitioner, such as a physician, familiar with autoimmune disorders and their symptoms.

    • For example, symptoms of multiple sclerosis can include the following: visual symptoms, such as, optic neuritis (blurred vision, eye pain, loss of color vision, blindness); diplopia (double vision); nystagmus (jerky eye movements); ocular dysmetria (constant under- or overshooting eye movements); internuclear opthalmoplegia (lack of coordination between the two eyes, nystagmus, diplopia); movement and sound phosphenes (flashing lights when moving eyes or in response to a sudden noise); afferent pupillary defect (abnormal pupil responses);
    • motor symptoms, such as, paresis, monoparesis, paraparesis, hemiparesis, quadraparesis (muscle weakness—partial or mild paralysis); plegia, paraplegia, hemiplegia, tetraplegia, quadraplegia (paralysis—total or near total loss of muscle strength); spasticity (loss of muscle tone causing stiffness, pain and restricting free movement of affected limbs); dysarthria (slurred speech and related speech problems); muscle atrophy (wasting of muscles due to lack of use); spasms, cramps (involuntary contraction of muscles); hypotonia, clonus (problems with posture); myoclonus, myokymia (jerking and twitching muscles, tics); restless leg syndrome (involuntary leg movements, especially bothersome at night); footdrop (foot drags along floor during walking); dysfunctional reflexes (MSRs, Babinski's, Hoffman's, Chaddock's);
    • sensory symptoms, such as, paraesthesia (partial numbness, tingling, buzzing and vibration sensations); anaesthesia (complete numbness/loss of sensation); neuralgia, neuropathic and neurogenic pain (pain without apparent cause, burning, itching and electrical shock sensations); L'Hermitte's (electric shocks and buzzing sensations when moving head); proprioceptive dysfunction (loss of awareness of location of body parts); trigeminal neuralgia (facial pain);
    • coordination and balance symptoms, such as, ataxia (loss of coordination); intention tremor (shaking when performing fine movements); dysmetria (constant under- or overshooting limb movements); vestibular ataxia (abnormal balance function in the inner ear); vertigo (nausea/vomitting/sensitivity to travel sickness from vestibular ataxia); speech ataxia (problems coordinating speech, stuttering); dystonia (slow limb position feedback); dysdiadochokinesia (loss of ability to produce rapidly alternating movements, for example to move to a rhythm);
    • bowel, bladder and sexual symptoms, such as, frequent micturation, bladder spasticity (urinary urgency and incontinence); flaccid bladder, detrusor-sphincter dyssynergia (urinary hesitancy and retention); erectile dysfunction (male and female impotence); anorgasmy (inability to achieve orgasm); retrograde ejaculation (ejaculating into the bladder); frigidity (inability to become sexually aroused); constipation (infrequent or irregular bowel movements); fecal urgency (bowel urgency); fecal incontinence (bowel incontinence);
    • cognitive symptoms, such as, depression; cognitive dysfunction (short-term and long-term memory problems, forgetfulness, slow word recall); dementia; mood swings, emotional lability, euphoria; bipolar syndrome; anxiety; aphasia, dysphasia (impairments to speech comprehension and production); and
    • other symptoms, such as, fatigue; Uhthoff's Symptom (increase in severity of symptoms with heat); gastroesophageal reflux (acid reflux); impaired sense of taste and smell; epileptic seizures; swallowing problems, respiratory problems; and sleeping disorders.


By way of further example, symptoms of inflammatory bowel disease can include the following symptoms of Crohn's Disease or ulcerative colitis:


A. Symptoms of Crohn's disease can include:

    • Abdominal pain. The pain often is described as cramping and intermittent, and the abdomen may be sore when touched. Abdominal pain may turn to a dull, constant ache as the condition progresses.
    • Diarrhea. Some patients may have diarrhea 10 to 20 times per day. They may wake up at night and need to go to the bathroom. Crohn's disease may cause blood in stools, but not always.
    • Loss of appetite.
    • Fever. In severe cases, fever or other symptoms that affect the entire body may develop. A high fever may indicate a complication involving infection, such as an abscess.
    • Weight loss. Ongoing symptoms, such as diarrhea, can lead to weight loss. Too few red blood cells (anemia). Some patients with Crohn's disease develop anemia because of low iron levels caused by bloody stools or the intestinal inflammation itself.


B. The symptoms of ulcerative colitis may include:

    • Diarrhea or rectal urgency. Some patients may have diarrhea 10 to 20 times per day. The urge to defecate may wake patients at night.
    • Rectal bleeding. Ulcerative colitis usually causes bloody diarrhea and mucus. Patients also may have rectal pain and an urgent need to empty the bowels.
    • Abdominal pain, often described as cramping. The patient's abdomen may be sore when touched.
    • Constipation. This symptom may develop depending on what part of the colon is affected.
    • Loss of appetite.
    • Fever. In severe cases, fever or other symptoms that affect the entire body may develop.
    • Weight loss. Ongoing (chronic) symptoms, such as diarrhea, can lead to weight loss.
    • Too few red blood cells (anemia). Some patients develop anemia because of low iron levels caused by bloody stools or intestinal inflammation.


The symptoms of multiple sclerosis and inflammatory bowel disease (including Crohn's Disease and ulcerative colitis) enumerated above, are merely illustrative and are not intended to be an exhaustive description of all possible symptoms experienced by a single patient or by several sufferers in composite, and to which the present invention is directed. Those skilled in the art are aware of various clinical symptoms and constellations of symptoms of autoimmune disorders suffered by individual patients, and to those symptoms are also directed the present inventive methods of treating an autoimmune disorder or of preventing or mitigating a relapse of a symptom of multiple sclerosis.


The therapeutically effective amount, prophylactically effective amount, and dosage regimen involved in the inventive methods of treating an autoimmune disorder or of preventing or mitigating a relapse of a symptom of multiple sclerosis, will be determined by the attending physician, considering various factors which modify the action of therapeutic agents, such as the age, condition, body weight, sex and diet of the patient, the severity of the condition being treated, time of administration, and other clinical factors. Generally, the daily amount or regimen should be in the range of about 1 to about 10,000 micrograms (ag) of the vehicle-conjugated peptide per kilogram (kg) of body mass, preferably about 1 to about 5000 μg per kilogram of body mass, and most preferably about 1 to about 1000 μg per kilogram of body mass.


Molecules of this invention incorporating peptide antagonists of the voltage-gated potassium channel Kv2.1 can be used to treat type II diabetes.


Molecules of this invention incorporating peptide antagonists of the M current (e.g., BeKm-1) can be used to treat Alzheimer's disease and enhance cognition.


Molecules of this invention incorporating peptide antagonists of the voltage-gated potassium channel Kv4.3 can be used to treat Alzheimer's disease.


Molecules of this invention incorporating peptide antagonists of the calcium-activated potassium channel of small conductance, SKCa can be used to treat epilepsy, memory, learning, neuropsychiatric, neurological, neuromuscular, and immunological disorders, schizophrenia, bipolar disorder, sleep apnea, neurodegeneration, and smooth muscle disorders.


Molecules of this invention incorporating N-type calcium channel antagonist peptides are useful in alleviating pain. Peptides with such activity (e.g., Ziconotide™, ω-conotoxin-MVIIA) have been clinically validated.


Molecules of this invention incorporating T-type calcium channel antagonist peptides are useful in alleviating pain. Several lines of evidence have converged to indicate that inhibition of Cav3.2 in dorsal root ganglia may bring relief from chronic pain. T-type calcium channels are found at extremely high levels in the cell bodies of a subset of neurons in the DRG; these are likely mechanoreceptors adapted to detect slowly-moving stimuli (Shin et al., Nature Neuroscience 6:724-730, 2003), and T-type channel activity is likely responsible for burst spiking (Nelson et al., J Neurosci 25:8766-8775, 2005). Inhibition of T-type channels by either mibefradil or ethosuximide reverses mechanical allodynia in animals induced by nerve injury (Dogrul et al., Pain 105:159-168, 2003) or by chemotherapy (Flatters and Bennett, Pain 109:150-161, 2004). Antisense to Cav3.2, but not Cav3.1 or Cav3.3, increases pain thresholds in animals and also reduces expression of Cav3.2 protein in the DRG (Bourinet et al., EMBO J 24:315-324, 2005). Similarly, locally injected reducing agents produce pain and increase Cav3.2 currents, oxidizing agents reduce pain and inhibit Cav3.2 currents, and peripherally administered neurosteroids are analgesic and inhibit T-type currents from DRG (Todorovic et al., Pain 109:328-339, 2004; Pathirathna et al., Pain 114:429-443, 2005). Accordingly, it is thought that inhibition of Cav3.2 in the cell bodies of DRG neurons can inhibit the repetitive spiking of these neurons associated with chronic pain states.


Molecules of this invention incorporating L-type calcium channel antagonist peptides are useful in treating hypertension. Small molecules with such activity (e.g., DHP) have been clinically validated.


Molecules of this invention incorporating peptide antagonists of the NaV1 (TTXs-type) channel can be used to alleviate pain. Local anesthetics and tricyclic antidepressants with such activity have been clinically validated. Such molecules of this invention can in particular be useful as muscle relaxants.


Molecules of this invention incorporating peptide antagonists of the NaV1 (TTXR-type) channel can be used to alleviate pain arising from nerve and or tissue injury.


Molecules of this invention incorporating peptide antagonists of glial & epithelial cell Ca2+-activated chloride channel can be used to treat cancer and diabetes.


Molecules of this invention incorporating peptide antagonists of NMDA receptors can be used to treat pain, epilepsy, brain and spinal cord injury.


Molecules of this invention incorporating peptide antagonists of nicotinic receptors can be used as muscle relaxants. Such molecules can be used to treat pain, gastric motility disorders, urinary incontinence, nicotine addiction, and mood disorders.


Molecules of this invention incorporating peptide antagonists of 5HT3 receptor can be used to treat Nausea, pain, and anxiety.


Molecules of this invention incorporating peptide antagonists of the norepinephrine transporter can be used to treat pain, anti-depressant, learning, memory, and urinary incontinence.


Molecules of this invention incorporating peptide antagonists of the Neurotensin receptor can be used to treat pain.


In addition to therapeutic uses, the compounds of the present invention can be useful in diagnosing diseases characterized by dysfunction of their associated protein of interest. In one embodiment, a method of detecting in a biological sample a protein of interest (e.g., a receptor) that is capable of being activated comprising the steps of: (a) contacting the sample with a compound of this invention; and (b) detecting activation of the protein of interest by the compound. The biological samples include tissue specimens, intact cells, or extracts thereof. The compounds of this invention can be used as part of a diagnostic kit to detect the presence of their associated proteins of interest in a biological sample. Such kits employ the compounds of the invention having an attached label to allow for detection. The compounds are useful for identifying normal or abnormal proteins of interest.


The therapeutic methods, compositions and compounds of the present invention can also be employed, alone or in combination with other molecules in the treatment of disease.


Pharmaceutical Compositions


In General. The present invention also provides pharmaceutical compositions comprising the inventive composition of matter and a pharmaceutically acceptable carrier. Such pharmaceutical compositions can be configured for administration to a patient by a wide variety of delivery routes, e.g., an intravascular delivery route such as by injection or infusion, subcutaneous, intramuscular, intraperitoneal, epidural, or intrathecal delivery routes, or for oral, enteral, pulmonary (e.g., inhalant), intranasal, transmucosal (e.g., sublingual administration), transdermal or other delivery routes and/or forms of administration known in the art. The inventive pharmaceutical compositions may be prepared in liquid form, or may be in dried powder form, such as lyophilized form. For oral or enteral use, the pharmaceutical compositions can be configured, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups, elixirs or enteral formulas.


In the practice of this invention the “pharmaceutically acceptable carrier” is any physiologically tolerated substance known to those of ordinary skill in the art useful in formulating pharmaceutical compositions, including, any pharmaceutically acceptable diluents, excipients, dispersants, binders, fillers, glidants, anti-frictional agents, compression aids, tablet-disintegrating agents (disintegrants), suspending agents, lubricants, flavorants, odorants, sweeteners, permeation or penetration enhancers, preservatives, surfactants, solubilizers, emulsifiers, thickeners, adjuvants, dyes, coatings, encapsulating material(s), and/or other additives singly or in combination. Such pharmaceutical compositions can include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween® 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol®, benzyl alcohol) and bulking substances (e.g., lactose, mannitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes. Hyaluronic acid can also be used, and this can have the effect of promoting sustained duration in the circulation. Such compositions can influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the present proteins and derivatives. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712, which are herein incorporated by reference in their entirety. The compositions can be prepared in liquid form, or can be in dried powder, such as lyophilized form. Implantable sustained release formulations are also useful, as are transdermal or transmucosal formulations. Additionally (or alternatively), the present invention provides compositions for use in any of the various slow or sustained release formulations or microparticle formulations known to the skilled artisan, for example, sustained release microparticle formulations, which can be administered via pulmonary, intranasal, or subcutaneous delivery routes.


One can dilute the inventive compositions or increase the volume of the pharmaceutical compositions of the invention with an inert material. Such diluents can include carbohydrates, especially, mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts may also be used as fillers, including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.


A variety of conventional thickeners are useful in creams, ointments, suppository and gel configurations of the pharmaceutical composition, such as, but not limited to, alginate, xanthan gum, or petrolatum, may also be employed in such configurations of the pharmaceutical composition of the present invention. A permeation or penetration enhancer, such as polyethylene glycol monolaurate, dimethyl sulfoxide, N-vinyl-2-pyrrolidone, N-(2-hydroxyethyl)-pyrrolidone, or 3-hydroxy-N-methyl-2-pyrrolidone can also be employed. Useful techniques for producing hydrogel matrices are known. (E.g., Feijen, Biodegradable hydrogel matrices for the controlled release of pharmacologically active agents, U.S. Pat. No. 4,925,677; Shah et al., Biodegradable pH/thermosensitive hydrogels for sustained delivery of biologically active agents, WO 00/38651 A1). Such biodegradable gel matrices can be formed, for example, by crosslinking a proteinaceous component and a polysaccharide or mucopolysaccharide component, then loading with the inventive composition of matter to be delivered.


Liquid pharmaceutical compositions of the present invention that are sterile solutions or suspensions can be administered to a patient by injection, for example, intramuscularly, intrathecally, epidurally, intravascularly (e.g., intravenously or intraarterially), intraperitoneally or subcutaneously. (See, e.g., Goldenberg et al., Suspensions for the sustained release of proteins, U.S. Pat. No. 6,245,740 and WO 00/38652 A1). Sterile solutions can also be administered by intravenous infusion. The inventive composition can be included in a sterile solid pharmaceutical composition, such as a lyophilized powder, which can be dissolved or suspended at a convenient time before administration to a patient using sterile water, saline, buffered saline or other appropriate sterile injectable medium.


Implantable sustained release formulations are also useful embodiments of the inventive pharmaceutical compositions. For example, the pharmaceutically acceptable carrier, being a biodegradable matrix implanted within the body or under the skin of a human or non-human vertebrate, can be a hydrogel similar to those described above. Alternatively, it may be formed from a poly-alpha-amino acid component. (Sidman, Biodegradable, implantable drug delivery device, and process for preparing and using same, U.S. Pat. No. 4,351,337). Other techniques for making implants for delivery of drugs are also known and useful in accordance with the present invention.


In powder forms, the pharmaceutically acceptable carrier is a finely divided solid, which is in admixture with finely divided active ingredient(s), including the inventive composition. For example, in some embodiments, a powder form is useful when the pharmaceutical composition is configured as an inhalant. (See, e.g., Zeng et al., Method of preparing dry powder inhalation compositions, WO 2004/017918; Trunk et al., Salts of the CGRP antagonist BIBN4096 and inhalable powdered medicaments containing them, U.S. Pat. No. 6,900,317).


One can dilute or increase the volume of the compound of the invention with an inert material. These diluents could include carbohydrates, especially mannitol, α-lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts can also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo™, Emdex™, STA-Rx™ 1500, Emcompress™ and Avicell™.


Disintegrants can be included in the formulation of the pharmaceutical composition into a solid dosage form. Materials used as disintegrants include but are not limited to starch including the commercial disintegrant based on starch, Explotab™. Sodium starch glycolate, Amberlite™, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite can all be used. Insoluble cationic exchange resin is another form of disintegrant. Powdered gums can be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.


Binders can be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.


An antifrictional agent can be included in the formulation of the therapeutic to prevent sticking during the formulation process. Lubricants can be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants can also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.


Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression might be added. The glidants can include starch, talc, pyrogenic silica and hydrated silicoaluminate.


To aid dissolution of the compound of this invention into the aqueous environment a surfactant might be added as a wetting agent. Surfactants can include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents might be used and could include benzalkonium chloride or benzethonium chloride. The list of potential nonionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of the protein or derivative either alone or as a mixture in different ratios.


Oral dosage forms. Also useful are oral dosage forms of the inventive compositions. If necessary, the composition can be chemically modified so that oral delivery is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the molecule itself, where said moiety permits (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the compound and increase in circulation time in the body. Moieties useful as covalently attached half-life extending moieties in this invention can also be used for this purpose. Examples of such moieties include: PEG, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. See, for example, Abuchowski and Davis (1981), Soluble Polymer-Enzyme Adducts, Enzymes as Drugs (Hocenberg and Roberts, eds.), Wiley-Interscience, New York, N.Y., pp 367-83; Newmark, et al. (1982), J. Appl. Biochem. 4:185-9. Other polymers that could be used are poly-1,3-dioxolane and poly-1,3,6-tioxocane. Preferred for pharmaceutical usage, as indicated above, are PEG moieties.


For oral delivery dosage forms, it is also possible to use a salt of a modified aliphatic amino acid, such as sodium N-(8-[2-hydroxybenzoyl]amino) caprylate (SNAC), as a carrier to enhance absorption of the therapeutic compounds of this invention. The clinical efficacy of a heparin formulation using SNAC has been demonstrated in a Phase II trial conducted by Emisphere Technologies. See U.S. Pat. No. 5,792,451, “Oral drug delivery composition and methods.”


In one embodiment, the pharmaceutically acceptable carrier can be a liquid and the pharmaceutical composition is prepared in the form of a solution, suspension, emulsion, syrup, elixir or pressurized composition. The active ingredient(s) (e.g., the inventive composition of matter) can be dissolved, diluted or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both, or pharmaceutically acceptable oils or fats. The liquid carrier can contain other suitable pharmaceutical additives such as detergents and/or solubilizers (e.g., Tween 80, Polysorbate 80), emulsifiers, buffers at appropriate pH (e.g., Tris-HCl, acetate, phosphate), adjuvants, anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol), sweeteners, flavoring agents, suspending agents, thickening agents, bulking substances (e.g., lactose, mannitol), colors, viscosity regulators, stabilizers, electrolytes, osmolutes or osmo-regulators. Additives can also be included in the formulation to enhance uptake of the inventive composition. Additives potentially having this property are for instance the fatty acids oleic acid, linoleic acid and linolenic acid.


Useful are oral solid dosage forms, which are described generally in Remington's Pharmaceutical Sciences (1990), supra, in Chapter 89, which is hereby incorporated by reference in its entirety. Solid dosage forms include tablets, capsules, pills, troches or lozenges, cachets or pellets. Also, liposomal or proteinoid encapsulation can be used to formulate the present compositions (as, for example, proteinoid microspheres reported in U.S. Pat. No. 4,925,673). Liposomal encapsulation can be used and the liposomes can be derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556). A description of possible solid dosage forms for the therapeutic is given in Marshall, K., Modern Pharmaceutics (1979), edited by G. S. Banker and C. T. Rhodes, in Chapter 10, which is hereby incorporated by reference in its entirety. In general, the formulation will include the inventive compound, and inert ingredients that allow for protection against the stomach environment, and release of the biologically active material in the intestine.


The composition of this invention can be included in the formulation as fine multiparticulates in the form of granules or pellets of particle size about 1 mm. The formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets. The therapeutic could be prepared by compression.


Colorants and flavoring agents can all be included. For example, the protein (or derivative) can be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.


In tablet form, the active ingredient(s) are mixed with a pharmaceutically acceptable carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired.


The powders and tablets preferably contain up to 99% of the active ingredient(s). Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.


Controlled release formulation can be desirable. The composition of this invention could be incorporated into an inert matrix that permits release by either diffusion or leaching mechanisms e.g., gums. Slowly degenerating matrices can also be incorporated into the formulation, e.g., alginates, polysaccharides. Another form of a controlled release of the compositions of this invention is by a method based on the Oros™ therapeutic system (Alza Corp.), i.e., the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects. Some enteric coatings also have a delayed release effect.


Other coatings can be used for the formulation. These include a variety of sugars that could be applied in a coating pan. The therapeutic agent could also be given in a film-coated tablet and the materials used in this instance are divided into 2 groups. The first are the nonenteric materials and include methylcellulose, ethyl cellulose, hydroxyethyl cellulose, methylhydroxy-ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl-methyl cellulose, sodium carboxymethyl cellulose, providone and the polyethylene glycols. The second group consists of the enteric materials that are commonly esters of phthalic acid.


A mix of materials might be used to provide the optimum film coating. Film coating can be carried out in a pan coater or in a fluidized bed or by compression coating.


Pulmonarv delivery forms. Pulmonary delivery of the inventive compositions is also useful. The protein (or derivative) is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream. (Other reports of this include Adjei et al., Pharma. Res. (1990) 7: 565-9; Adjei et al. (1990), Internatl. J. Pharmaceutics 63: 135-44 (leuprolide acetate); Braquet et al. (1989), J. Cardiovasc. Pharmacol. 13 (supp1.5): s.143-146 (endothelin-1); Hubbard et al. (1989), Annals Int. Med. 3: 206-12 (α1-antitrypsin); Smith et al. (1989), J. Clin. Invest. 84: 1145-6 (α1-proteinase); Oswein et al. (March 1990), “Aerosolization of Proteins,” Proc. Symp. Resp. Drug Delivery II, Keystone, Colo. (recombinant human growth hormone); Debs et al. (1988), J. Immunol. 140: 3482-8 (interferon-γ and tumor necrosis factor α) and Platz et al., U.S. Pat. No. 5,284,656 (granulocyte colony stimulating factor).


Useful in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. Some specific examples of commercially available devices suitable for the practice of this invention are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Mo.; the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colo.; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, N.C.; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Mass. (See, e.g., Helgesson et al., Inhalation device, U.S. Pat. No. 6,892,728; McDerment et al., Dry powder inhaler, WO 02/11801 A1; Ohki et al., Inhalant medicator, U.S. Pat. No. 6,273,086).


All such devices require the use of formulations suitable for the dispensing of the inventive compound. Typically, each formulation is specific to the type of device employed and can involve the use of an appropriate propellant material, in addition to diluents, adjuvants and/or carriers useful in therapy.


The inventive compound should most advantageously be prepared in particulate form with an average particle size of less than 10 μm (or microns), most preferably 0.5 to 5 μm, for most effective delivery to the distal lung.


Pharmaceutically acceptable carriers include carbohydrates such as trehalose, mannitol, xylitol, sucrose, lactose, and sorbitol. Other ingredients for use in formulations can include DPPC, DOPE, DSPC and DOPC. Natural or synthetic surfactants can be used. PEG can be used (even apart from its use in derivatizing the protein or analog). Dextrans, such as cyclodextran, can be used. Bile salts and other related enhancers can be used. Cellulose and cellulose derivatives can be used. Amino acids can be used, such as use in a buffer formulation.


Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated.


Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise the inventive compound dissolved in water at a concentration of about 0.1 to 25 mg of biologically active protein per mL of solution. The formulation can also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure). The nebulizer formulation can also contain a surfactant, to reduce or prevent surface induced aggregation of the protein caused by atomization of the solution in forming the aerosol.


Formulations for use with a metered-dose inhaler device will generally comprise a finely divided powder containing the inventive compound suspended in a propellant with the aid of a surfactant. The propellant can be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid can also be useful as a surfactant. (See, e.g., Backström et al., Aerosol drug formulations containing hydrofluoroalkanes and alkyl saccharides, U.S. Pat. No. 6,932,962).


Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing the inventive compound and can also include a bulking agent, such as lactose, sorbitol, sucrose, mannitol, trehalose, or xylitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation.


Nasal delivery forms. In accordance with the present invention, intranasal delivery of the inventive composition of matter and/or pharmaceutical compositions is also useful, which allows passage thereof to the blood stream directly after administration to the inside of the nose, without the necessity for deposition of the product in the lung. Formulations suitable for intransal administration include those with dextran or cyclodextran, and intranasal delivery devices are known. (See, e.g, Freezer, Inhaler, U.S. Pat. No. 4,083,368).


Transdermal and transmucosal (e.g., buccal) delivery forms). In some embodiments, the inventive composition is configured as a part of a pharmaceutically acceptable transdermal or transmucosal patch or a troche. Transdermal patch drug delivery systems, for example, matrix type transdermal patches, are known and useful for practicing some embodiments of the present pharmaceutical compositions. (E.g., Chien et al., Transdermal estrogen/progestin dosage unit, system and process, U.S. Pat. Nos. 4,906,169 and 5,023,084; Cleary et al., Diffusion matrix for transdermal drug administration and transdermal drug delivery devices including same, U.S. Pat. No. 4,911,916; Teillaud et al., EVA-based transdermal matrix system for the administration of an estrogen and/or a progestogen, U.S. Pat. No. 5,605,702; Venkateshwaran et al., Transdermal drug delivery matrix for coadministering estradiol and another steroid, U.S. Pat. No. 5,783,208; Ebert et al., Methods for providing testosterone and optionally estrogen replacement therapy to women, U.S. Pat. No. 5,460,820). A variety of pharmaceutically acceptable systems for transmucosal delivery of therapeutic agents are also known in the art and are compatible with the practice of the present invention. (E.g., Heiber et al., Transmucosal delivery of macromolecular drugs, U.S. Pat. Nos. 5,346,701 and 5,516,523; Longenecker et al., Transmembrane formulations for drug administration, U.S. Pat. No. 4,994,439).


Buccal delivery of the inventive compositions is also useful. Buccal delivery formulations are known in the art for use with peptides. For example, known tablet or patch systems configured for drug delivery through the oral mucosa (e.g., sublingual mucosa), include some embodiments that comprise an inner layer containing the drug, a permeation enhancer, such as a bile salt or fusidate, and a hydrophilic polymer, such as hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, dextran, pectin, polyvinyl pyrrolidone, starch, gelatin, or any number of other polymers known to be useful for this purpose. This inner layer can have one surface adapted to contact and adhere to the moist mucosal tissue of the oral cavity and can have an opposing surface adhering to an overlying non-adhesive inert layer. Optionally, such a transmucosal delivery system can be in the form of a bilayer tablet, in which the inner layer also contains additional binding agents, flavoring agents, or fillers. Some useful systems employ a non-ionic detergent along with a permeation enhancer. Transmucosal delivery devices may be in free form, such as a cream, gel, or ointment, or may comprise a determinate form such as a tablet, patch or troche. For example, delivery of the inventive composition can be via a transmucosal delivery system comprising a laminated composite of, for example, an adhesive layer, a backing layer, a permeable membrane defining a reservoir containing the inventive composition, a peel seal disc underlying the membrane, one or more heat seals, and a removable release liner. (E.g., Ebert et al., Transdermal delivery system with adhesive overlay and peel seal disc, U.S. Pat. No. 5,662,925; Chang et al., Device for administering an active agent to the skin or mucosa, U.S. Pat. Nos. 4,849,224 and 4,983,395). These examples are merely illustrative of available transmucosal drug delivery technology and are not limiting of the present invention.


Dosages. The dosage regimen involved in a method for treating the above-described conditions will be determined by the attending physician, considering various factors which modify the action of drugs, e.g. the age, condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. Generally, the daily regimen should be in the range of 0.1-1000 micrograms of the inventive compound per kilogram of body weight, preferably 0.1-150 micrograms per kilogram.


WORKING EXAMPLES

The compositions described above can be prepared as described below. These examples are not to be construed in any way as limiting the scope of the present invention.


Example 1
Fc-L10-ShK[1-35] Mammalian Expression

Fc-L10-ShK[1-35], also referred to as “Fc-2xL-ShK[1-35]”, an inhibitor of Kv1.3. A DNA sequence coding for the Fc region of human IgG1 fused in-frame to a linker sequence and a monomer of the Kv1.3 inhibitor peptide ShK[1-35] was constructed as described below. Methods for expressing and purifying the peptibody from mammalian cells (HEK 293 and Chinese Hamster Ovary cells) are disclosed herein.


The expression vector pcDNA3.1(+) CMVi (FIG. 13A) was constructed by replacing the CMV promoter between MluI and HindIII in pcDNA3.1(+) with the CMV promoter plus intron (Invitrogen). The expression vector pcDNA3.1 (+) CMVi-hFc-ActivinRIIB (FIG. 13B) was generated by cloning a HindIII-NotI digested PCR product containing a 5′ Kozak sequence, a signal peptide and the human Fc-linker-ActivinRIIB fusion protein with the large fragment of HindIII-NotI digested pcDNA3.1(+) CMVi. The nucleotide and amino acid sequence of the human IgG1 Fc region in pcDNA3.1(+) CMVi-hFc-ActivinRIIB is shown in FIG. 3A-3B. This vector also has a GGGGSGGGGS (“L10”; SEQ ID NO:79) linker split by a BamHI site thus enabling with the oligo below formation of the 10 amino acid linker between Fc and the ShK[1-35] peptide (see FIG. 14A-14B) for the final Fc-L10-ShK[1-35] nucleotide and amino acid sequence (FIG. 14A-14B and SEQ ID NO: 77 and SEQ ID NO:78).


The Fc-L10-ShK[1-35] expression vector was constructing using PCR stategies to generate the full length ShK gene linked to a four glycine and one serine amino acid linker (lower case letters here indicate linker sequence of L-form amino acid residues) with two stop codons and flanked by BamHI and NotI restriction sites as shown below.












BamHI





GGATCCGGAGGAGGAGGAAGCCGCAGC

SEQ ID NO: 657



      g  g  g  g  s  R  S



TGCATCGACACCATCCCCAAG



C  I  D  T  I  P  K



AGCCGCTGCACCGCCTTCCAG//



S  R  C  T  A  F  Q







TGCAAGCACAGCATGAAGTACCGC
SEQ ID NO: 658



C  K  H  S  M  K  Y  R



CTGAGCTTCTGCCGCAAGACC



L  S  F  C  R  K  T



TGCGGCACCTGCTAATGAGCGGCCGC



C  G  T  C        NotI//






Two oligos with the sequence as depicted below were used in a PCR reaction with Herculase™ polymerase (Stratagene) at 94° C.-30 sec, 50° C.-30 sec, and 72° C.-1 min for 30 cycles.










cat gga tcc gga gga gga gga agc
SEQ ID NO: 659


cgc agc tgc atc gac acc atc ccc


aag agc cgc tgc acc gcc ttc cag


tgc aag cac//





cat gcg gcc gct cat tag cag gtg
SEQ ID NO: 660


ccg cag gtc ttg cgg cag aag ctc


agg cgg tac ttc atg ctg tgc ttg


cac tgg aag g//






The resulting PCR products were resolved as the 150 bp bands on a one percent agarose gel. The 150 bp PCR product was digested with BamHI and NotI (Roche) restriction enzymes and agarose gel purified by Gel Purification Kit (Qiagen). At the same time, the pcDNA3.1 (+) CMVi-hFc-ActivinRIIB vector (FIG. 13B) was digested with BamHI and NotI restriction enzymes and the large fragment was purified by Gel Purification Kit. The gel purified PCR fragment was ligated to the purified large fragment and transformed into XL-1 blue bacteria (Stratagene). DNAs from transformed bacterial colonies were isolated and digested with BamHI and NotI restriction enzyme digestion and resolved on a one percent agarose gel. DNAs resulting in an expected pattern were submitted for sequencing. Although, analysis of several sequences of clones yielded a 100% percent match with the above sequence, only one clone was selected for large scaled plasmid purification. The DNA from Fc-2xL-ShK in pcDNA3.1(+) CMVi clone was resequenced to confirm the Fc and linker regions and the sequence was 100% identical to the predicted coding sequence, which is shown in FIG. 14A-14B.


HEK-293 cells used in transient transfection expression of Fc-2xL-ShK[1-35] in pcDNA3.1(+) CMVi protein were cultured in growth medium containing DMEM High Glucose (Gibco), 10% fetal bovine serum (FBS from Gibco) and 1× non-essential amino acid (NEAA from Gibco). 5.6 ug of Fc-2xL-ShK[1-35] in pcDNA3.1(+) CMVi plasmid that had been phenol/chloroform extracted was transfected into HEK-293 cells using Fugene 6 (Roche). The cells recovered for 24 hours, and then placed in DMEM High Glucose and 1×NEAA medium for 48 hours. The conditioned medium was concentrated 50× by running 30 ml through Centriprep YM-10 filter (Amicon) and further concentrated by a Centricon YM-10 (Amicon) filter. Various amounts of concentrated medium were mixed with an in-house 4× Loading Buffer (without B-mercaptoethanol) and electrophoresed on a Novex 4-20% tris-glycine gel using a Novex Xcell II apparatus at 101V/46 mA for 2 hours in a 5× Tank buffer solution (0.123 Tris Base, 0.96M Glycine) along with 10 ul of BenchMark Pre-Stained Protein ladder (Invitrogen). The gel was then soaked in Electroblot buffer (35 mM Tris base, 20% methanol, 192 mM glycine) for 30 minutes. A PVDF membrane from Novex (Cat. No. LC2002, 0.2 um pores size) was soaked in methanol for 30 seconds to activate the PVDF, rinsed with deionized water, and soaked in Electroblot buffer. The pre-soaked gel was blotted to the PVDF membrane using the XCell II Blot module according to the manufacturer instructions (Novex) at 40 mA for 2 hours. Then, the blot was first soaked in a 5% milk (Carnation) in Tris buffered saline solution pH7.5 (TBS) for 1 hour at room temperature and incubated with 1:500 dilution in TBS with 0.1% Tween-20 (TBST Sigma) and 1% milk buffer of the HRP-conjugated murine anti-human Fc antibody (Zymed Laboratores Cat. no. 05-3320) for two hours shaking at room temperature. The blot was then washed three times in TBST for 15 minutes per wash at room temperature. The primary antibody was detected using Amersham Pharmacia Biotech's ECL western blotting detection reagents according to manufacturer's instructions. Upon ECL detection, the western blot analysis displayed the expected size of 66 kDa under non-reducing gel conditions (FIG. 24A).


AM1 CHOd- (Amgen Proprietary) cells used in the stable expression of Fc-L10-ShK[1-35] protein were cultured in AM1 CHOd- growth medium containing DMEM High Glucose, 10% fetal bovine serum, 1× hypoxantine/thymidine (HT from Gibco) and 1×NEAA. 6.5 ug of pcDNA3.1(+) CMVi-Fc-ShK plasmid was also transfected into AM1 CHOd- cells using Fugene 6. The following day, the transfected cells were plated into twenty 15 cm dishes and selected using DMEM high glucose, 10% FBS, 1×HT, 1×NEAA and Geneticin (800 μg/ml G418 from Gibco) for thirteen days. Forty-eight surviving colonies were picked into two 24-well plates. The plates were allowed to grow up for a week and then replicated for freezing. One set of each plate was transferred to AM1 CHOd- growth medium without 10% FBS for 48 hours and the conditioned media were harvested. Western Blot analysis similar to the transient Western blot analysis with detection by the same anti-human Fc antibody was used to screen 15 ul of conditioned medium for expressing stable CHO clones. Of the 48 stable clones, more than 50% gave ShK expression at the expected size of 66 kDa. The BB6, BD5 and BD6 clones were selected with BD5 and BD6 as a backup to the primary clone BB6 (FIG. 24B).


The BB6 clone was scaled up into ten roller bottles (Corning) using AM1 CHOd- growth medium and grown to confluency as judged under the microscope. Then, the medium was exchanged with a serum-free medium containing to 50% DMEM high glucose and 50% Ham's F12 (Gibco) with 1×HT and 1×NEAA and let incubate for one week. The conditioned medium was harvested at the one-week incubation time, filtered through 0.45 μm filter (Corning) and frozen. Fresh serum-free medium was added and incubated for an additional week. The conditioned serum-free medium was harvested like the first time and frozen.


Purification of monovalent and bivalent dimeric Fc-L10-ShK(1-35). Approximately 4 L of conditioned medium was thawed in a water bath at room temperature. The medium was concentrated to about 450 ml using a Satorius Sartocon Polysulfon 10 tangential flow ultra-filtration cassette (0.1 m2) at room temperature. The retentate was then filtered through a 0.22 μm cellulose acetate filter with a pre-filter. The retentate was then loaded on to a 5 ml Amersham HiTrap Protein A column at 5 ml/min 7° C., and the column was washed with several column volumes of Dulbecco's phosphate buffered saline without divalent cations (PBS) and sample was eluted with a step to 100 mM glycine pH 3.0. The protein A elution pool (approximately 9 ml) was diluted to 50 ml with water and loaded on to a 5 ml Amersham HiTrap SP-HP column in S-Buffer A (20 mM NaH2PO4, pH 7.0) at 5 ml/min and 7° C. The column was then washed with several column volumes S-Buffer A, and then developed using a linear gradient from 25% to 75% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.0) at 5 ml/min followed by a step to 100% S-Buffer B at 7° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data. The pooled material was then concentrated to about 3.4 ml using a Pall Life Sciences Macrosep 10K Omega centrifugal ultra-filtration device and then filtered though a Costar 0.22 μm cellulose acetate syringe filter.


A spectral scan was then conducted on 10 μl of the filtered material diluted in 700111 PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 26A). The concentration of the filtered material was determined to be 5.4 mg/ml using a calculated molecular mass of 32,420 g/mol and extinction coefficient of 47,900 M−1 cm−1.


The purity of the filtered bivalent dimeric Fc-L10-ShK(1-35) product was assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 26B). The monvalent dimeric Fc-L10-ShK(1-35) product was analyzed using reducing and non-reducing sample buffers by SDS-PAGE on a 1.0 mm TRIS-glycine 4-20% gel developed at 220 V and stained with Boston Biologicals QuickBlue (FIG. 26E). The endotoxin levels were then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 108-fold dilution of the sample in PBS yielding a result of <1 EU/mg protein. The macromolecular state of the products was then determined using size exclusion chromatography on 20 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 26C, bivalent dimeric Fc-L10-ShK(1-35); FIG. 26F, monovalent dimeric Fc-L10-SHK(1-35)). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). The resultant solution (1 μl) was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses (FIG. 26D) and confirmed (within experimental error) the integrity of the purified peptibody. The product was then stored at −80° C.


Purified bivalent dimeric Fc-L10-ShK[1-35] potently blocked human Kv1.3 (FIG. 30A and FIG. 30B) as determined by electrophysiology (see Example 36). The purified bivalent dimeric Fc-L10-ShK[1-35] molecule also blocked T cell proliferation (FIG. 36A and FIG. 36B) and production of the cytokines IL-2 (FIG. 35A and FIG. 37A) and IFN-g (FIG. 35B and FIG. 37B).


Example 2
Fc-L-ShK[2-35] Mammalian Expression

A DNA sequence coding for the Fc region of human IgG1 fused in-frame to a monomer of the Kv1.3 inhibitor peptide ShK[2-35] was constructed using standard PCR technology. The ShK[2-35] and the 5, 10, or 25 amino acid linker portion of the molecule were generated in a PCR reaction using the original Fc-2xL-ShK[1-35] in pcDNA3.1(+) CMVi as a template (Example 1, FIG. 14A-14B). All ShK constructs should have the following amino acid sequence of











SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
(SEQ ID NO: 92)








with the first amino acid of the wild-type sequence deleted.


The sequences of the primers used to generate Fc-L5-ShK[2-35], also referred to as “Fc-1xL-ShK[2-35]”, are shown below:










SEQ ID NO: 661











cat gga tcc agc tgc atc gac acc atc//;













SEQ ID NO: 662











cat gcg gcc gct cat tag c//;







The sequences of the primers used to generate Fc-L10-ShK[2-35], also referred to as “Fc-2xL-ShK[2-35]” are shown below:










SEQ ID NO: 663











cat gga tcc gga gga gga gga agc agc tgc a //;













SEQ ID NO: 664











cat gcg gcc gct cat tag cag gtg c //;







The sequences of the primers used to generate Fc-L25-ShK[2-35], also referred to as “Fc-5xL-ShK[2-35]”, are shown below:










SEQ ID NO: 665









cat gga tcc ggg ggt ggg ggt tct ggg ggt ggg ggt



tct gga gga gga gga agc gga gga gga gga agc agc


tgc a//;











SEQ ID NO: 666









cat gcg gcc gct cat tag cag gtg c//;







The PCR products were digested with BamHI and NotI (Roche) restriction enzymes and agarose gel purified by Gel Purification Kit. At the same time, the pcDNA3.1(+) CMVi-hFc-ActivinRIIB vector was digested with BamHI and NotI restriction enzymes and the large fragment was purified by Gel Purification Kit. Each purified PCR product was ligated to the large fragment and transformed into XL-1 blue bacteria. DNAs from transformed bacterial colonies were isolated and subjected to BamHI and NotI restriction enzyme digestions and resolved on a one percent agarose gel. DNAs resulting in an expected pattern were submitted for sequencing. Although, analysis of several sequences of clones yielded a 100% percent match with the above sequence, only one clone was selected for large scaled plasmid purification. The DNA from this clone was resequenced to confirm the Fc and linker regions and the sequence was 100% identical to the expected sequence.


Plasmids containing the Fc-1xL-Shk[2-35], Fc-2xL-Shk[2-35] and Fc-5xL-Shk[2-35] inserts in pcDNA3.1 (+) CMVi vector were digested with Xba1 and Xho1 (Roche) restriction enzymes and gel purified. The inserts were individually ligated into Not1 and SalI (Roche) digested pDSRα-22 (Amgen Proprietary) expression vector. Integrity of the resulting constructs were confirmed by DNA sequencing. The final plasmid DNA expression vector constructs were pDSRα-22-Fc-1xL-Shk[2-35], pDSRα-22-Fc-2xL-Shk[2-35] (FIG. 13C and FIG. 15A-15B) and pDSRα-22-Fc-5xL-Shk[2-35] (FIG. 16A-16B) and contained 5, 10 and 25 amino acid linkers, respectively.


Twenty-four hours prior to transfection, 1.2e7 AM-1/D CHOd- (Amgen Proprietary) cells were plated into a T-175 cm sterile tissue culture flask, to allow 70-80% confluency on the day of transfection. The cells had been maintained in the AM-1/D CHOd- culture medium containing DMEM High Glucose, 5% FBS, 1× Glutamine Pen/Strep (Gibco), 1×HT, 1×NEAA's and 1× sodium pyruvate (Gibco). The following day, eighteen micrograms of each of the linearized pDSRα22:Fc-1xL-ShK[2-35], pDSRα22:Fc-2xL-ShK[2-35] and pDSRα22:Fc-5xL-ShK[2-35] (RDS's #20050037685, 20050053709, 20050073295) plasmids were mixed with 72 μg of linearized Selexis MAR plasmid and pPAGO1 (RDS 20042009896) and diluted into 6 ml of OptiMEM in a 50 ml conical tube and incubate for five minutes. LF2000 (210 μl) was added to 6 ml of OptiMEM and incubated for five minutes. The diluted DNA and LF2000 were mixed together and incubated for 20 minutes at room temperature. In the meantime, the cells were washed one time with PBS and then 30 ml OptiMEM without antibiotics were added to the cells. The OptiMEM was aspirated off, and the cells were incubated with 12 ml of DNA/LF2000 mixture for 6 hours or overnight in the 37° C. incubator with shaking. Twenty-four hours post transfection, the cells were split 1:5 into AM-1/D CHOd- culture medium and at differing dilutions for colony selection. Seventy-two hours post transfection, the cell medium was replaced with DHFR selection medium containing 10% Dialyzed FBS (Gibco) in DMEM High Glucose, plus 1× Glutamine Pen/Strep, 1×NEAA's and 1× Na Pyr to allow expression and secretion of protein into the cell medium. The selection medium was changed two times a week until the colonies are big enough to pick. The pDSRa22 expression vector contains a DHFR expression cassette, which allows transfected cells to grow in the absence of hypoxanthine and thymidine. The five T-175 pools of the resulting colonies were scaled up into roller bottles and cultured under serum free conditions. The conditioned media were harvested and replaced at one-week intervals. The resulting 3 liters of conditioned medium was filtered through a 0.45 μm cellulose acetate filter (Corning, Acton, Mass.) and transferred to Protein Chemistry for purification. As a backup, twelve colonies were selected from the 10 cm plates after 10-14 days on DHFR selection medium and expression levels evaluated by western blot using HRP conjugated anti human IgGFc as a probe. The three best clones expressing the highest level of each of the different linker length Fc-L-ShK[2-35] fusion proteins were expanded and frozen for future use.


Purification of Fc-L10-ShK(2-35). Approximately 1 L of conditioned medium was thawed in a water bath at room temperature. The medium was loaded on to a 5 ml Amersham HiTrap Protein A column at 5 ml/min 7° C., and the column was washed with several column volumes of Dulbecco's phosphate buffered saline without divalent cations (PBS) and sample was eluted with a step to 100 mM glycine pH 3.0. The protein A elution pool (approximately 8.5 ml) combined with 71 μl 3 M sodium acetate and then diluted to 50 ml with water. The diluted material was then loaded on to a 5 ml Amersham HiTrap SP-HP column in S-Buffer A (20 mM NaH2PO4, pH 7.0) at 5 ml/min 7° C. The column was then washed with several column volumes S-Buffer A, and then developed using a linear gradient from 0% to 75% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.0) at 5 ml/min followed by a step to 100% S-Buffer B at 7° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data. The pooled material was then filtered through a 0.22 μm cellulose acetate filter and concentrated to about 3.9 ml using a Pall Life Sciences Macrosep 10K Omega centrifugal ultra-filtration device. The concentrated material was then filtered though a Pall Life Sciences Acrodisc with a 0.22 μm, 25 mm Mustang E membrane at 2 ml/min room temperature. A spectral scan was then conducted on 10 μl of the filtered material diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 27E). The concentration of the filtered material was determined to be 2.76 mg/ml using a calculated molecular mass of 30,008 g/mol and extinction coefficient of 36,900 M−1 cm−1. Since material was found in the permeate, repeated concentration step on the permeate using a new Macrosep cartridge. The new batch of concentrated material was then filtered though a Pall Life Sciences Acrodisc with a 0.22 μm, 25 mm Mustang E membrane at 2 ml/min room temperature. Both lots of concentrated material were combined into one pool.


A spectral scan was then conducted on 10 μl of the combined pool diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer. The concentration of the filtered material was determined to be 3.33 mg/ml using a calculated molecular mass of 30,008 g/mol and extinction coefficient of 36,900 M−1 cm−1. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 27A). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 67-fold dilution of the sample in PBS yielding a result of <1 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 50 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 27B). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). The resultant solution (1 μl) was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses (FIG. 27F) and the experiment confirmed the integrity of the peptibody, within experimental error. The product was then stored at −80° C.



FIG. 31B shows that purified Fc-L10-ShK[2-35] potently blocks human Kv1.3 current (electrophysiology was done as described in Example 36). The purified Fc-L10-ShK[2-35] molecule also blocked IL-2 (FIG. 64A and FIG. 64B) and IFN-g (FIG. 65A and FIG. 65B) production in human whole blood, as well as, upregulation of CD40L (FIG. 66A and FIG. 66B) and IL-2R (FIG. 67A and FIG. 67B) on T cells.


Purification of Fc-L5-ShK(2-35). Approximately 1 L of conditioned medium was loaded on to a 5 ml Amersham HiTrap Protein A column at 5 ml/min 7° C., and the column was washed with several column volumes of Dulbecco's phosphate buffered saline without divalent cations (PBS) and sample was eluted with a step to 100 mM glycine pH 3.0. The protein A elution pool (approximately 9 ml) combined with 450 μl 1 M tris HCl pH 8.5 followed by 230 μl 2 M acetic acid then diluted to 50 ml with water. The pH adjusted material was then filtered through a 0.22 μm cellulose acetate filter and loaded on to a 5 ml Amersham HiTrap SP-HP column in S-Buffer A (20 mM NaH2PO4, pH 7.0) at 5 ml/min 7° C. The column was then washed with several column volumes S-Buffer A, and then developed using a linear gradient from 0% to 75% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.0) at 5 ml/min followed by a step to 100% S-Buffer B at 7° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data. The pooled material was then concentrated to about 5.5 ml using a Pall Life Sciences Macrosep 10K Omega centrifugal ultra-filtration device. The concentrated material was then filtered though a Pall Life Sciences Acrodisc with a 0.22 μm, 25 mm Mustang E membrane at 2 ml/min room temperature.


A spectral scan was then conducted on 10 μl of the combined pool diluted in 700111 PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 27G). The concentration of the filtered material was determined to be 4.59 mg/ml using a calculated molecular mass of 29,750 g/mol and extinction coefficient of 36,900 M−1 cm−1. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 27C). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 92-fold dilution of the sample in Charles Rivers Endotoxin Specific Buffer BG120 yielding a result of <1 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 50 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 27H). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). The resultant solution (1 μl) was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses (FIG. 271) and confirmed the integrity of the peptibody, within experimental error. The product was then stored at 80° C.



FIG. 31C shows that purified Fc-L5-ShK[2-35] is highly active and blocks human Kv1.3 as determined by whole cell patch clamp electrophysiology (see Example 36).


Purification of Fc-L25-ShK(2-35). Approximately 1 L of conditioned medium was loaded on to a 5 ml Amersham HiTrap Protein A column at 5 ml/min 7° C., and the column was washed with several column volumes of Dulbecco's phosphate buffered saline without divalent cations (PBS) and sample was eluted with a step to 100 mM glycine pH 3.0. The protein A elution pool (approximately 9.5 ml) combined with 119 μl 3 M sodium acetate and then diluted to 50 ml with water. The pH adjusted material was then loaded on to a 5 ml Amersham HiTrap SP-HP column in S-Buffer A (20 mM NaH2PO4, pH 7.0) at 5 ml/min 7° C. The column was then washed with several column volumes S-Buffer A, and then developed using a linear gradient from 0% to 75% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.0) at 5 ml/min followed by a step to 100% S-Buffer B at 7° C. Fractions containing the main peak from the chromatogram were pooled and filtered through a 0.22 μm cellulose acetate filter.


A spectral scan was then conducted on 20 μl of the combined pool diluted in 700111 PBS using a Hewlett Packard 8453 spectrophotometer FIG. 27J. The concentration of the filtered material was determined to be 1.40 mg/ml using a calculated molecular mass of 31,011 g/mol and extinction coefficient of 36,900 M−1 cm−1. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 27D). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 28-fold dilution of the sample in Charles Rivers Endotoxin Specific Buffer BG120 yielding a result of <1 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 50 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 27K). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). The resultant solution (1 μl) was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses (FIG. 27L) and this confirmed the integrity of the peptibody, within experimental error. The product was then stored at −80° C.


Purified Fc-L25-ShK[2-35] inhibited human Kv1.3 with an IC50 of ˜150 pM by whole cell patch clamp electrophysiology on HEK293/Kv1.3 cells (Example 36).


Example 3
Fc-L-ShK[1-35] Bacterial Expression

Description of bacterial peptibody expression vectors and procedures for cloning and expression of peptibodies. The cloning vector used for bacterial expression (Examples 3-30) is based on pAMG21 (originally described in U.S. Patent 2004/0044188). It has been modified in that the kanamycin resistance component has been replaced with ampicillin resistance by excising the DNA between the unique BstBI and NsiI sites of the vector and replacing with an appropriately digested PCR fragment bearing the beta-lactamase gene using PCR primers CCA ACA CAC TTC GAA AGA CGT TGA TCG GCA C (SEQ ID NO: 667) and CAC CCA ACA ATG CAT CCT TAA AAA AAT TAC GCC C (SEQ ID NO: 668) with pUC19 DNA as the template source of the beta-lactamase gene conferring resistance to ampicillin. The new version is called pAMG21ampR.


Description of cloning vector pAMG21ampR-Fc-Pep used in examples 3 to 30, excluding 15 and 16. FIG. 11A-C and FIG. 11D (schematic diagram) show the ds-DNA that has been added to the basic vector pAMG21ampR to permit the cloning of peptide fusions to the C-terminus of the Fc gene. The DNA has been introduced between the unique NdeI and BamHI sites in the pAMG21ampR vector. This entire region of DNA is shown in FIG. 11A-C. The coding region for Fc extends from nt 5134 to 5817 and the protein sequence appears below the DNA sequence. This is followed in frame by a glyX5 linker (nt's 5818-5832). A BsmBI site (GAGACG) spansnucleotides 5834-5839. DNA cleavage occurs between nucleotides 5828 and 5829 on the upper DNA strand and between nucleotides 5832 and 5833 on the lower DNA strand. Digestion creates 4 bp cohesive termini as shown here. The BsmBI site is underlined.












AGGTGG
TGGTTGAGACG SEQ ID NO: 683







TCCACCACCA
    ACTCTGC



SEQ ID NO: 684






A second BsmBI site occurs at nucleotides 6643 through 6648; viz., CGTCTC. DNA cleavage occurs between nucleotides 6650 and 6651 on the upper strand and between 6654 and 6655 on the lower strand.













CGTCTCT

TAAGGATCCG SEQ ID NO: 685








GCAGAGAATTC

    CTAGGC



SEQ ID NO: 686






Between the two BsmBI sites is a dispensable chloramphenicol resistance cassette constitutively expressing chloramphenicol acetyltransferase (cat gene). The cat protein sequence:












1
MEKKITGYTT VDISQWHRKE HFEAFQSVAQ CTYNQTVQLD ITAFLKTVKK
SEQ ID NO: 1337






51
NKHKFYPAFI HILARLMNAH PEFRMAMKDG ELVIWDSVHP CYTVFHEQTE





101
TFSSLWSEYH DDFRQFLHIY SQDVACYGEN LAYFPKGFIE NMFFVSANPW





151
VSFTSFDLNV ANMDNFFAPV FTMGKYYTQG DKVLMPLAIQ VHHAVCDGFH





201
VGRMLNELQQ YCDEWQGGA//







is shown in FIG. 11A-C and extends from nucleotides 5954 to 6610. The peptide encoding duplexes in each example (except Examples 15 and 16) bear cohesive ends complementary to those presented by the vector.


Description of the cloning vector pAMG21ampR-Pep-Fc used in examples 15 and 16. FIG. 12A-C, and the schematic diagram in FIG. 12D, shows the ds-DNA sequence that has been added to the basic vector pAMG21ampR to permit the cloning of peptide fusions to the N-terminus of the Fc gene. The DNA has been introduced between the unique NdeI and BamHI sites in the pAMG21ampR vector. The coding region for Fc extends from nt 5640 to 6309 and the protein sequence appears below the DNA sequence. This is preceded in frame by a glyX5 linker (nt's 5614-5628). A BsmBI site spans nucleotides 5138 to 5143; viz., GAGACG. The cutting occurs between nucleotides 5132 and 5133 on the upper DNA strand and between 5136 and 5137 on the lower DNA strand.


Digestion creates 4 bp cohesive termini as shown. The BsmBI site is underlined.












AATAACA
TATGCGAGACG SEQ ID NO: 687







TTATTGTATAC
    GCTCTGC



SEQ ID NO: 688






A second BsmBI site occurs at nucleotides 5607 through 5612; viz., CGTCTC. Cutting occurs between nucleotides 5613 and 5614 on the upper strand and between 5617 and 5618 on the lower strand.













CGTCTCA

GGTGGTGGT




GCAGAGTCCAC

    CACCA



SEQ ID NO: 689






Between the BsmBI sites is a dispensable zeocin resistance cassette constitutively expressing the Shigella ble protein. The ble protein sequence:












1
MAKLTSAVPV LTARDVAGAV EFWTDRLGFS RDFVEDDFAG VVRDDVTLFI
SEQ ID NO: 1338






51
SAVQDQVVPD NTLAWVWVRG LDELYAEWSE VVSTNFRDAS GPAMTEIGEQ





101
PWGREFALRD PAGNCVHFVA EEQD//







is shown extending from nucleotides 5217 to 5588 in FIG. 12A-C. The peptide encoding duplexes in Examples 15 and 16 bear cohesive ends complementary to those presented by the vector.


Description of the cloning vector DAMG21ampR-Pep-Fc used in Examples 52 and 53. FIG. 12E-G shows the ds-DNA sequence that has been added to the basic vector pAMG21ampR to permit the cloning of peptide fusions to the N-terminus of the Fc gene in which the first two codons of the peptide are to be met-gly. The DNA has been introduced between the unique NdeI and BamHI sites in the pAMG21ampR vector. The coding region for Fc extends from nt 5632 to 6312 and the protein sequence appears below the DNA sequence. This is preceded in frame by a glyX5 linker (nt's 5617-5631). A BsmBI site spans nucleotides 5141 to 5146; viz., GAGACG. The cutting occurs between nucleotides 5135 and 5136 on the upper DNA strand and between 5139 and 5140 on the lower DNA strand.


Digestion creates 4 bp cohesive termini as shown. The BsmBI site is underlined.












AATAACATAT
GGGTCGAGACG SEQ ID NO: 1344



SEQ ID NO: 1343







TTATTGTATACCCA
    GCTCTGC



SEQ ID NO: 1345







A second BsmBI site occurs at nucleotides 5607 through 5612; viz., CGTCTC. Cutting occurs between nucleotides 5613 and 5614 on the upper strand and between 5617 and 5618 on the lower strand.













CGTCTCA

GGTGGTGGT








GCAGAGTCCAC SEQ ID NO: 1346

    CACCA







Between the BsmBI sites is a dispensable zeocin resistance cassette constitutively expressing the Shigella ble protein. The ble protein sequence, as described above, is shown extending from nucleotide positions 5220 to 5591. The peptide encoding duplexes in Examples 52 and 53 herein below bear cohesive ends complementary to those presented by the vector.


For Examples 3 to 30 for which all are for bacterial expression, cloned peptide sequences are all derived from the annealing of oligonucleotides to create a DNA duplex that is directly ligated into the appropriate vector. Two oligos suffice for Example 20, four are required for all other examples. When the duplex is to be inserted at the N-terminus of Fc (see, Examples 15, 16, 52, and 53 herein) the design is as follows with the ordinal numbers matching the listing of oligos in each example:













    First Oligo
         Second Oligo



TATG

                


                       













                      


                 

CCAC



       Fourth Oligo
       Third Oligo






When the duplex is to be inserted at the C-terminus of Fc (Examples 3, 4, 5, 10, 11, 12, 13, and 30) the design is as follows:













      First Oligo
 Second Oligo



TGGT

                      


                 













                   


                     

ATTC



     Fourth Oligo
     Third Oligo






All remaining examples have the duplex inserted at the C-terminus of Fc and utilize the following design.













    First Oligo
         Second Oligo



TGGT

                


                       













                      


                 

ATTC



       Fourth Oligo
       Third Oligo






No kinasing step is required for the phosphorylation of any of the oligos. A successful insertion of a duplex results in the replacement of the dispensable antibiotic resistance cassette (Zeocin resistance for pAMG21ampR-Pep-Fc and chloramphenicol resistance for pAMG21ampR-Fc-Pep). The resulting change in phenotype is useful for discriminating recombinant from nonrecombinant clones.


The following description gives the uniform method for carrying out the cloning of all 30 bacterially expressed recombinant proteins exemplified herein. Only the set of oligonucleotides and the vector are varied. These specifications are given below in each example.


An oligonucleotide duplex containing the coding region for a given peptide was formed by annealing the oligonucleotides listed in each example. Ten picomoles of each oligo was mixed in a final volume of 10 μl containing 1× ligation buffer along with 0.3 μg of appropriate vector that had been previously digested with restriction endonuclease BsmBI. The mix was heated to 80° C. and allowed to cool at 0.1 degree/sec to room temperature. To this was added 10 μl of 1× ligase buffer plus 400 units of T4 DNA ligase. The sample was incubated at 14 C for 20 min. Ligase was inactivated by heating at 65° C. for 10 minutes. Next, 10 units of restriction endonucleases BsmBI were added followed by incubation at 55 C for one hour to cleave any reformed parental vector molecules. Fifty ul of chemically competent E. coli cells were added and held at 2 C for 20 minutes followed by heat shock at 42 C for 5 second. The entire volume was spread onto Luria Agar plates supplemented with carbenicillin at 200 μg/ml and incubated overnight at 37 C. Colonies were tested for the loss of resistance to the replaceable antibiotic resistance marker. A standard PCR test can be used to confirm the expected size of the duplex insert. Plasmid preparations were obtained and the recombinant insert was verified by DNA sequencing. Half liter cultures of a sequence confirmed construct were grown in Terrific Broth, expression of the peptibody was induced by addition of N-(3-oxo-hexanoyl)-homoserine lactone at 50 ng/ml and after 4-6 hours of shaking at 37 C the cells were centrifuged and the cell paste stored at −20 C.


The following gives for each example the cloning vector and the set of oligonucleotides used for constructing each fusion protein. Also shown is a DNA/protein map.


Bacterial expression of Fc-L-ShK[1-35] inhibitor of Kv1.3. The methods to clone and express the peptibody in bacteria are described above. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-ShK[1-35].


Oligos used to form the duplex:










TGGTTCCGGTGGTGGTGGTTCCCGTTCCTGCATCGA
SEQ ID NO: 669


CACCAT//;





CCCGAAATCCCGTTGCACCGCTTTCCAGTGCAAACA
SEQ ID NO: 670


CTCCATGAAATACCGTCTGTCCTTCTGCCGTAAAAC


CTGCGGTACCTGC//;





CTTAGCAGGTACCGCAGGTTTTACGGCAGAAGGACA
SEQ ID NO: 671


GACGGT//;





ATTTCATGGAGTGTTTGCACTGGAAAGCGGTGCAAC
SEQ ID NO: 672


GGGATTTCGGGATGGTGTCGATGCAGGAACGGGAA


CCACCACCACCGGA//;






The oligo duplex is shown below:













TGGTTCCGGTGGTGGTGGTTCCCGTTCCTGCATCGACACCATCCCGAAATCCCGTTGCAC




1
---------+---------+---------+---------+---------+---------+
 60



    AGGCCACCACCACCAAGGGCAAGGACGTAGCTGTGGTAGGGCTTTAGGGCAACGTG






 G  S  G  G  G  G  S  R  S  C  I  D  T  I  P  K  S  R  C  T
-






CGCTTTCCAGTGCAAACACTCCATGAAATACCGTCTGTCCTTCTGCCGTAAAACCTGCGG


61
---------+---------+---------+---------+---------+---------+
120



GCGAAAGGTCACGTTTGTGAGGTACTTTATGGCAGACAGGAAGACGGCATTTTGGACGCC






 A  F  Q  C  K  H  S  M  K  Y  R  L  S  F  C  R  K  T  C  G
-






TACCTGC//
SEQ ID NO: 673


121
-------



ATGGACGATTC//
SEQ ID NO: 675






 T  C   -//
SEQ ID NO: 674






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen. Purification of bacterially expressed Fc-L10-ShK(1-35) is further described in Example 38 herein below.


Example 4
Fc-L-ShK[2-35] Bacterial Expression

Bacterial expression of Fc-L-ShK[2-35]. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-ShK[2-35].


Oligos used to form duplex are shown below:










TGGTTCCGGTGGTGGTGGTTCCTGCATCGACACCAT
SEQ ID NO: 676


CCCGAAATCCCGTTGCACCGCTTTCCAGTGCAAAC


ACTCCATGAAAT//;





ACCGTCTGTCCTTCTGCCGTAAAACCTGCGGTAC
SEQ ID NO: 677


CTGC//;





CTTAGCAGGTACCGCAGGTTTTACGGCAGAAGGA
SEQ ID NO: 678


CAGACGGTATTTCATGGAGTGTTTGCACTGGAAAG


CGGTGCAACGGGA//;





TTTCGGGATGGTGTCGATGCAGGAACCACCACCA
SEQ ID NO: 679


CCGGA//;






The oligo duplex formed is shown below:













TGGTTCCGGTGGTGGTGGTTCCTGCATCGACACCATCCCGAAATCCCGTTGCACCGCTTT




1
---------+---------+---------+---------+---------+---------+
 60



    AGGCCACCACCACCAAGGACGTAGCTGTGGTAGGGCTTTAGGGCAACGTGGCGAAA






 G  S  G  G  G  G  S  C  I  D  T  I  P  K  S  R  C  T  A  F
-






CCAGTGCAAACACTCCATGAAATACCGTCTGTCCTTCTGCCGTAAAACCTGCGGTACCTG


61
---------+---------+---------+---------+---------+---------+
120



GGTCACGTTTGTGAGGTACTTTATGGCAGACAGGAAGACGGCATTTTGGACGCCATGGAC






 Q  C  K  H  S  M  K  Y  R  L  S  F  C  R  K  T  C  G  T  C
- //SEQ ID NO: 681






C//
SEQ ID NO: 680


121
-



GATTC//
SEQ ID NO: 682






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen. Purification of bacterially expressed Fc-L10-ShK(2-35) is further described in Example 39 herein below.


Example 5
Fc-L-HmK Bacterial Expression

Bacterial expression of Fc-L-HmK. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-HmK.


Oligos used to form duplex are shown below:










TGGTTCCGGTGGTGGTGGTTCCCGTACCTGCAAAGA
SEQ ID NO: 690


CCTGAT//;





CCCGGTTTCCGAATGCACCGACATCCGTTGCCGTAC
SEQ ID NO: 692


CTCCATGAAATACCGTCTGAACCTGTGCCGTAAAAC


CTGCGGTTCCTGC;





CTTAGCAGGAACCGCAGGTTTTACGGCACAGGTTCA
SEQ ID NO: 693


GACGGT//;





ATTTCATGGAGGTACGGCAACGGATGTCGGTGCATT
SEQ ID NO: 694


CGGAAACCGGGATCAGGTCTTTGCAGGTACGGGAA


CCACCACCACCGGA//






The oligo duplex formed is shown below:













TGGTTCCGGTGGTGGTGGTTCCCGTACCTGCAAAGACCTGATCCCGGTTTCCGAATGCAC




1
---------+---------+---------+---------+---------+---------+
 60



    AGGCCACCACCACCAAGGGCATGGACGTTTCTGGACTAGGGCCAAAGGCTTACGTG






 G  S  G  G  G  G  S  R  T  C  K  D  L  I  P  V  S  E  C  T
-






CGACATCCGTTGCCGTACCTCCATGAAATACCGTCTGAACCTGTGCCGTAAAACCTGCGG


61
---------+---------+---------+---------+---------+---------+
120



GCTGTAGGCAACGGCATGGAGGTACTTTATGGCAGACTTGGACACGGCATTTTGGACGCC






 D  I  R  C  R  T  S  M  K  Y  R  L  N  L  C  R  K  T  C  G
- //SEQ ID NO: 696






TTCCTGC//
SEQ ID NO: 695


121
---------



AAGGACGATTC//
SEQ ID NO: 697






 S  C   -






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.


Example 6
Fc-L-KTX1 Bacterial Expression

Bacterial expression of Fc-L-KTX1. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-KTX1.


Oligos used to form duplex are shown below:










TGGTTCCGGTGGTGGTGGTTCCGGTGTTGAAATCAA
SEQ ID NO: 698


CGTTAAATGCT//;





CCGGTTCCCCGCAGTGCCTGAAACCGTGCAAAGAC
SEQ ID NO: 699


GCTGGTATGCGTTTCGGTAAATGCATGAACCGTAAA


TGCCACTGCACCCCGAAA//;





CTTATTTCGGGGTGCAGTGGCATTTACGGTTCATGC
SEQ ID NO: 700


ATTTACCGAAA//;





CGCATACCAGCGTCTTTGCACGGTTTCAGGCACTGC
SEQ ID NO: 701


GGGGAACCGGAGCATTTAACGTTGATTTCAACACCG


GAACCACCACCACCGGA//;






The oligo duplex formed is shown below:













TGGTTCCGGTGGTGGTGGTTCCGGTGTTGAAATCAACGTTAAATGCTCCGGTTCCCCGCA




1
---------+---------+---------+---------+---------+---------+
 60



    AGGCCACCACCACCAAGGCCACAACTTTAGTTGCAATTTACGAGGCCAAGGGGCGT






 G  S  G  G  G  G  S  G  V  E  I  N  V  K  C  S  G  S  P  Q
-






GTGCCTGAAACCGTGCAAAGACGCTGGTATGCGTTTCGGTAAATGCATGAACCGTAAATG


61
---------+---------+---------+---------+---------+---------+
120



CACGGACTTTGGCACGTTTCTGCGACCATACGCAAAGCCATTTACGTACTTGGCATTTAC






 C  L  K  P  C  K  D  A  G M  R  F  G  K  C  M  N  R  K  C
-






CCACTGCACCCCGAAA//
SEQ ID NO: 702


121
---------+------



GGTGACGTGGGGCTTTATTC//
SEQ ID NO: 704






 H  C  T  P  K   -//
SEQ ID NO: 703






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.


Purification and refolding of Fc-L-KTX1 expressed in bacteria. Frozen, E. coli paste (28 g) was combined with 210 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 8.0 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 22,000 g for 20 min at 4° C. The pellet was then resuspended in 200 ml 1% deoxycholic acid using a tissue grinder and then centrifuged at 22,000 g for 20 min at 4° C. The pellet was then resuspended in 200 ml water using a tissue grinder and then centrifuged at 22,000 g for 20 min at 4° C. The pellet (4.8 g) was then dissolved in 48 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. The dissolved pellet was then reduced by adding 30 μl 1 M dithiothreitol to 3 ml of the solution and incubating at 37° C. for 30 minutes. The reduced pellet solution was then centrifuged at 14,000 g for 5 min at room temperature, and then 2.5 ml of the supernatant was transferred to 250 ml of the refolding buffer (2 M urea, 50 mM tris, 160 mM arginine HCl, 5 mM EDTA, 1 mM cystamine HCl, 4 mM cysteine, pH 8.5) at 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for 2 days at 4° C. The refolding solution was then filtered through a 0.22 μm cellulose acetate filter and stored at 4° C. for 3 days.


The stored refold was then diluted with 1 L of water and the pH was adjusted to 7.5 using 1 M H3PO4. The pH adjusted material was then loaded on to a 10 ml Amersham SP-HP HiTrap column at 10 ml/min in S-Buffer A (20 mM NaH2PO4, pH 7.3) at 7° C. The column was then washed with several column volumes of S-Buffer A, followed by elution with a linear gradient from 0% to 60% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.3) followed by a step to 100% S-Buffer B at 5 ml/min 7° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data (45 ml). The pool was then loaded on to a 1 ml Amersham rProtein A HiTrap column in PBS at 2 ml/min 7° C. Then column was then washed with several column volumes of PBS and eluted with 100 mM glycine pH 3.0. To the elution peak (2.5 ml), 62.5 μl 2 M tris base was added, and then the pH adjusted material was filtered though a Pall Life Sciences Acrodisc with a 0.22 μm, 25 mm Mustang E membrane at 2 ml/min room temperature.


A spectral scan was then conducted on 20 μl of the combined pool diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 28C). The concentration of the filtered material was determined to be 2.49 mg/ml using a calculated molecular mass of 30,504 g/mol and extinction coefficient of 35,410 M−1 cm−1. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 28A). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 50-fold dilution of the sample in Charles Rivers Endotoxin Specific Buffer BG120 yielding a result of <1 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 45 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 28B). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). The resultant solution (1 μl) was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses (FIG. 28D) and these studies confirmed the integrity of the purified peptibody, within experimental error. The product was then stored at −80° C.


Purified Fc-L-KTX1 blocked the human Kv1.3 current in a dose-dependent fashion (FIG. 32A and FIG. 32B) by electrophysiology (method was as described in Example 36).


Example 7
Fc-L-HsTx1 Bacterial Expression

Bacterial expression of Fc-L-HsT1. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-HsTx1.


Oligos used to form duplex are shown below:










TGGTTCCGGTGGTGGTGGTTCCGCTTCCTGCCGTA
SEQ ID NO: 705


CCCCGAAAGAC//;





TGCGCTGACCCGTGCCGTAAAGAAACCGGTTGCC
SEQ ID NO: 706


CGTACGGTAAATGCATGAACCGTAAATGCAAATGC


AACCGTTGC//;





CTTAGCAACGGTTGCATTTGCATTTACGGTTCATGC
SEQ ID NO: 707


ATTTACCGTACG//;





GGCAACCGGTTTCTTTACGGCACGGGTCAGCGCAGT
SEQ ID NO: 708


CTTTCGGGGTACGGCAGGAAGCGGAACCACCACCA


CCGGA//;






The duplex formed by the oligos above is shown below:













TGGTTCCGGTGGTGGTGGTTCCGCTTCCTGCCGTACCCCGAAAGACTGCGCTGACCCGTG




1
---------+---------+---------+---------+---------+---------+
 60



    AGGCCACCACCACCAAGGCGAAGGACGGCATGGGGCTTTCTGACGCGACTGGGCAC






 G  S  G  G  G  G  S  A  S  C  R  T  P  K  D  C  A  D  P  C
-






CCGTAAAGAAACCGGTTGCCCGTACGGTAAATGCATGAACCGTAAATGCAAATGCAACCG


61
---------+---------+---------+---------+---------+---------+
120



GGCATTTCTTTGGCCAACGGGCATGCCATTTACGTACTTGGCATTTACGTTTACGTTGGC






 R  K  E  T  G  C  P  Y  G  K  C  M  N  R  K  C  K  C  N  R
-






TTGC
SEQ ID NO: 709


121
----
124



AACGATTC
SEQ ID NO: 711






 C   -
SEQ ID NO: 710






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.


Example 8
Fc-L-MgTx Bacterial Expression

Bacterial expression of Fc-L-MgTx. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-MgTx.


Oligos used to form duplex are shown below:










TGGTTCCGGTGGTGGTGGTTCCACCATCATCAACGT
SEQ ID NO: 712


TAAATGCACCTC//;





CCCGAAACAGTGCCTGCCGCCGTGCAAAGCTCAGT
SEQ ID NO: 713


TCGGTCAGTCCGCTGGTGCTAAATGCATGAACGGTA


AATGCAAATGCTACCCGCAC//;





CTTAGTGCGGGTAGCATTTGCATTTACCGTTCATGC
SEQ ID NO: 714


ATTTAGCACCAG//;





CGGACTGACCGAACTGAGCTTTGCACGGCGGCAGG
SEQ ID NO: 715


CACTGTTTCGGGGAGGTGCATTTAACGTTGATGATG


GTGGAACCACCACCACCGGA//;






The oligos above were used to form the duplex shown below:














TGGTTCCGGTGGTGGTGGTTCCACCATCATCAACGTTAAATGCACCTCCCCGAAACAGTG

SEQ ID NO: 716



1
---------+---------+---------+---------+---------+---------+
60




    AGGCCACCACCACCAAGGTGGTAGTAGTTGCAATTTACGTGGAGGGGCTTTGTCAC

SEQ ID NO: 718















 G  S  G  G  G  G  S  T  I  I  N  V  K  C  T  S  P  K  Q  C
-
SEQ ID NO: 717







CCTGCCGCCGTGCAAAGCTCAGTTCGGTCAGTCCGCTGGTGCTAAATGCATGAACGGTAA










61
---------+---------+---------+---------+---------+---------+
120




GGACGGCGGCACGTTTCGAGTCAAGCCAGTCAGGCGACCACGATTTACGTACTTGCCATT















 L  P  P  C  K  A  Q  F  G  Q  S  A  G  A  K  C  M  N  G  K
-















ATGCAAATGCTACCCGCAC



121
---------+---------



TACGTTTACGATGGGCGTGATTC






 C  K  C  Y  P  H   -






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.


Example 9
Fc-L-AgTx2 Bacterial Expression

Bacterial expression of Fc-L-AgTx2. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-AgTx2.


Oligos used to form duplex are shown below:










SEQ ID NO: 719









TGGTTCCGGTGGTGGTGGTTCCGGTGTTCCGATCAACGTTTCCTGCACCG



GT //;











SEQ ID NO: 720









TCCCCGCAGTGCATCAAACCGTGCAAAGACGCTGGTATGCGTTTCGGTAA



ATGCATGAACCGTAAATGCCACTGCACCCCGAAA //;











SEQ ID NO: 721









CTTATTTCGGGGTGCAGTGGCATTTACGGTTCATGCATTTACCGAAACGC



ATA //;











SEQ ID NO: 722









CCAGCGTCTTTGCACGGTTTGATGCACTGCGGGGAACCGGTGCAGGAAAC



GTTGATCGGAACACCGGAACCACCACCACCGGA //;






The oligos listed above were used to form the duplex shown below:














TGGTTCCGGTGGTGGTGGTTCCGGTGTTCCGATCAACGTTTCCTGCACCGGTTCCCCGCA

SEQ ID NO: 723



1
---------+---------+---------+---------+---------+---------+
60




    AGGCCACCACCACCAAGGCCACAAGGCTAGTTGCAAAGGACGTGGCCAAGGGGCGT

SEQ ID NO: 725















 G  S  G  G  G  G  S  G  V  P  I  N  V  S  C  T  G  S  P  Q
-
SEQ ID NO: 724







GTGCATCAAACCGTGCAAAGACGCTGGTATGCGTTTCGGTAAATGCATGAACCGTAAATG


61
---------+---------+---------+---------+---------+---------+
120




CACGTAGTTTGGCACGTTTCTGCGACCATACGCAAAGCCATTTACGTACTTGGCATTTAC






 C  I  K  P  C  K  D  A  G  M  R  F  G  K  C  M  N  R  K  C
-








CCACTGCACCCCGAAA_


121
---------+------



GGTGACGTGGGGCTTTATTC






 H  C  T  P  K   -






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.


Refolding and purification of Fc-L-AgTx2 expressed in bacteria. Frozen, E. coli paste (15 g) was combined with 120 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 8.0 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 22,000 g for 20 min at 4° C. The pellet was then resuspended in 200 ml 1% deoxycholic acid using a tissue grinder and then centrifuged at 22,000 g for 20 min at 4° C. The pellet was then resuspended in 200 ml water using a tissue grinder and then centrifuged at 22,000 g for 20 min at 4° C. The pellet (4.6 g) was then dissolved in 46 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. The dissolved pellet was then reduced by adding 30 μl 1 M dithiothreitol to 3 ml of the solution and incubating at 37° C. for 30 minutes. The reduced pellet solution was then centrifuged at 14,000 g for 5 min at room temperature, and then 2.5 ml of the supernatant was transferred to 250 ml of the refolding buffer (2 M urea, 50 mM tris, 160 mM arginine HCl, 5 mM EDTA, 1 mM cystamine HCl, 4 mM cysteine, pH 9.5) at 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for 2 days at 4° C. The refolding solution was then filtered through a 0.22 μm cellulose acetate filter and stored at −70° C.


The stored refold was defrosted and then diluted with 1 L of water and the pH was adjusted to 7.5 using 1 M H3PO4. The pH adjusted material was then filtered through a 0.22 μm cellulose acetate filter and loaded on to a 10 ml Amersham SP-HP HiTrap column at 10 ml/min in S-Buffer A (20 mM NaH2PO4, pH 7.3) at 7° C. The column was then washed with several column volumes of S-Buffer A, followed by elution with a linear gradient from 0% to 60% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.3) followed by a step to 100% S-Buffer B at 5 ml/min 7° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data (15 ml). The pool was then loaded on to a 1 ml Amersham rProtein A HiTrap column in PBS at 2 ml/min 7° C. Then column was then washed with several column volumes of 20 mM NaH2PO4 pH 6.5, 1 M NaCl and eluted with 100 mM glycine pH 3.0. To the elution peak (1.5 ml), 70 μl 1 M tris HCl pH 8.5 was added, and then the pH-adjusted material was filtered though a 0.22 μm cellulose acetate filter.


A spectral scan was then conducted on 20 μl of the combined pool diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 29C). The concentration of the filtered material was determined to be 1.65 mg/ml using a calculated molecular mass of 30,446 g/mol and extinction coefficient of 35,410 M−1 cm−1. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 29A). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 33-fold dilution of the sample in Charles Rivers Endotoxin Specific Buffer BG120 yielding a result of <4 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 20 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 29D). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μL of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). The resultant solution (1 μl) was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses (FIG. 29E) and these studies confirmed the integrity of the purified peptibody, within experimental error. The product was then stored at −80° C.


Example 10
Fc-L-OSK1 Bacterial Expression

Bacterial expression of Fc-L-OSK1. The methods used to clone and express the peptibody in bacteria were as described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-OSK1.


Oligos used to form duplex are shown below:










SEQ ID NO: 726









TGGTTCCGGTGGTGGTGGTTCCGGTGTTATCATCAACGTTAAATGCAAAA



TCTCCCGTCAGTGCCTGGAACCGTGCAAAAAAG //;











SEQ ID NO: 727









CTGGTATGCGTTTCGGTAAATGCATGAACGGTAAATGCCACTGCACCCCG



AAA //;











SEQ ID NO: 728









CTTATTTCGGGGTGCAGTGGCATTTACCGTTCATGCATTTACCGAAACGC



ATACCAGCTTTTTTGCACGGTTCCAGGCACTGA //;











SEQ ID NO: 729









CGGGAGATTTTGCATTTAACGTTGATGATAACACCGGAACCACCACCACC



GGA //;






The oligos shown above were used to form the duplex below:














TGGTTCCGGTGGTGGTGGTTCCGGTGTTATCATCAACGTTAAATGCAAAATCTCCCGTCA

SEQ ID NO: 730



1
---------+---------+---------+---------+---------+---------+
60




    AGGCCACCACCACCAAGGCCACAATAGTAGTTGCAATTTACGTTTTAGAGGGCAGT

SEQ ID NO: 732















 G  S  G  G  G  G  S  G  V  I  I  N  V  K  C  K  I  S  R  Q
-
SEQ ID NO: 731







GTGCCTGGAACCGTGCAAAAAAGCTGGTATGCGTTTCGGTAAATGCATGAACGGTAAATG


61
---------+---------+---------+---------+---------+---------+
120




CACGGACCTTGGCACGTTTTTTCGACCATACGCAAAGCCATTTACGTACTTGCCATTTAC






 C  L  E  P  C  K  K  A  G  M  R  F  G  K  C  M  N  G  K  C
-








CCACTGCACCCCGAAA


121
---------+------



GGTGACGTGGGGCTTTATTC






 H  C  T  P  K   -






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen for later use. Purification of Fc-L10-OSK1 from E. coli paste is described in Example 40 herein below.


Example 11
Fc-L-OSK1(E16K, K20D) Bacterial Expression

Bacterial expression of Fc-L-OSK1(E16K, K20D). The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-OSK1(E16K,K20D).


Oligos used to form duplex are shown below:










SEQ ID NO:733









TGGTTCCGGTGGTGGTGGTTCCGGTGTTATCATCAACGTTAAATGCAAAA



TCTCCCGTCAGTGCCTGAAACCGTGCAAAGACG //;











SEQ ID NO: 734









CTGGTATGCGTTTCGGTAAATGCATGAACGGTAAATGCCACTGCACCCCG



AAA //;











SEQ ID NO: 735









CTTATTTCGGGGTGCAGTGGCATTTACCGTTCATGCATTTACCGAAACGC



ATACCAGCGTCTTTGCACGGTTTCAGGCACTGA //;











SEQ ID NO: 736









CGGGAGATTTTGCATTTAACGTTGATGATAACACCGGAACCACCACCACC



GGA //;






The oligos shown above were used to form the duplex below:














TGGTTCCGGTGGTGGTGGTTCCGGTGTTATCATCAACGTTAAATGCAAAATCTCCCGTCA

SEQ ID NO: 737



1
---------+---------+---------+---------+---------+---------+
60




    AGGCCACCACCACCAAGGCCACAATAGTAGTTGCAATTTACGTTTTAGAGGGCAGT

SEQ ID NO: 739















 G  S  G  G  G  G  S  G  V  I  I  N  V  K  C  K  I  S  R  Q
-
SEQ ID NO: 738







GTGCCTGAAACCGTGCAAAGACGCTGGTATGCGTTTCGGTAAATGCATGAACGGTAAATG


61
---------+---------+---------+---------+---------+---------+
120




CACGGACTTTGGCACGTTTCTGCGACCATACGCAAAGCCATTTACGTACTTGCCATTTAC






 C  L  K  P  C  K  D  A  G  M  R  F  G  K  C  M  N  G  K  C
-








CCACTGCACCCCGAAA


121
---------+------



GGTGACGTGGGGCTTTATTC






 H  C  T  P  K   -






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen for later use.


Example 12
Fc-L-Anuroctoxin Bacterial Expression

Bacterial expression of Fc-L-Anuroctoxin. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-Anuroctoxin.


Oligos used to form duplex are shown below:










SEQ ID NO: 740









TGGTTCCGGTGGTGGTGGTTCCAAAGAATGCACCGGTCCGCAGCACTGCA



CCAACTTCTGCCGTAAAAACAAATGCACCCACG //;











SEQ ID NO: 741









GTAAATGCATGAACCGTAAATGCAAATGCTTCAACTGCAAA //;












SEQ ID NO: 742









CTTATTTGCAGTTGAAGCATTTGCATTTACGGTTCATGCATTTACCGTGG



GTGCATTTGTTTTTACGGCAGAAGTTGGTGCAG //;











SEQ ID NO: 743









TGCTGCGGACCGGTGCATTCTTTGGAACCACCACCACCGGA //;







The oligos shown above were used to form the duplex below:














TGGTTCCGGTGGTGGTGGTTCCAAAGAATGCACCGGTCCGCAGCACTGCACCAACTTCTG

SEQ ID NO: 744



1
---------+---------+---------+---------+---------+---------+
60




    AGGCCACCACCACCAAGGTTTCTTACGTGGCCAGGCGTCGTGACGTGGTTGAAGAC

SEQ ID NO: 746















 G  S  G  G  G  G  S  K  E  C  T  G  P  Q  H  C  T  N  F  C
-
SEQ ID NO: 745







CCGTAAAAACAAATGCACCCACGGTAAATGCATGAACCGTAAATGCAAATGCTTCAACTG


61
---------+---------+---------+---------+---------+---------+
120




GGCATTTTTGTTTACGTGGGTGCCATTTACGTACTTGGCATTTACGTTTACGAAGTTGAC






 R  K  N  K  C  T  H  G  K  C  M  N  R  K  C  K  C  F  N  C
-








CAAA


121
----



GTTTATTC






 K   -






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.


Example 13
Fc-L-Noxiustoxin Bacterial Expression

Bacterial expression of Fc-L-Noxiustoxin or Fc-L-NTX. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-NTX.


Oligos used to form duplex are shown below:










SEQ ID NO: 747









TGGTTCCGGTGGTGGTGGTTCCACCATCATCAACGTTAAATGCACCTCCC



CGAAACAGTGCTCCAAACCGTGCAAAGAACTGT //;











SEQ ID NO: 748









ACGGTTCCTCCGCTGGTGCTAAATGCATGAACGGTAAATGCAAATGCTAC



AACAAC //;











SEQ ID NO: 749









CTTAGTTGTTGTAGCATTTGCATTTACCGTTCATGCATTTAGCACCAGCG



GAGGAACCGTACAGTTCTTTGCACGGTTTGGAG //;











SEQ ID NO: 750









CACTGTTTCGGGGAGGTGCATTTAACGTTGATGATGGTGGAACCACCACC



ACCGGA //;






The oligos shown above were used to form the duplex below:














TGGTTCCGGTGGTGGTGGTTCCACCATCATCAACGTTAAATGCACCTCCCCGAAACAGTG

SEQ ID NO: 751



1
---------+---------+---------+---------+---------+---------+
60




    AGGCCACCACCACCAAGGTGGTAGTAGTTGCAATTTACGTGGAGGGGCTTTGTCAC

SEQ ID NO: 753












 G  S  G  G  G  G  S  T  I  I  N  V  K  C  T  S  P  K  Q  C
-
SEQ ID NO: 752







CTCCAAACCGTGCAAAGAACTGTACGGTTCCTCCGCTGGTGCTAAATGCATGAACGGTAA


61
---------+---------+---------+---------+---------+---------+
120




GAGGTTTGGCACGTTTCTTGACATGCCAAGGAGGCGACCACGATTTACGTACTTGCCATT






 S  K  P  C  K  E  L  Y  G  S  S  A  G  A  K  C  M  N  G  K
-








ATGCAAATGCTACAACAAC


121
---------+---------



TACGTTTACGATGTTGTTGATTC






 C  K  C  Y  N  N   -






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.


Example 14
Fc-L-Pi2 Bacterial Expression

Bacterial expression of Fc-L-Pi2. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-Pi2.


Oligos used to form duplex are shown below:










SEQ ID NO: 754









TGGTTCCGGTGGTGGTGGTTCCACCATCTCCTGCACCAACCCG //;












SEQ ID NO: 755









AAACAGTGCTACCCGCACTGCAAAAAAGAAACCGGTTACCCGAACGCTAA



ATGCATGAACCGTAAATGCAAATGCTTCGGTCGT //;











SEQ ID NO: 756









CTTAACGACCGAAGCATTTGCATTTACGGTTCATGCATTTAGCG //;












SEQ ID NO: 757









TTCGGGTAACCGGTTTCTTTTTTGCAGTGCGGGTAGCACTGTTTCGGGTT



GGTGCAGGAGATGGTGGAACCACCACCACCGGA //;






The oligos above were used to form the duplex below:














TGGTTCCGGTGGTGGTGGTTCCACCATCTCCTGCACCAACCCGAAACAGTGCTACCCGCA

SEQ ID NO: 758



1
---------+---------+---------+---------+---------+---------+
60




    AGGCCACCACCACCAAGGTGGTAGAGGACGTGGTTGGGCTTTGTCACGATGGGCGT

SEQ ID NO: 760















 G  S  G  G  G  G  S  T  I  S  C  T  N  P  K  Q  C  Y  P  H
-
SEQ ID NO: 759







CTGCAAAAAAGAAACCGGTTACCCGAACGCTAAATGCATGAACCGTAAATGCAAATGCTT


61
---------+---------+---------+---------+---------+---------+
120




GACGTTTTTTCTTTGGCCAATGGGCTTGCGATTTACGTACTTGGCATTTACGTTTACGAA






 C  K  K  E  T  G  Y  P  N  A  K  C  M  N  R  K  C  K  C  F
-








CGGTCGT


121
-------



GCCAGCAATTC






 G  R   -






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.


Example 15
ShK[1-35]-L-Fc Bacterial Expression

Bacterial expression of ShK[1-35]-L-Fc. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Pep-Fc and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of ShK[1-35]-L-Fc.


Oligos used to form duplex are shown below:










SEQ ID NO: 761









TATGCGTTCTTGTATTGATACTATTCCAAAATCTCGTTGTACTGCTTTTC



AATGTAAACATTCTATGAAATATCGTCTTTCTT //;











SEQ ID NO: 762









TTTGTCGTAAAACTTGTGGTACTTGTTCTGGTGGTGGTGGTTCT //;












SEQ ID NO: 763









CACCAGAACCACCACCACCAGAACAAGTACCACAAGTTTTACGACAAAAA



GAAAGACGATATTTCATAGAATGTTTACATTGA //;











SEQ ID NO: 764









AAAGCAGTACAACGAGATTTTGGAATAGTATCAATACAAGAACG //;







The oligos shown above were used to form the duplex shown below:














TATGCGTTCTTGTATTGATACTATTCCAAAATCTCGTTGTACTGCTTTTCAATGTAAACA

SEQ ID NO: 765



1
---------+---------+---------+---------+---------+---------+
60




    GCAAGAACATAACTATGATAAGGTTTTAGAGCAACATGACGAAAAGTTACATTTGT

SEQ ID NO: 767















 M  R  S  C  I  D  T  I  P  K  S  R  C  T  A  F  Q  C  K  H
-
SEQ ID NO: 766







TTCTATGAAATATCGTCTTTCTTTTTGTCGTAAAACTTGTGGTACTTGTTCTGGTGGTGG


61
---------+---------+---------+---------+---------+---------+
120




AAGATACTTTATAGCAGAAAGAAAAACAGCATTTTGAACACCATGAACAAGACCACCACC






 S  M  K  Y  R  L  S  F  C  R  K  T  C  G  T  C  S  G  G  G
-








TGGTTCT


121
-------
127



ACCAAGACCAC






 G  S   -






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen. Purification of met-ShK[1-35]-Fc was as described in Example 51 herein below.


Example 16
ShK[2-35]-L-Fc Bacterial Expression

Bacterial expression of ShK[2-35]-L-Fc. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Pep-Fc and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of ShK[2-35]-L-Fc.


Oligos used to form duplex are shown below:










TATGTCTTGTATTGATACTATTCCAAAATCT
SEQ ID NO: 768


CGTTGTACTGCTTTTCAATGTAAACATTCT


ATGAAATATCGTCTTTCTT//;





TTTGTCGTAAAACTTGTGGTACTTGTTCTGGT
SEQ ID NO: 769


GGTGGTGGTTCT//;





CACCAGAACCACCACCACCAGAACAAGTA
SEQ ID NO: 770


CCACAAGTTTTACGACAAAAAGAAAGACGA


TATTTCATAGAATGTTTACATTGA//;





AAAGCAGTACAACGAGATTTTGGAATAGTA
SEQ ID NO: 771


TCAATACAAGA;






The oligos above were used to form the duplex shown below:













TATGTCTTGTATTGATACTATTCCAAAATCTCGTTGTACTGCTTTTCAATGTAAACATTC




1
---------+---------+---------+---------+---------+---------+
 60



    AGAACATAACTATGATAAGGTTTTAGAGCAACATGACGAAAAGTTACATTTGTAAG



 M  S  C  I  D  T  I  P  K  S  R  C  T  A  F  Q  C  K  H  S
-






TATGAAATATCGTCTTTCTTTTTGTCGTAAAACTTGTGGTACTTGTTCTGGTGGTGGTGG


61
---------+---------+---------+---------+---------+---------+
120



ATACTTTATAGCAGAAAGAAAAACAGCATTTTGAACACCATGAACAAGACCACCACCACC



 M  K  Y  R  L  S  F  C  R  K  T  C  G  T  C  S  G  G  G  G
-






TTCT
SEQ ID NO: 772


121
----



AAGACCAC
SEQ ID NO: 774



 S   -
SEQ ID NO: 773






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen. Purification of the ShK[2-35]-Fc was as described in Example 50 herein below.


Example 17
Fc-L-ChTx Bacterial Expression

Bacterial expression of Fc-L-ChTx. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-ChTx.


Oligos used to form duplex are shown below:










TGGTTCCGGTGGTGGTGGTTCCCAGTTCACC
SEQ ID NO: 775


AACGTT//;





TCCTGCACCACCTCCAAAGAATGCTGGTCCG
SEQ ID NO: 776


TTTGCCAGCGTCTGCACAACACCTCCCGTGGT


AAATGCATGAACAAAAAATGCCGTTGCTACTCC;





CTTAGGAGTAGCAACGGCATTTTTTGTTCATGC
SEQ ID NO: 777


ATTTA//;





CCACGGGAGGTGTTGTGCAGACGCTGGCAAA
SEQ ID NO: 778


CGGACCAGCATTCTTTGGAGGTGGTGCAGGA


AACGTTGGTGAACTGGGAACCACCACCACC


GGA//;






The oligos shown above were used to form the duplex below:













TGGTTCCGGTGGTGGTGGTTCCCAGTTCACCAACGTTTCCTGCACCACCTCCAAAGAATG




1
---------+---------+---------+---------+---------+---------+
 60



    AGGCCACCACCACCAAGGGTCAAGTGGTTGCAAAGGACGTGGTGGAGGTTTCTTAC



 G  S  G  G  G  G  S  Q  F  T  N  V  S  C  T  T  S  K  E  C
-






CTGGTCCGTTTGCCAGCGTCTGCACAACACCTCCCGTGGTAAATGCATGAACAAAAAATG


61
---------+---------+---------+---------+---------+---------+
120



GACCAGGCAAACGGTCGCAGACGTGTTGTGGAGGGCACCATTTACGTACTTGTTTTTTAC



 W  S  V  C  Q  R  L  H  N  T  S  R  G  K  C  M  N  K  K  C
-






CCGTTGCTACTCC
SEQ ID NO: 779


121
---------+---



GGCAACGATGAGGATTC
SEQ ID NO: 781



 R  C  Y  S   -
SEQ ID NO: 780






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.


Example 18
Fc-L-MTX Bacterial Expression

Bacterial expression of Fc-L-MTX. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-MTX.


Oligos used to form duplex are shown below:










TGGTTCCGGTGGTGGTGGTTCGGTTTCCTGCA
SEQ ID NO: 782


CCGGT//;





TCCAAAGACTGCTACGCTCCGTGCCGTAAACA
SEQ ID NO: 783


GACCGGTTGCCCGAACGCTAAATGCATCAACA


AATCCTGCAAATGCTACGGTTGC//;





CTTAGCAACCGTAGCATTTGCAGGATTTGTTGAT
SEQ ID NO: 784


GCAT//;





TTAGCGTTCGGGCAACCGGTCTGTTTACGGCAC
SEQ ID NO: 785


GGAGCGTAGCAGTCTTTGGAACCGGTGCAGGA


AACGGAACCACCACCACCGGA//;






The oligos above were used to form the duplex shown below:













TGGTTCCGGTGGTGGTGGTTCCGTTTCCTGCACCGGTTCCAAAGACTGCTACGCTCCGTG




1
---------+---------+---------+---------+---------+---------+
 60



    AGGCCACCACCACCAAGGCAAAGGACGTGGCCAAGGTTTCTGACGATGCGAGGCAC



 G  S  G  G  G  G  S  V  S  C  T  G  S  K  D  C  Y  A  P  C
-






CCGTAAACAGACCGGTTGCCCGAACGCTAAATGCATCAACAAATCCTGCAAATGCTACGG


61
---------+---------+---------+---------+---------+---------+
120



GGCATTTGTCTGGCCAACGGGCTTGCGATTTACGTAGTTGTTTAGGACGTTTACGATGCC



 R  K  Q  T  G  C  P  N  A  K  C  I  N  K  S  C  K  C  Y  G
-






TTGC
SEQ ID NO: 786


121
----



AACGATTC
SEQ ID NO: 788



 C   -
SEQ ID NO: 787






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.


Example 19
Fc-L-ChTx (K32E) Bacterial Expression

Bacterial expression of Fc-L-ChTx (K32E). The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-ChTx (K32E).


Oligos used to form duplex are shown below:










TGGTTCCGGTGGTGGTGGTTCCCAGTTCACCA
SEQ ID NO: 789


ACGTTTCCTG//;





CACCACCTCCAAAGAATGCTGGTCCGTTTGCC
SEQ ID NO: 790


AGCGTCTGCACAACACCTCCCGTGGTAAATGC


ATGAACAAAGAATGCCGTTGCTACTCC//;





CTTAGGAGTAGCAACGGCATTCTTTGTTCATGCA
SEQ ID NO: 791


TTTACCACG//;





GGAGGTGTTGTGCAGACGCTGGCAAACGGACCA
SEQ ID NO: 792


GCATTCTTTGGAGGTGGTGCAGGAAACGTTGGTG


AACTGGGAACCACCACCACCGGA//;






The oligos shown above were used to form the duplex below:













TGGTTCCGGTGGTGGTGGTTCCCAGTTCACCAACGTTTCCTGCACCACCTCCAAAGAATG




1
---------+---------+---------+---------+---------+---------+
 60



    AGGCCACCACCACCAAGGGTCAAGTGGTTGCAAAGGACGTGGTGGAGGTTTCTTAC



 G  S  G  G  G  G  S  Q  F  T  N  V  S  C  T  T  S  K  E  C
-






CTGGTCCGTTTGCCAGCGTCTGCACAACACCTCCCGTGGTAAATGCATGAACAAAGAATG


61
---------+---------+---------+---------+---------+---------+
120



GACCAGGCAAACGGTCGCAGACGTGTTGTGGAGGGCACCATTTACGTACTTGTTTCTTAC



 W  S  V  C  Q  R  L  H  N  T  S  R  G  K  C  M  N  K  E  C
-






CCGTTGCTACTCC
SEQ ID NO: 793


121
---------+---



GGCAACGATGAGGATTC
SEQ ID NO: 795



 R  C  Y  S   -
SEQ ID NO: 794






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.


Example 20
Fc-L-Apamin Bacterial Expression

Bacterial expression of Fc-L-Apamin. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-Apamin.


Oligos used to form duplex are shown below:










TGGTTCCGGTGGTGGTGGTTCCTGCAACTGCA
SEQ ID NO: 796


AAGCTCCGGAAACCGCTCTGTGCGCTCGTCG


TTGCCAGCAGCACGGT//;





CTTAACCGTGCTGCTGGCAACGACGAGCGCA
SEQ ID NO: 797


CAGAGCGGTTTCCGGAGCTTTGCAGTTGCAGG


AACCACCACCACCGGA//;






The oligos above were used to form the duplex shown below:













TGGTTCCGGTGGTGGTGGTTCCTGCAACTGCAAAGCTCCGGAAACCGCTCTGTGCGCTCG




1
---------+---------+---------+---------+---------+---------+
60



    AGGCCACCACCACCAAGGACGTTGACGTTTCGAGGCCTTTGGCGAGACACGCGAGC



 G  S  G  G  G  G  S  C  N  C  K  A  P  E  T  A  L  C  A  R
-






TCGTTGCCAGCAGCACGGT
SEQ ID NO: 798


61
---------+---------



AGCAACGGTCGTCGTGCCAATTC
SEQ ID NO: 800



 R  C  Q  Q  H  G   -
SEQ ID NO: 799






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.


Example 21
Fc-L-Scyllatoxin Bacterial Expression

Bacterial expression of Fc-L-Scyllatoxin or Fc-L-ScyTx. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-ScyTx.


Oligos used to form duplex are shown below:










TGGTTCCGGTGGTGGTGGTTCCGCTTTCTGCA
SEQ ID NO: 801


ACCTGCG//;





TATGTGCCAGCTGTCCTGCCGTTCCCTGGGTC
SEQ ID NO: 802


TGCTGGGTAAATGCATCGGTGACAAATGCGAA


TGCGTTAAACAC//;





CTTAGTGTTTAACGCATTCGCATTTGTCACCGAT
SEQ ID NO: 803


GCATTT//;





ACCCAGCAGACCCAGGGAACGGCAGGACAGC
SEQ ID NO: 804


TGGCACATACGCAGGTTGCAGAAAGCGGAACC


ACCACCACCGGA//;






The oligos above were used to form the duplex below:













TGGTTCCGGTGGTGGTGGTTCCGCTTTCTGCAACCTGCGTATGTGCCAGCTGTCCTGCCG




1
---------+---------+---------+---------+---------+---------+
60



    AGGCCACCACCACCAAGGCGAAAGACGTTGGACGCATACACGGTCGACAGGACGGC



 G  S  G  G  G  G  S  A  F  C  N  L  R  M  C  Q  L  S  C  R
-






TTCCCTGGGTCTGCTGGGTAAATGCATCGGTGACAAATGCGAATGCGTTAAACAC
SEQ ID NO: 805


61
---------+---------+---------+---------+---------+---------



AAGGGACCCAGACGACCCATTTACGTAGCCACTGTTTACGCTTACGCAATTTGTGATTC
SEQ ID NO: 807



 S  L  G  L  L  G  K  C  I  G  D  K  C  E  C  V  K  H   -
SEQ ID NO: 806






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.


Example 22
Fc-L-IbTx Bacterial Expression

Bacterial expression of Fc-L-lbTx. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-lbTx.


Oligos used to form duplex are shown below:










TGGTTCCGGTGGTGGTGGTTCCCAGTTCACCG
SEQ ID NO: 808


ACGTTGACTGCTCCGT//;





TTCCAAAGAATGCTGGTCCGTTTGCAAAGACC
SEQ ID NO: 809


TGTTCGGTGTTGACCGTGGTAAATGCATGGGTA


AAAAATGCCGTTGCTACCAG//;





CTTACTGGTAGCAACGGCATTTTTTACCCATGCA
SEQ ID NO: 810


TTTACCACGGTCAA//;





CACCGAACAGGTCTTTGCAAACGGACCAGCATT
SEQ ID NO: 811


CTTTGGAAACGGAGCAGTCAACGTCGGTGAACT


GGGAACCACCACCACCGGA//;






The oligos above were used to form the duplex below:













TGGTTCCGGTGGTGGTGGTTCCCAGTTCACCGACGTTGACTGCTCCGTTTCCAAAGAATG




1
---------+---------+---------+---------+---------+---------+
60



    AGGCCACCACCACCAAGGGTCAAGTGGCTGCAACTGACGAGGCAAAGGTTTCTTAC



 G  S  G  G  G  G  S  Q  F  T  D  V  D  C  S  V  S  K  E  C
-






CTGGTCCGTTTGCAAAGACCTGTTCGGTGTTGACCGTGGTAAATGCATGGGTAAAAAATG


61
---------+---------+---------+---------+---------+---------+
120



GACCAGGCAAACGTTTCTGGACAAGCCACAACTGGCACCATTTACGTACCCATTTTTTAC



 W  S  V  C  K  D  L  F  G  V  D  R  G  K  C  M  G  K  K  C
-






CCGTTGCTACCAG
SEQ ID NO: 812


121
---------+---



GGCAACGATGGTCATTC
SEQ ID NO: 814



 R  C  Y  Q   -
SEQ ID NO: 813






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.


Example 23
Fc-L-HaTx1 Bacterial Expression

Bacterial expression of Fc-L-HaTx1. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-HaTx1.


Oligos used to form duplex are shown below:










TGGTTCCGGTGGTGGTGGTTCCGAATGCCGTT
SEQ ID NO: 815


ACCTGTTCGGTGGTTG//;





CAAAACCACCTCCGACTGCTGCAAACACCTG
SEQ ID NO: 816


GGTTGCAAATTCCGTGACAAATACTGCGCTTG


GGACTTCACCTTCTCC//;





CTTAGGAGAAGGTGAAGTCCCAAGCGCAGTAT
SEQ ID NO: 817


TTGTCACGGAATTTGC//;





AACCCAGGTGTTTGCAGCAGTCGGAGGTGGTT
SEQ ID NO: 818


TTGCAACCACCGAACAGGTAACGGCATTCGGA


ACCACCACCACCGGA//;






The oligos above were used to form the duplex below:














TGGTTCCGGTGGTGGTGGTTCCGAATGCCGTTACCTGTTCGGTGGTTGCAAAACCACCTC

SEQ ID NO: 819



1
---------+---------+---------+---------+---------+---------+
60




    AGGCCACCACCACCAAGGCTTACGGCAATGGACAAGCCACCAACGTTTTGGTGGAG

SEQ ID NO: 821















 G  S  G  G  G  G  S  E  C  R  Y  L  F  G  G  C  K  T  T  S
-
SEQ ID NO: 820







CGACTGCTGCAAACACCTGGGTTGCAAATTCCGTGACAAATACTGCGCTTGGGACTTCAC


61
---------+---------+---------+---------+---------+---------+
120




GCTGACGACGTTTGTGGACCCAACGTTTAAGGCACTGTTTATGACGCGAACCCTGAAGTG














 D  C  C  K  H  L  G  C  K  F  R  D  K  Y  C  A  W  D  F  T
-







CTTCTCC


121
-------



GAAGAGGATTC






 F  S   -






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.


Refolding and purification of Fc-L-HaTx1 expressed in bacteria. Frozen, E. coli paste (13 g) was combined with 100 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 8.0 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 22,000 g for 20 min at 4° C. The pellet was then resuspended in 200 ml 1% deoxycholic acid using a tissue grinder and then centrifuged at 22,000 g for 20 min at 4° C. The pellet was then resuspended in 200 ml water using a tissue grinder and then centrifuged at 22,000 g for 20 min at 4° C. The pellet (2.6 g) was then dissolved in 26 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. The dissolved pellet was then reduced by adding 30 μl 1 M dithiothreitol to 3 ml of the solution and incubating at 37° C. for 30 minutes. The reduced pellet solution was then centrifuged at 14,000 g for 5 min at room temperature, and then 2.5 ml of the supernatant was transferred to 250 ml of the refolding buffer (2 M urea, 50 mM tris, 160 mM arginine HCl, 5 mM EDTA, 1 mM cystamine HCl, 4 mM cysteine, pH 8.5) at 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for 2 days at 4° C. The refolding solution was then filtered through a 0.22 μm cellulose acetate filter and stored at −70° C.


The stored refold was defrosted and then diluted with 1 L of water and the pH was adjusted to 7.5 using 1 M H3PO4. The pH adjusted material was then filtered through a 0.22 μm cellulose acetate filter and loaded on to a 10 ml Amersham SP-HP HiTrap column at 10 ml/min in S-Buffer A (20 mM NaH2PO4, pH 7.3) at 7° C. The column was then washed with several column volumes of S-Buffer A, followed by elution with a linear gradient from 0% to 60% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.3) followed by a step to 100% S-Buffer B at 5 ml/min 7° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data (15 ml). The pool was then loaded on to a 1 ml Amersham rProtein A HiTrap column in PBS at 2 ml/min 7° C. Then column was then washed with several column volumes of 20 mM NaH2PO4 pH 6.5, 1 M NaCl and eluted with 100 mM glycine pH 3.0. To the elution peak (1.4 ml), 70 μl 1 M tris HCl pH 8.5 was added, and then the pH adjusted material was filtered though a 0.22 μm cellulose acetate filter.


A spectral scan was then conducted on 20 μl of the combined pool diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 29F). The concentration of the filtered material was determined to be 1.44 mg/ml using a calculated molecular mass of 30,469 g/mol and extinction coefficient of 43,890 M−1 cm−1. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 29B). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 33-fold dilution of the sample in Charles Rivers Endotoxin Specific Buffer BG120 yielding a result of <4 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 20 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 29G). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). The resultant solution (1 μl) was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses (FIG. 29H) and these studies confirmed the integrity of the purified peptibody, within experimental error. The product was then stored at −80° C.


Example 24
Fc-L-PaTx2 Bacterial Expression

Bacterial expression of Fc-L-PaTx2. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-PaTx2.


Oligos used to form duplex are shown below:










SEQ ID NO: 822









TGGTTCCGGTGGTGGTGGTTCCTACTGCCAGAAATGGA //;












SEQ ID NO: 823









TGTGGACCTGCGACGAAGAACGTAAATGCTGCGAAGGTCTGGTTTGCCGT



CTGTGGTGCAAACGTATCATCAACATG //;











SEQ ID NO: 824









CTTACATGTTGATGATACGTTTGCACCACAGACGGCAAA //;












SEQ ID NO: 825









CCAGACCTTCGCAGCATTTACGTTCTTCGTCGCAGGTCCACATCCATTTC



TGGCAGTAGGAACCACCACCACCGGA //;






The oligos above were used to form the duplex below:













1
TGGTTCCGGTGGTGGTGGTTCCTACTGCCAGAAATGGATGTGGACCTGCGACGAAGAACG

SEQ ID NO: 826




---------+---------+---------+---------+---------+---------+
60




    AGGCCACCACCACCAAGGATGACGGTCTTTACCTACACCTGGACGCTGCTTCTTGC

SEQ ID NO: 828















 G  S  G  G  G  G  S  Y  C  Q  K  W  M  W  T  C  D  E  E  R
-
SEQ ID NO: 827







TAAATGCTGCGAAGGTCTGGTTTGCCGTCTGTGGTGCAAACGTATCATCAACATG


61
---------+---------+---------+---------+---------+-----



ATTTACGACGCTTCCAGACCAAACGGCAGACACCACGTTTGCATAGTAGTTGTACATTC






 K  C  C  E  G  L  V  C  R  L  W  C  K  R  I  I  N  M   -






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.


Example 25
Fc-L-wGVIA Bacterial Expression

Bacterial expression of Fc-L-wGVIA. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-wGVIA.


Oligos used to form duplex are shown below:










SEQ ID NO: 829









TGGTTCCGGTGGTGGTGGTTCCTGCAAATCCCCGGGTT //;












SEQ ID NO: 830









CCTCCTGCTCCCCGACCTCCTACAACTGCTGCCGTTCCTGCAACCCGTAC



ACCAAACGTTGCTACGGT;











SEQ ID NO: 831









CTTAACCGTAGCAACGTTTGGTGTACGGGTTGCAGGAA //;












SEQ ID NO: 832









CGGCAGCAGTTGTAGGAGGTCGGGGAGCAGGAGGAACCCGGGGATTTGCA



GGAACCACCACCACCGGA //;






The oligos above were used to form the duplex below:














TGGTTCCGGTGGTGGTGGTTCCTGCAAATCCCCGGGTTCCTCCTGCTCCCCGACCTCCTA

SEQ ID NO: 833



1
---------+---------+---------+---------+---------+---------+
60




    AGGCCACCACCACCAAGGACGTTTAGGGGCCCAAGGAGGACGAGGGGCTGGAGGAT

SEQ ID NO: 835















 G  S  G  G  G  G  S  C  K  S  P  G  S  S  C  S  P  T  S  Y
-
SEQ ID NO: 834







CAACTGCTGCCGTTCCTGCAACCCGTACACCAAACGTTGCTACGGT


61
---------+---------+---------+---------+------



GTTGACGACGGCAAGGACGTTGGGCATGTGGTTTGCAACGATGCCAATTC






 N  C  C  R  S  C  N  P  Y  T  K  R  C  Y  G






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.


Example 26
Fc-L-ωMVIIA Bacterial Expression

Bacterial expression of Fc-L-ωMVIIA. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-ωMVIIA.


Oligos used to form duplex are shown below:










SEQ ID NO: 836









TGGTTCCGGTGGTGGTGGTTCCTGCAAAGGTAAA //;












SEQ ID NO: 837









GGTGCTAAATGCTCCCGTCTGATGTACGACTGCTGCACCGGTTCCTGCCG



TTCCGGTAAATGCGGT //;











SEQ ID NO: 836









CTTAACCGCATTTACCGGAACGGCAGGAACCGGT //;












SEQ ID NO: 839









GCAGCAGTCGTACATCAGACGGGAGCATTTAGCACCTTTACCTTTGCAGG



AACCACCACCACCGGA //;






The oligos above were used to form the duplex below:














TGGTTCCGGTGGTGGTGGTTCCTGCAAAGGTAAAGGTGCTAAATGCTCCCGTCTGATGTA

SEQ ID NO: 840



1
---------+---------+---------+---------+---------+---------+
60




    AGGCCACCACCACCAAGGACGTTTCCATTTCCACGATTTACGAGGGCAGACTACAT

SEQ ID NO: 842















 G  S  G  G  G  G  S  C  K  G  K  G  A  K  C  S  R  L  M  Y
-
SEQ ID NO: 841







CGACTGCTGCACCGGTTCCTGCCGTTCCGGTAAATGCGGT


61
---------+---------+---------+---------+



GCTGACGACGTGGCCAAGGACGGCAAGGCCATTTACGCCAATTC






 D  C  C  T  G  S  C  R  S  G  K  C  G   -






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.


Example 27
Fc-L-PtuI Bacterial Expression

Bacterial expression of Fc-L-Ptu1. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-Ptu1.


Oligos used to form duplex are shown below:










SEQ ID NO: 843









TGGTTCCGGTGGTGGTGGTTCCGCTGAAAAAGACTGCATC //;












SEQ ID NO: 844









GCTCCGGGTGCTCCGTGCTTCGGTACCGACAAACCGTGCTGCAACCCGCG



TGCTTGGTGCTCCTCCTACGCTAACAAATGCCTG //;











SEQ ID NO: 845









CTTACAGGCATTTGTTAGCGTAGGAGGAGCACCAAGCACG //;












SEQ ID NO: 846









CGGGTTGCAGCACGGTTTGTCGGTACCGAAGCACGGAGCACCCGGAGCGA



TGCAGTCTTTTTCAGCGGAACCACCACCACCGGA //;






The oligos above were used to form the duplex below:












1
TGGTTCCGGTGGTGGTGGTTCCGCTGAAAAAGACTGCATCGCTCCGGGTGCTCCGTGCTT

SEQ ID NO: 847



---------+---------+---------+---------+---------+---------+
60




    AGGCCACCACCACCAAGGCGACTTTTTCTGACGTAGCGAGGCCCACGAGGCACGAA

SEQ ID NO: 849















 G  S  G  G  G  G  S  A  E  K  D  C  I  A  P  G  A  P  C  F
-
SEQ ID NO: 848







CGGTACCGACAAACCGTGCTGCAACCCGCGTGCTTGGTGCTCCTCCTACGCTAACAAATG


61
---------+---------+---------+---------+---------+---------+
120




GCCATGGCTGTTTGGCACGACGTTGGGCGCACGAACCACGAGGAGGATGCGATTGTTTAC














 G  T  D  K  P  C  C  N  P  R  A  W  C  S  S  Y  A  N  K  C
-







CCTG


121
----



GGACATTC






 L   -






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.


Example 28
Fc-L-ProTx1 Bacterial Expression

Bacterial expression of Fc-L-ProTx1. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-ProTx1.


Oligos used to form duplex are shown below:










SEQ ID NO: 850









TGGTTCCGGTGGTGGTGGTTCCGAATGCCGTTACTGGCTGG //;












SEQ ID NO: 851









GTGGTTGCTCCGCTGGTCAGACCTGCTGCAAACACCTGGTTTGCTCCCGT



CGTCACGGTTGGTGCGTTTGGGACGGTACCTTCTCC //;











SEQ ID NO: 852









CTTAGGAGAAGGTACCGTCCCAAACGCACCAACCGTGACGA //;












SEQ ID NO: 853









CGGGAGCAAACCAGGTGTTTGCAGCAGGTCTGACCAGCGGAGCAACCACC



CAGCCAGTAACGGCATTCGGAACCACCACCACCGGA //;






The oligos above were used to form the duplex below:













1
TGGTTCCGGTGGTGGTGGTTCCGAATGCCGTTACTGGCTGGGTGGTTGCTCCGCTGGTCA

SEQ ID HO: 854




---------+---------+---------+---------+---------+---------+
60




    AGGCCACCACCACCAAGGCTTACGGCAATGACCGACCCACCAACGAGGCGACCAGT

SEQ ID NO: 856















 G  S  G  G  G  G  S  E  C  R  Y  W  L  G  G  C  S  A  G  Q
-
SEQ ID NO: 855







GACCTGCTGCAAACACCTGGTTTGCTCCCGTCGTCACGGTTGGTGCGTTTGGGACGGTAC


61
---------+---------+---------+---------+---------+---------+
120




CTGGACGACGTTTGTGGACCAAACGAGGGCAGCAGTGCCAACCACGCAAACCCTGCCATG














 T  C  C  K  H  L  V  C  S  R  R  H  G  W  C  V  W  D  G  T
-







CTTCTCC


121
-------



GAAGAGGATTC






 F  S   -






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.


Example 29
Fc-L-BeKM1 Bacterial Expression

Bacterial expression of Fc-L-BeKM1. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-BeKM1.


Oligos used to form duplex are shown below:










SEQ ID NO: 857









TGGTTCCGGTGGTGGTGGTTCCCGTCCGACCGACATCAAATG //;












SEQ ID NO: 858









CTCCGAATCCTACCAGTGCTTCCCGGTTTGCATCCCGTTTCGGTAAAACC



AACGGTCGTTGCGTTAACGGTTTCTGCGACTGCTTC //;











SEQ ID NO: 859









CTTAGAAGCAGTCGCAGAAACCGTTAACGCAACGACCGTTGG //;












SEQ ID NO: 860









TTTTACCGAAACGGGATTTGCAAACCGGGAAGCACTGGTAGGATTCGGAG



CATTTGATGTCGGTCGGACGGGAACCACCACCACCGGA //;






The oligos above were used to form the duplex below:














TGGTTCCGGTGGTGGTGGTTCCCGTCCGACCGACATCAAATGCTCCGAATCCTACCAGTG

SEQ ID NO: 861



1
---------+---------+---------+---------+---------+---------+
60




    AGGCCACCACCACCAAGGGCAGGCTGGCTGTAGTTTACGAGGCTTAGGATGGTCAC

SEQ ID NO: 863















 G  S  G  G  G  G  S  R  P  T  D  I  K  C  S  E  S  Y  Q  C
-
SEQ ID NO: 862







CTTCCCGGTTTGCAAATCCCGTTTCGGTAAAACCAACGGTCGTTGCGTTAACGGTTTCTG


61
---------+---------+---------+---------+---------+---------+
120




GAAGGGCCAAACGTTTAGGGCAAAGCCATTTTGGTTGCCAGCAACGCAATTGCCAAAGAC














 F  P  V  C  K  S  R  F  G  K  T  N  G  R  C  V  N  G  F  C
-







CGACTGCTTC


121
---------+



GCTGACGAAGATTC






 D  C  F   -






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.


Example 30
Fc-L-CTX Bacterial Expression

Bacterial expression of Fc-L-CTX. The methods to clone and express the peptibody in bacteria are described in Example 3. The vector used was pAMG21ampR-Fc-Pep and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Fc-L-CTX.


Oligos used to form duplex are shown below:










SEQ ID NO: 864









TGGTTCCGGTGGTGGTGGTTCCATGTGCATGCCGTGCTTCAC //;












SEQ ID NO: 865









CACCGACCACCAGATGGCTCGTAAATGCGACGACTGCTGCGGTGGTAAAG



GTCGTGGTAAATGCTACGGTCCGCAGTGCCTGTGCCGT //;











SEQ ID NO: 866









CTTAACGGCACAGGCACTGCGGACCGTAGCATTTACCACGAC //;












SEQ ID NO: 867









CTTTACCACCGCAGCAGTCGTCGCATTTACGAGCCATCTGGTGGTCGGTG



GTGAAGCACGGCATGCACATGGAACCACCACCACCGGA //;






The oligos above were used to form the duplex below:














TGGTTCCGGTGGTGGTGGTTCCATGTGCATGCCGTGCTTCACCACCGACCACCAGATGGC

SEQ ID NO: 868



1
---------+---------+---------+---------+---------+---------+
60




    AGGCCACCACCACCAAGGTACACGTACGGCACGAAGTGGTGGCTGGTGGTCTACCG

SEQ ID NO: 870















 G  S  G  G  G  G  S  M  C  M  P  C  F  T  T  D  H  Q  M  A
-
SEQ ID NO: 869







TCGTAAATGCGACGACTGCTGCGGTGGTAAAGGTCGTGGTAAATGCTACGGTCCGCAGTG


61
---------+---------+---------+---------+---------+---------+
120




AGCATTTACGCTGCTGACGACGCCACCATTTCCAGCACCATTTACGATGCCAGGCGTCAC














 R  K  C  D  D  C  C  G  G  K  G  R  G  K  C  Y  G  P  Q  C
-







CCTGTGCCGT


121
---------+



GGACACGGCACCAC






 L  C  R   -






Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.


Example 31
N-Terminally PEGylated-Des-Arg1-ShK

Peptide Synthesis of reduced Des-Arq1-ShK. Des-Arg1-ShK, having the sequence





SCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC  (Peptide 1, SEQ ID NO: 92)


was synthesized in a stepwise manner on a Symphony™ multi-peptide synthesizer by solid-phase peptide synthesis (SPPS) using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)/N-methyl morpholine (NMM)/N,N-dimethyl-formamide (DMF) coupling chemistry at 0.1 mmol equivalent resin scale on Tentagel™-S PHB Fmoc-Cys(Trt)-resin. N-alpha-(9-fluorenylmethyloxycarbonyl)- and side-chain protected amino acids were purchased from Midwest Biotech Incorporated. Fmoc-Cys(Trt)-Tentagel™ resin was purchased from Fluka. The following side-chain protection strategy was employed: Asp(OtBu), Arg(Pbf), Cys(Trt), Gln(Trt), His(Trt), Lys(Nε-Boc), Ser(OtBu), Thr(OtBu) and Tyr(OtBu). Two Oxazolidine dipeptides, Fmoc-Gly-Thr(ψMe,MePro)-OH and Fmoc-Leu-Ser(ψMe,MePro)-OH, were used in the chain assembly and were obtained from NovaBiochem and used in the synthesis of the sequence. The protected amino acid derivatives (20 mmol) were dissolved in 100 ml 20% dimethyl sulfoxide (DMSO) in DMF (v/v). Protected amino acids were activated with 20 mM HBTU, 400 mM NMM in 20% DMSO in DMF, and coupling were carried out using two treatments with 0.5 mmol protected amino acid, 0.5 mmol HBTU, 1 mmol NMM in 20% DMF/DMSO for 25 minutes and then 40 minutes. Fmoc deprotection reactions were carried out with two treatments using a 20% piperidine in DMF (v/v) solution for 10 minutes and then 15 minutes. Following synthesis, the resin was then drained, and washed with DCM, DMF, DCM, and then dried in vacuo. The peptide-resin was deprotected and released from the resin by treatment with a TFA/EDT/TIS/H2O (92.5:2.5:2.5:2.5 (v/v)) solution at room temperature for 1 hour. The volatiles were then removed with a stream of nitrogen gas, the crude peptide precipitated twice with cold diethyl ether and collected by centrifugation. The crude peptide was then analyzed on a Waters 2795 analytical RP-HPLC system using a linear gradient (0-60% buffer B in 12 minutes, A: 0.1% TFA in water, B: 0.1% TFA in acetonitrile) on a Jupiter 4 μm Proteo™ 90 Å column. A PE-Sciex™ API Electro-spray mass spectrometer was used to confirm correct peptide product mass. Crude peptide was obtained in 143 mg yield at approximately 70% pure based as estimated by analytical RP-HPLC analysis. Reduced Des-Arg1-ShK (Peptide 1) Retention time (Rt)=5.31 minutes, calculated molecular weight=3904.6917 Da (average); Experimental observed molecular weight 3907.0 Da.


Folding of Des-Arq1-ShK (Disulphide bond formation). Following TFA cleavage and peptide precipitation, reduced Des-Arg1-ShK was then air-oxidized to give the folded peptide. The crude cleaved peptide was extracted using 20% AcOH in water (v/v) and then diluted with water to a concentration of approximately 0.15 mg reduced Des-Arg1-ShK per mL, the pH adjusted to about 8.0 using NH4OH (28-30%), and gently stirred at room temperature for 36 hours. Folding process was monitored by LC-MS analysis. Following this, folded Des-Arg1-ShK peptide was purified using reversed phase HPLC using a 1  Luna 5 μm C18 100 Å Proteo™ column with a linear gradient 0-40% buffer B in 120 min (A=0.1% TFA in water, B=0.1% TFA in acetonitrile). Folded Des-Arg1-ShK crude peptide eluted earlier (when compared to the elution time in its reduced form) at approximately 25% buffer B. Folded Des-Arg1-ShK (Peptide 2) was obtained in 23.2 mg yield in >97% purity as estimated by analytical RP-HPLC analysis (FIG. 20A). Calculated molecular weight=3895.7693 Da (monoisotopic), experimental observed molecular weight=3896.5 Da(analyzed on a Waters LCT Premier Micromass MS Technologies). (FIG. 20B). Des-Arg1-ShK disulfide connectivity was C1-C6, C2-C4, C3-C5.







N-terminal PEGylation of Folded Des-Arq1-ShK. Folded Des-Arg1-ShK, (Peptide 2) was dissolved in water at 1 mg/ml concentration. A 2 M MeO-PEG-Aldehyde, CH3O—[CH2CH2O]n-CH2CH2CHO (average molecular weight 20 kDa), solution in 50 mM NaOAc, pH 4.5, and a separate 1 M solution of NaCNBH3 were freshly prepared. The peptide solution was then added to the MeO-PEG-Aldehyde containing solution and was followed by the addition of the NaCNBH3 solution. The reaction stoichiometry was peptide:PEG:NaCNBH3 (1:2:0.02), respectively. The reaction was left for 48 hours, and was analyzed on an Agilent 1100 RP-HPLC system using Zorbax™ 300SB-C8 5 μm column at 40° C. with a linear gradient (6-60% B in 16 minutes, A: 0.1% TFA in water, B: 0.1% TFA/90% ACN in water). Mono-pegylated folded Des-Arg1-ShK constituted approximately 58% of the crude product by analytical RP-HPLC. Mono Pegylated Des-Arg1-ShK was then isolated using a HiTrap™ 5 ml SP HP cation exchange column on AKTA FPLC system at 4° C. at 1 mL/min using a gradient of 0-50% B in 25 column volumes (Buffers: A=20 mM sodium acetate pH 4.0, B=1 M NaCl, 20 mM sodium acetate, pH 4.0). The fractions were analyzed using a 4-20 tris-Gly SDS-PAGE gel and RP-HPLC (as described for the crude). SDS-PAGE gels were run for 1.5 hours at 125 V, 35 mA, 5 W. Pooled product was then dialyzed at 4° C. in 3 changes of 1 L of A4S buffer (10 mM NaOAc, 5% sorbitol, pH 4.0). The dialyzed product was then concentrated in 10 K microcentrifuge filter to 2 mL volume and sterile-filtered using 0.2 μM syringe filter to give the final product. N-Terminally PEGylated-Des-Arg1-ShK (Peptide 3) was isolated in 1.7 mg yield with 85% purity as estimated by analytical RP-HPLC analysis (FIG. 23).


The N-Terminally PEGylated-Des-Arg1-ShK, also referred to as “PEG-ShK[2-35]”, was active in blocking human Kv1.3 (FIG. 38A and FIG. 38B) as determined by patch clamp electrophysiology (Example 36).


Example 32
N-Terminally PEGylated ShK

The experimental procedures of this working example correspond to the results shown in FIG. 17.


Peptide Synthesis of reduced ShK. ShK, having the amino acid sequence





RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC  (Peptide 4, SEQ ID NO:5)


was synthesized in a stepwise manner on a Symphony™ multi-peptide synthesizer by solid-phase peptide synthesis (SPPS) using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)/N-methyl morpholine (NMM)/N,N-dimethyl-formamide (DMF) coupling chemistry at 0.1 mmol equivalent resin scale on Tentagel™-S PHB Fmoc-Cys(Trt)-resin. N-alpha-9-fluorenylmethyloxycarbonyl) and side-chain protected amino acids were purchased from Midwest Biotech Incorporated. Fmoc-Cys(Trt)-Tentagel™ resin was purchased from Fluka. The following side-chain protection strategy was employed: Asp(OtBu), Arg(Pbf), Cys(Trt), Gln(Trt), His(Trt), Lys(Nε-Boc), Ser(OtBu), Thr(OtBu) and Tyr(OtBu). Two Oxazolidine dipeptides, Fmoc-Gly-Thr(ψMe,MePro)-OH and Fmoc-Leu-Ser(ψMe,MePro)-OH, were used in the chain assembly and were obtained from NovaBiochem and used in the synthesis of the sequence. The protected amino acid derivatives (20 mmol) were dissolved in 100 ml 20% dimethyl sulfoxide (DMSO) in DMF (v/v). Protected amino acids were activated with 200 mM HBTU, 400 mM NMM in 20% DMSO in DMF, and coupling were carried out using two treatments with 0.5 mmol protected amino acid, 0.5 mmol HBTU, 1 mmol NMM in 20% DMF/DMSO for 25 minutes and then 40 minutes. Fmoc deprotections were carried out with two treatments using a 20% piperidine in DMF (v/v) solution for 10 minutes and then 15 minutes. Following synthesis, the resin was then drained, and washed with DCM, DMF, DCM, and then dried in vacuo. The peptide-resin was deprotected and released from the resin by treatment with a TFA/EDT/TIS/H2O (92.5:2.5:2.5:2.5 (v/v)) solution at room temperature for 1 hour. The volatiles were then removed with a stream of nitrogen gas, the crude peptide precipitated twice with cold diethyl ether and collected by centrifugation. The crude peptide was then analyzed on a Waters 2795 analytical RP-HPLC system using a linear gradient (0-60% buffer B in 12 minutes, A: 0.1% TFA in water, B: 0.1% TFA in acetonitrile) on a Jupiter 4 μm Proteo™ 90 Å column. A PE-Sciex API Electro-spray mass spectrometer was used to confirm correct peptide product mass. Crude peptide was approximately was obtained 170 mg yield at about 45% purity as estimated by analytical RP-HPLC analysis. Reduced ShK (Peptide 4) Retention time (Rt)=5.054 minutes, calculated molecular weight=4060.8793 Da (average); experimental observed molecular weight=4063.0 Da.


Folding of ShK (Disulphide bond formation). Following TFA cleavage and peptide precipitation, reduced ShK was then air oxidized to give the folded peptide. The crude cleaved peptide was extracted using 20% AcOH in water (v/v) and then diluted with water to a concentration of approximately 0.15 mg reduced ShK per mL, the pH adjusted to about 8.0 using NH4OH (28-30%), and gently stirred at room temperature for 36 hours. Folding process was monitored by LC-MS analysis. Following this, folded ShK peptide was purified by reversed phase HPLC using a 1″ Luna 5 μm C18 100 Å Proteo™ column with a linear gradient 0-40% buffer B in 120 min (A=0.1% TFA in water, B=0.1% TFA in acetonitrile). Folded ShK crude peptide eluted earlier (when compared to the elution time in its reduced form) at approximately 25% buffer B. Folded ShK (Peptide 5) was obtained in 25.5 mg yield in >97% purity as estimated by analytical RP-HPLC analysis. See FIG. 60. Calculated molecular weight=4051.8764 Da (monoisotopic); experimental observed molecular weight=4052.5 Da (analyzed on Waters LCT Premier Micromass MS Technologies). ShK disulfide connectivity was C1-C6, C2-C4, and C3-C5.







N-terminal PEGylation of Folded ShK. Folded ShK, having the amino acid sequence











RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
(SEQ ID NO: 5)








can be dissolved in water at 1 mg/ml concentration. A 2 M MeO-PEG-Aldehyde, CH3O—CH2CH2O]n-CH2CH2CHO (average molecular weight 20 kDa), solution in 50 mM NaOAc, pH 4.5 and a separate 1 M solution of NaCNBH3 can be freshly prepared. The peptide solution can be then added to the MeO-PEG-Aldehyde containing solution and can be followed by the addition of the NaCNBH3 solution. The reaction stoichiometry can be peptide:PEG:NaCNBH3 (1:2:0.02), respectively. The reaction can be left for 48 hours, and can be analyzed on an Agilent™ 1100 RP-HPLC system using Zorbax™ 300SB-C8 5 μm column at 40° C. with a linear gradient (6-60% B in 16 minutes, A: 0.1% TFA in water, B: 0.1% TFA/90% ACN in water). Mono-pegylated Shk (Peptide 6) can be then isolated using a HiTrap™ 5 mL SP HP cation exchange column on AKTA FPLC system at 4° C. at 1 mL/min using a gradient of 0-50% B in 25 column volumes (Buffers: A=20 mM sodium acetate pH 4.0, B=1 M NaCl, 20 mM sodium acetate, pH 4.0). The fractions can be analyzed using a 4-20 tris-Gly SDS-PAGE gel and RP-HPLC. SDS-PAGE gels can be run for 1.5 hours at 125 V, 35 mA, 5 W. Pooled product can be then dialyzed at 4° C. in 3 changes of 1 L of A4S buffer (10 mM sodium acetate, 5% sorbitol, pH 4.0). The dialyzed product can be then concentrated in 10 K microcentrifuge filter to 2 mL volume and sterile-filtered using 0.2 μM syringe filter to give the final product.


Example 33
N-Terminally PEGylated ShK by Oxime Formation

Peptide Synthesis of reduced ShK. ShK, having the sequence











RSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTC
(SEQ ID NO: 5)








can be synthesized in a stepwise manner on a Symphony™ multi-peptide synthesizer by solid-phase peptide synthesis (SPPS) using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)/N-methyl morpholine (NMM)/N,N-dimethyl-formamide (DMF) coupling chemistry at 0.1 mmol equivalent resin scale on Tentagel™-S PHB Fmoc-Cys(Trt)-resin. N-alpha-(9-fluorenylmethyloxycarbonyl)- and side-chain protected amino acids can be purchased from Midwest Biotech Incorporated. Fmoc-Cys(Trt)-Tentagel™ resin can be purchased from Fluka. The following side-chain protection strategy can be employed: Asp(OtBu), Arg(Pbf), Cys(Trt), Gln(Trt), His(Trt), Lys(Nε-Boc), Ser(OtBu), Thr(OtBu) and Tyr(OtBu). Two Oxazolidine dipeptides, Fmoc-Gly-Thr(ΨMe,MePro)-OH and Fmoc-Leu-Ser(ψMe,MePro)-OH, can be used in the chain assembly and can be obtained from NovaBiochem and used in the synthesis of the sequence. The protected amino acid derivatives (20 mmol) can be dissolved in 100 ml 20% dimethyl sulfoxide (DMSO) in DMF (v/v). Protected amino acids can be activated with 200 mM HBTU, 400 mM NMM in 20% DMSO in DMF, and coupling can be carried out using two treatments with 0.5 mmol protected amino acid, 0.5 mmol HBTU, 1 mmol NMM in 20% DMF/DMSO for 25 minutes and then 40 minutes. Fmoc deprotection reactions can be carried out with two treatments using a 20% piperidine in DMF (v/v) solution for 10 minutes and then 15 minutes. Following the chain-assembly of the Shk peptide, Boc-amionooxyacetic acid (1.2 equiv) can be coupled at the N-terminus using 0.5 M HBTU in DMF with 4 equiv collidine for 5 minutes. Following synthesis, the resin can be then drained, and washed with DCM, DMF, DCM, and then dried in vacuo. The peptide-resin can be deprotected and released from the resin by treatment with a TFA/amionooxyacetic acid/TIS/EDT/H2O (90:2.5:2.5:2.5:2.5) solution at room temperature for 1 hour. The volatiles can be then removed with a stream of nitrogen gas, the crude peptide precipitated twice with cold diethyl ether and collected by centrifugation. The aminooxy-Shk peptide (Peptide 7) can be then analyzed on a Waters 2795 analytical RP-HPLC system using a linear gradient (0-60% buffer B in 12 minutes, A: 0.1% TFA in water also containing 0.1% aminooxyacetic acid, B: 0.1% TFA in acetonitrile) on a Jupiter 4 μm Proteo™ 90 Å column.


Reversed-Phase HPLC Purification. Preparative Reversed-phase high-performance liquid chromatography can be performed on C18, 5 μm, 2.2 cm×25 cm) column. Chromatographic separations can be achieved using linear gradients of buffer B in A (A=0.1% aqueous TFA; B=90% aq. ACN containing 0.09% TFA and 0.1% aminooxyacetic acid), typically 5-95% over 90 minutes at 15 mL/min. Preparative HPLC fractions can be characterized by ESMS and photodiode array (PDA) HPLC, combined and lyophilized.


N-Terminal PEGylation of Shk by Oxime Formation. Lyophilized aminooxy-Shk (Peptide 7) can be dissolved in 50% HPLC buffer A/B (5 mg/mL) and added to a two-fold molar excess of MeO-PEG-Aldehyde, CH3O—[CH2CH2O]n—CH2CH2CHO (average molecular weight 20 kDa). The reaction can be left for 24 hours, and can be analyzed on an Agilent™ 1100 RP-HPLC system using Zorbax™ 300SB-C8 5 μm column at 40° C. with a linear gradient (6-60% B in 16 minutes, A: 0.1% TFA in water, B: 0.1% TFA/90% ACN in water). Mono-pegylated reduced Shk constituted approximately 58% of the crude product by analytical RP-HPLC. Mono PEGylated (oximated) Shk (Peptide 8) can be then isolated using a HiTrap™ 5 mL SP HP cation exchange column on AKTA FPLC system at 4° C. at 1 mL/min using a gradient of 0-50% B in 25 column volumes (Buffers: A 20 mM sodium acetate pH 4.0, B=1 M NaCl, 20 mM sodium acetate, pH 4.0). The fractions can be analyzed using a 4-20 tris-Gly SDS-PAGE gel and RP-HPLC. SDS-PAGE gels can be run for 1.5 hours at 125 V, 35 mA, 5 W. Pooled product can be then dialyzed at 4° C. in 3 changes of 1 L of A4S buffer (10 mM NaOAc, 5% sorbitol, pH 4.0). The dialyzed product can be then concentrated in 10 K microcentrifuge filter to 2 mL volume and sterile-filtered using 0.2 μM syringe filter to give the final product.


Folding of ShK (Disulphide bond formation). The mono-PEGylated (oximated) Shk can be dissolved in 20% AcOH in water (v/v) and can be then diluted with water to a concentration of approximately 0.15 mg peptide mL, the pH adjusted to about 8.0 using NH4OH (28-30%), and gently stirred at room temperature for 36 hours. Folding process can be monitored by LC-MS analysis. Following this, folded mono-PEGylated (oximated) Shk (Peptide 9) can be purified using by reversed phase HPLC using a 1′ Luna 5 μm C18 100 Å Proteo™ column with a linear gradient 0-40% buffer B in 120 min (A=0.1% TFA in water, B=0.1% TFA in acetonitrile). Mono-PEGylated (oximated) ShK disulfide connectivity can be C1-C6, C2-C4, and C3-C5.







Example 34
N-Terminally PEGylated ShK (Amidation)

The experimental procedures of this working example correspond to the results shown in FIG. 18.


N-Terminal PEGylation of Shk by Amide Formation. A 10 mg/mL solution of folded Shk (Peptide 5), in 100 mM Bicine pH 8.0, can be added to solid succinimidyl ester of 20 kDa PEG propionic acid (mPEG-SPA; CH3O—[CH2CH2O]n-CH2CH2CO—NHS) at room temperature using a 1.5 molar excess of the mPEG-SPA to Shk. After one hour with gentle stirring, the mixture can be diluted to 2 mg/mL with water, and the pH can be adjusted to 4.0 with dilute HCl. The extent of mono-pegylated Shk (Peptide 10), some di-PEGylated Shk or tri-PEGylated Shk, unmodified Shk and succinimidyl ester hydrolysis can be determined by SEC HPLC using a Superdex™ 75 HR 10/30 column (Amersham) eluted with 0.05 M NaH2PO4, 0.05 M Na2HPO4, 0.15 M NaCl, 0.01 M NaN3, pH 6.8, at 1 mL/min. The fractions can be analyzed using a 4-20 tris-Gly SDS-PAGE gel and RP-HPLC. SDS-PAGE gels can be run for 1.5 hours at 125 V, 35 mA, 5 W. Pooled product can be then dialyzed at 4° C. in 3 changes of 1 L of A4S buffer (10 mM NaOAc, 5% sorbitol, pH 4.0). The dialyzed N-terminally PEGylated (amidated) ShK (Peptide 10) can be then concentrated in 10 K microcentrifuge filter to 2 mL volume and sterile-filtered using 0.2 μM syringe filter to give the final product.







Example 35
Fc-L-SmIIIA

Fc-SmIIIA expression vector. A 104 bp BamHI-NotI fragment containing partial linker sequence and SmIIIA peptide encoded with human high frequency codons was assembled by PCR with overlapping primers 3654-50 and 3654-51 and cloned into to the 7.1 kb NotI-BamHI back bone to generate pcDNA3.1(+) CMVi-hFc-SmIIIA as described in Example 1.










  BamHI










5′ GGATCCGGAGGAGGAGGAAGCTGCTGCAACGGC
SEQ ID NO: 872



                        C  C  N  G
SEQ ID NO:873


CGCCGCGGCTGCAGCAGCCGCTGGTGCCGCGACCAC


R  R  G  C  S  S  R  W  C  R  D  H


AGCCGCTGCTGCTGAGCGGCCGC3′ //


S  R  C  C       NotI





Forward 5′-3′


GGAGGAGGATCCGGAGGAGGAGGAAGCTGCTGCAAC
SEQ ID NO: 874


GGCCGCCGCGGCTGCAGCAGC CGC //





Reverse 5′-3′


ATTATTGCGGCCGCTCAGCAGCAGCGGCTGTGGTCG
SEQ ID NO: 875


CGGCACCAGCGGCTGCTGCAG CCGC







The sequences of the BamHI to NotI fragments in the final constructs were verified by sequencing.


Transient expression of Fc-L-SmIIIa. 7.5 ug of the toxin peptide Fc fusion construct pcDNA3.1(+) CMVi-hFc-SmIIIA were transfected into 293-T cells in 10 cm tissue culture plate with FuGENE 6 as transfection reagent. Culture medium was replaced with serum-free medium at 24 hours post-transfection and the conditioned medium was harvested at day 5 post-transfection. Transient expression of Fc-SmIIIA from 293-T cells was analyzed by Western blot probed with anti-hFc antibody (FIG. 25A and FIG. 25B). Single band of expressed protein with estimated MW was shown in both reduced and non-reduced samples. Transient expression level of Fc-SmIIIA was further determined to be 73.4 μg/ml according to ELISA.


Example 36
Electrophysiology Experiments

Cell Culture. Stable cell line expressing human Kv1.3 channel was licensed from Biofocus. Cells were kept at 37° C. in 5% CO2 environment. Culture medium contains DMEM with GlutaMax™ (Invitrogen), 1× non-essential amino acid, 10% fetal bovine serum and 500 μg/mL geneticin. Cells were plated and grown at low confluence on 35 mm culture dishes for at least 24 hours prior to electrophysiology experiments.


Electrophysiology Recording by Patch Clamping. Whole-cell currents were recorded from single cells by using tight seal configuration of the patch-clamp technique. A 35 mm culture dish was transferred to the recording stage after rinsing and replacing the culture medium with recording buffer containing 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 10 mM HEPES, and 5 mM Glucose. pH was adjusted to 7.4 with NaOH and the osmolarity was set at 300 mOsm. Cells were perfused continuously with the recording buffer via one of the glass capillaries arranged in parallel and attached to a motorized rod, which places the glass capillary directly on top of the cell being recorded. Recording pipette solution contained 90 mM K-gluconate, 20 mM KF, 10 mM NaCl, 1 mM MgCl2-6H2O, 10 mM EGTA, 5 mM K2-ATP, and 10 mM HEPES. The pH for the internal solution was adjusted to 7.4 with KOH and the osmolarity was set at 280 mOsm. Experiments were performed at room temperature (20-22° C.) and recorded using Multiclamp™ 700 Å amplifier (Molecular Devices Inc.). Pipette resistances were typically 2-3 MΩ.


Protein toxin potency determination on Kv1.3 current: HEK293 cells stably expressing human Kv1.3 channel were voltage clamped at −80 mV holding potential. Outward Kv1.3 currents were activated by giving 200 msec long depolarizing steps to +30 mV from the holding potential of −80 mV and filtered at 3 kHz. Each depolarizing step was separated from the subsequent one with a 10 s interval. Analogue signals were digitized by Digidata™ 1322A digitizer (Molecular Devices) subsequently stored on computer disk for offline analyses using Clampfit™ 9 (Molecular Devices Inc.). In all studies, stable baseline Kv1.3 current amplitudes were established for 4 minutes before starting the perfusion of the protein toxin at incremental concentrations. A steady state block was always achieved before starting the perfusion of the subsequent concentration of the protein toxin.


Data analysis. Percent of control (POC) is calculated based on the following equation: (Kv1.3 current after protein toxin addition/Kv1.3 current in control)*100. At least 5 concentrations of the protein toxin (e.g. 0.003, 0.01, 0.03, 0.1, 0.3, 100 nM) were used to calculate the IC50 value. IC50 values and curve fits were estimated using the four parameter logistic fit of XLfit software (Microsoft Corp.). IC50 values are presented as mean value±s.e.m. (standard error of the mean).


Drug preparations. Protein toxins (typically 10-100 μM) were dissolved in distilled water and kept frozen at −80° C. Serial dilutions of the stock protein toxins were mixed into the recording buffer containing 0.1% bovine serum albumin (BSA) and subsequently transferred to glass perfusion reservoirs. Electronic pinch valves controlled the flow of the protein toxin from the reservoirs onto the cell being recorded.


Example 37
Immunobiology and Channel Binding

Inhibition of T cell cytokine Production following PMA and anti-CD3 antibody stimulation of PBMCs. PBMC's were previously isolated from normal human donor Leukophoresis packs, purified by density gradient centrifugation (Ficoll Hypaque), cryopreserved in CPZ Cryopreservation Medium Complete (INCELL, MCPZF-100 plus 10% DMSO final). PBMC's were thawed (95% viability), washed, and seeded at 2×105 cells per well in culture medium (RPMI medium 1640; GIBCO) supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin 2 mM L-glutamine, 100 uM non-essential amino acids, and 20 uM 2-ME) in 96-well flat-bottom tissue culture plates. Cells were pre-incubated with serially diluted (100 nM-0.001 nM final) ShK[1-35], Fc-L10-ShK[1-35] or fc control for 90 min before stimulating for 48 hr with PMA/anti-CD3 (1 ng/ml and 50 ng/ml, respectively) in a final assay volume of 200 ul. Analysis of the assay samples was performed using the Meso Scale Discovery (MSD) SECTOR™ Imager 6000 (Meso Scale Discovery, Gaithersbury, Md.) to measure the IL-2 and IFNg protein levels by utilizing electrochemiluminescence (ECL). The conditioned medium (50 ul) was added to the MSD Multi-spot 96-well plates (each well containing three capture antibodies; IL-2, TNF, IFNγ). The plates were sealed, wrapped in tin foil, and incubated at room temperature on a plate shaker for 2 hr. The wells were washed 1× with 200 ul PBST (BIOTEK, Elx405 Auto Plate Washer). For each well, 20 ul of Ruthenium-labeled detection antibodies (1 μg/ml final in Antibody Dilution Buffer; IL-1, TNF, IFNγ) and 130 ul of 2×MSD Read Buffer added, final volume 150 ul. The plates were sealed, wrapped in tin foil, and incubated at room temperature on a plate shaker for 1 hr. The plates were then read on the SECTOR™ Imager 6000. FIGS. 35A & 35B shows the CHO-derived Fc-L10-ShK[1-35] peptibody potently inhibits IL-2 and IFNg production from T cells in a dose-dependent manner. Compared to native ShK[1-35] peptide, the peptibody produces a greater extent of inhibition (POC=Percent Of Control of response in the absence of inhibitor).


Inhibition of T cell cytokine production following anti-CD3 and anti-CD28 antibody stimulation of PBMCs. PBMCs were previously isolated from normal human donor Leukopheresis packs, purified by density gradient centrifugation (Ficoll Hypaque), and cryopreserved using INCELL Freezing Medium. PBMCs were thawed (95% viability), washed, and seeded (in RPMI complete medium containing serum replacement, PSG) at 2×105 cells per well into 96-well flat bottom plates. Cells were pre-incubated with serially diluted (100 nM-0.003 nM final) ShK[1-35], Fc-L10-ShK[1-35], or Fc control for 1 hour before the addition of aCD3 and aCD28 (2.5 ng/mL and 100 ng/mL respectively) in a final assay volume of 200 mL. Supernatants were collected after 48 hours, and analyzed using the Meso Scale Discovery (MSD) SECTOR™ Imager 6000 (Meso Scale Discovery, Gaithersbury, Md.) to measure the IL-2 and IFNg protein levels by utilizing electrochemiluminescence (ECL). 20 mL of supernatant was added to the MSD multi-spot 96-well plates (each well containing IL-2, TNFa, and IFNg capture antibodies). The plates were sealed and incubated at room temperature on a plate shaker for 1 hour. Then 20 mL of Ruthenium-labeled detection antibodies (1 μg/ml final of IL-2, TNFα, and IFNγ in Antibody Dilution Buffer) and 110 mL of 2×MSD Read Buffer were added. The plates were sealed, covered with tin foil, and incubated at room temperature on a plate shaker for 1 hour. The plates were then read on the SECTOR™ Imager 6000. FIGS. 37A & 37B shows the CHO-derived Fc-L10-ShK[1-35] peptibody potently inhibits IL-2 and IFNg production from T cells in a dose-dependent manner. Compared to native ShK[1-35] peptide which shows only partial inhibition, the peptibody produces nearly complete inhibition of the inflammatory cytokine response. (POC=Percent Of Control of response in the absence of inhibitor).


Inhibition of T cell proliferation following anti-CD3 and anti-CD28 antibody stimulation of PBMCs. PBMC's were previously isolated from normal human donor Leukophoresis packs, purified by density gradient centrifugation (Ficoll Hypaque), cryopreserved in CPZ Cryopreservation Medium Complete (INCELL, MCPZF-100 plus 10% DMSO final). PBMC's were thawed (95% viability), washed, and seeded at 2×105 cells per well in culture medium (RPMI medium 1640; GIBCO) supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, 100 μM non-essential amino acids, and 20 μM 2-ME) in 96-well flat-bottom tissue culture plates. Cells were pre-incubated with either anti-human CD32 (FcyRII) blocking antibody (per manufacturers instructions EASY SEP Human Biotin Selection Kit #18553, StemCell Technologies Vancouver, BC) or Fc-L10-ShK (100 nM-0.001 nM final) for 45 min. Fc-L10-ShK (100 nM-0.001 nM final) was then added to the cells containing anti-human CD32 blocking antibody while medium was added to the cells containing Fc-L10-ShK. Both sets were incubated for an additional 45 min before stimulating for 48 hr with aCD3/aCD28 (0.2 ng/ml and 100 ng/ml, respectively). Final assay volume was 200 ul. [3H]TdR (1 uCi per well) was added and the plates were incubated for an additional 16 hrs. Cells were then harvested onto glass fiber filters and radioactivity was measured in a B-scintillation counter. FIGS. 36A & 36B shows the CHO-derived Fc-L10-ShK[1-35] peptibody potently inhibits proliferation of T cells in a dose-dependent manner. Pre-blocking with the anti-CD32 (FcR) blocking antibody has little effect on the peptibodies ability to inhibit T cell proliferation suggesting Kv1.3 inhibition and not FcR binding is the mechanism for the inhibition observed (POC=Percent Of Control of response in the absence of inhibitor).


Immunohistochemistry analysis of Fc-L10-ShK[1-35] binding to HEK 293 cells overexpressing human Kv1.3. HEK 293 cells overexpressing human Kv1.3 (HEK Kv1.3) were obtained from BioFocus plc (Cambridge, UK) and maintained per manufacturer's recommendation. The parental HEK 293 cell line was used as a control. Cells were plated on Poly-D-Lysine 24 well plates (#35-4414; Becton-Dickinson, Bedford, Mass.) and allowed to grow to approximately 70% confluence. HEK KV1.3 were plated at 0.5×10e5 cells/well in 1 ml/well of medium. HEK 293 cells were plated at a density of 1.5×10e5 cells/well in 1 ml/well of medium. Before staining, cells were fixed with formalin (Sigma HT50-1-1 Formalin solution, diluted 1:1 with PBS/0.5% BSA before use) by removing cell growth medium, adding 0.2 ml/well formalin solution and incubating at room temperature for ten minutes. Cells were stained by incubating with 0.2 ml/well of 5 μg/ml Fc-L10-ShK[1-35] in PBS/BSA for 30′ at room temperature. Fc-L10-ShK[1-35] was aspirated and then the cells were washed one time with PBS/0.5% BSA. Detection antibody (Goat F(ab)2 anti-human IgG-phycoerythrin; Southern Biotech Associates, Birmingham, Ala.) was added to the wells at 5 μg/ml in PBS/0.5% BSA and incubated for 30′ at room temperature. Wash cells once with PBS/0.5% BSA and examine using confocal microscopy (LSM 510 Meta Confocal Microscope; Carl Zeiss AG, Germany). FIG. 33B shows the Fc-L10-ShK[1-35] peptibody retains binds to Kv1.3 overexpressing HEK 293 cells but shows little binding to untransfected cells (FIG. 33A) indicating the Fc-L10-ShK[1-35] peptibody can be used as a reagent to detect cells overexpressing the Kv1.3 channel. In disease settings where activated T effector memory cells have been reported to overproduce Kv1.3, this reagent can find utility in both targeting these cells and in their detection.


An ELISA assay demonstrating Fc-L10-ShK[1-35] binding to fixed HEK 293 cells overexpressing Kv1.3. FIG. 34A shows a dose-dependent increase in the peptibody binding to fixed cells that overexpress Kv1.3, demonstrating that the peptibody shows high affinity binding to its target and the utility of the Fc-L10-ShK[1-35] molecule in detection of cells expressing the channel. Antigen specific T cells that cause disease in patients with multiple sclerosis have been shown to overexpress Kv1.3 by whole cell patch clamp electrophysiology,—a laborius approach. Our peptibody reagent can be a useful and convenient tool for monitoring Kv1.3 channel expression in patients and have utility in diagnostic applications. The procedure shown in FIG. 34A and FIG. 34B follows.



FIG. 34A. A whole cell immunoassay was performed to show binding of intact Fc-L10-ShK[1-35] to Kv1.3 transfected HEK 293 cells (BioFocus plc, Cambridge, UK). Parent HEK 293 cells or HEK Kv1.3 cells were plated at 3×10e4 cells/well in poly-D-Lysine coated ninety-six well plates (#35-4461; Becton-Dickinson, Bedford, Mass.). Cells were fixed with formalin (Sigma HT50-1-1 Formalin solution, diluted 1:1 with PBS/0.5% BSA before use) by removing cell growth medium, adding 0.2 ml/well formalin solution and incubating at room temperature for 25 minutes and then washing one time with 100 μl/well of PBS/0.5% BSA. Wells were blocked by addition of 0.3 ml/well of BSA blocker (50-61-00; KPL 10% BSA Diluent/Blocking Solution, diluted 1:1 with PBS; KPL, Gaithersburg, Md.) followed by incubation at room temperature, with shaking, for 3 hr. Plates were washed 2 times with 1×KP Wash Buffer (50-63-00; KPL). Samples were diluted in Dilution Buffer (PBS/0.5% Tween-20) or Dilution Buffer with 1% Male Lewis Rat Serum (RATSRM-M; Bioreclamation Inc., Hicksville, N.Y.) and 0.1 ml/well was added to blocked plates, incubating for 1 hr at room temperature with shaking. Plates were washed 3 times with 1xKP Wash Buffer and then incubated with HRP-Goat anti-human IgG Fc (#31416; Pierce, Rockford, Ill.) diluted 1:5000 in PBS/0.1% Tween-20 for 1 hr at room temperature, with shaking. Plates were washed plates 3 times with 1xKP Wash Buffer, and then 0.1 ml/well TMB substrate (52-00-01; KPL) was added. The reactions were stopped by addition of 0.1 ml/well 2 N Sulfuric Acid. Absorbance was read at 450 nm on a Molecular Devices SpectroMax 340 (Sunnyvale, Calif.).



FIG. 34B. Whole cell immunoassay was performed as above with the following modifications. HEK 293 cells were plated at 1×10e5 cells/well and HEK Kv1.3 cells were plated at 6×10e4 cells/well in poly-D-Lysine coated 96 well plates. Fc Control was added at 500 ng/ml in a volume of 0.05 ml/well. HRP-Goat anti-human IgG Fc (#31416; Pierce, Rockford, Ill.) was diluted 1:10,000 in PBS/0.1% Tween-20. ABTS (50-66-00, KPL) was used as the substrate. Absorbances were read at 405 nm after stopping reactions by addition of 0.1 ml/well of 1% SDS.


Example 38
Purification of Fc-L10-ShK(1-35)

Expression of Fc-L10-ShK[1-35] was as described in Example 3 herein above. Frozen, E. coli paste (18 g) was combined with 200 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 8.0 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 22,000 g for 15 min at 4° C. The pellet was then resuspended in 200 ml 1% deoxycholic acid using a tissue grinder and then centrifuged at 22,000 g for 15 min at 4° C. The pellet was then resuspended in 200 ml water using a tissue grinder and then centrifuged at 22,000 g for 15 min at 4° C. The pellet (3.2 g) was then dissolved in 32 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. The pellet solution was then centrifuged at 27,000 g for 15 min at room temperature, and then 5 ml of the supernatant was transferred to 500 ml of the refolding buffer (3 M urea, 20% glycerol, 50 mM tris, 160 mM arginine HCl, 5 mM EDTA, 1 mM cystamine HCl, 4 mM cysteine, pH 9.5) at 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for 2 days at 4° C. The refolding solution was then stored at −70° C.


The stored refold was defrosted and then diluted with 2 L of water and the pH was adjusted to 7.3 using 1 M H3PO4. The pH adjusted material was then filtered through a 0.22 μm cellulose acetate filter and loaded on to a 60 ml Amersham SP-FF (2.6 cm I.D.) column at 20 ml/min in S-Buffer A (20 mM NaH2PO4, pH 7.3) at 7° C. The column was then washed with several column volumes of S-Buffer A, followed by elution with a linear gradient from 0% to 60% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.3) followed by a step to 100% S-Buffer B at 10 ml/min 7° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data. The pool was then loaded on to a 1 ml Amersham rProtein A HiTrap column in PBS at 1 ml/min 7° C. Then column was then washed with several column volumes of 20 mM NaH2PO4 pH 6.5, 1 M NaCl and eluted with 100 mM glycine pH 3.0. To the elution peak, 0.0125 volumes (25 ml) of 3 M sodium acetate was added.


A spectral scan was then conducted on 50 μl of the combined pool diluted in 700 μl water using a Hewlett Packard 8453 spectrophotometer (FIG. 46A). The concentration of the filtered material was determined to be 2.56 mg/ml using a calculated molecular mass of 30,410 g/mol and extinction coefficient of 36,900 M−1 cm−1. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 46B). The macromolecular state of the product was then determined using size exclusion chromatography on 20 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 46C). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). One milliliter of the resultant solution was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses. The product was then stored at −80° C.


The IC50 for blockade of human Kv1.3 by purified E. coli-derived Fc-L10-ShK[1-35], also referred to as “Fc-L-ShK[1-35]”, is shown in Table 35 (in Example 50 herein below).


Example 39
Purification of Bacterially Expressed Fc-L10-ShK(2-35)

Expression of Fc-L10-ShK[2-35] was as described in Example 4 herein above. Frozen, E. coli paste (16.5 g) was combined with 200 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 8.0 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 22,000 g for 15 min at 4° C. The pellet was then resuspended in 200 ml 1% deoxycholic acid using a tissue grinder and then centrifuged at 22,000 g for 15 min at 4° C. The pellet was then resuspended in 200 ml water using a tissue grinder and then centrifuged at 22,000 g for 15 min at 4° C. The pellet (3.9 g) was then dissolved in 39 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. The pellet solution was then centrifuged at 27,000 g for 15 min at room temperature, and then 5 ml of the supernatant was transferred to 500 ml of the refolding buffer (3 M urea, 20% glycerol, 50 mM tris, 160 mM arginine HCl, 5 mM EDTA, 1 mM cystamine HCl, 4 mM cysteine, pH 9.5) at 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for 2 days at 4° C. The refolding solution was then stored at −70° C.


The stored refold was defrosted and then diluted with 2 L of water and the pH was adjusted to 7.3 using 1 M H3PO4. The pH adjusted material was then filtered through a 0.22 μm cellulose acetate filter and loaded on to a 60 ml Amersham SP-FF (2.6 cm I.D.) column at 20 ml/min in S-Buffer A (20 mM NaH2PO4, pH 7.3) at 7° C. The column was then washed with several column volumes of S-Buffer A, followed by elution with a linear gradient from 0% to 60% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.3) followed by a step to 100% S-Buffer B at 10 ml/min 7° C. The fractions containing the desired product were pooled and filtered through a 0.22 μm cellulose acetate filter. The pool was then loaded on to a 1 ml Amersham rProtein A HiTrap column in PBS at 2 ml/min 7° C. Then column was then washed with several column volumes of 20 mM NaH2PO4 pH 6.5, 1 M NaCl and eluted with 100 mM glycine pH 3.0. To the elution peak, 0.0125 volumes (18 ml) of 3 M sodium acetate was added, and the sample was filtered through a 0.22 μm cellulose acetate filter.


A spectral scan was then conducted on 20 μl of the combined pool diluted in 700 μl water using a Hewlett Packard 8453 spectrophotometer (FIG. 40A). The concentration of the filtered material was determined to be 3.20 mg/ml using a calculated molecular mass of 29,282 g/mol and extinction coefficient of 36,900 M−1 cm−1. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 40B). The macromolecular state of the product was then determined using size exclusion chromatography on 50 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 40C). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). One milliliter of the resultant solution was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses (FIG. 40D). The product was then stored at −80° C.


The IC50 for blockade of human Kv1.3 by purified E. coli-derived Fc-L10-ShK[2-35], also referred to as “Fc-L-ShK[2-35]”, is shown in Table 35 (in Example 50 herein below).


Example 40
Purification of Bacterially Expressed Fc-L10-OsK1

Frozen, E. coli paste (129 g; see Example 10) was combined with 1290 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 7.8 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 17,700 g for 15 min at 4° C. The pellet was then resuspended in 1290 ml 1% deoxycholic acid using a tissue grinder and then centrifuged at 17,700 g for 15 min at 4° C. The pellet was then resuspended in 1290 ml water using a tissue grinder and then centrifuged at 17,700 g for 15 min at 4° C. 8 g of the pellet (16.3 g total) was then dissolved in 160 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. 100 ml of the pellet solution was then incubated with 1 ml of 1 M DTT for 60 min at 37° C. The reduced material was transferred to 5000 ml of the refolding buffer (1 M urea, 50 mM tris, 160 mM arginine HCl, 2.5 mM EDTA, 1.2 mM cystamine HCl, 4 mM cysteine, pH 10.5) at 2 ml/min, 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for 3 days at 4° C.


The pH of the refold was adjusted to 8.0 using acetic acid. The pH adjusted material was then filtered through a 0.22 μm cellulose acetate filter and loaded on to a 50 ml Amersham Q Sepharose-FF (2.6 cm I.D.) column at 10 ml/min in Q-Buffer A (20 mM Tris, pH 8.5) at 8° C. with an inline 50 Amersham Protein A column (2.6 cm I.D.). After loading, the Q Sepharose column was removed from the circuit, and the remaining chromatography was carried out on the protein A column. The column was washed with several column volumes of Q-Buffer A, followed by elution using a step to 100 mM glycine pH 3.0. The fractions containing the desired product were pooled and immediately loaded on to a 50 ml Amersham SP-Sepharose HP column (2.6 cm I.D.) at 20 ml/min in S-Buffer A (20 mM NaH2PO4, pH 7.0) at 8° C. The column was then washed with several column volumes of S-Buffer A followed by a linear gradient from 5% to 60% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.0) followed by a step to 100% S-Buffer B. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE. The fractions containing the bulk of the desired product were pooled and then applied to a 75 ml MEP Hypercel column (2.6 cm I.D.) at 5 ml/min in MEP Buffer A (20 mM tris, 200 mM NaCl, pH 8.0) at 8° C. Column was eluted with a linear gradient from 5% to 50% MEP Buffer B (50 mM sodium citrate pH 4.0) followed by a step to 100% MEP Buffer B. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the bulk of the desired product were pooled.


The MEP pool was then concentrated to about 20 ml using a Pall Jumbo-Sep with a 10 kDa membrane followed by buffer exchange with Formulation Buffer (20 mM NaH2PO4, 200 mM NaCl, pH 7.0) using the same membrane. A spectral scan was then conducted on 50 μl of the combined pool diluted in 700 μl Formulation Buffer using a Hewlett Packard 8453 spectrophotometer (FIG. 41A). The concentration of the material was determined to be 4.12 mg/ml using a calculated molecular mass of 30,558 g/mol and extinction coefficient of 35,720 M−1 cm−1. The purity of the material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 41B). The macromolecular state of the product was then determined using size exclusion chromatography on 123 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 41C). The product was then subject to mass spectral analysis by chromatographing approximately 4 μg of the sample through a RP-HPLC column (Vydac C4, 1×150 mm). Solvent A was 0.1% trifluoroacetic acid in water and solvent B was 0.1% trifluoroacetic acid in 90% acetonitrile, 10% water. The column was pre-equilibrated in 10% solvent B at a flow rate of 80 μl per min. The protein was eluted using a linear gradient of 10% to 90% solvent B over 30 min. Part of the effluent was directed into a LCQ ion trap mass spectrometer. The mass spectrum was deconvoluted using the Bioworks software provided by the mass spectrometer manufacturer. (FIG. 41D). The product was filtered through a 0.22 μm cellulose acetate filter and then stored at −80° C.


The yield for the E. coli-expressed Fc-L10-OSK1 prep was 81 mg from 40 g of cell paste (129 g×(8 g/16.3 g)×(100 ml/160 ml)=39.6 g which was rounded to 40 g), the purity was greater than 80% judging by SDS-PAGE, it is running as the expected dimer judging by SEC-HPLC, and the mass was within the expected molecular weight range judging by MS.


The IC50 for blockade of human Kv1.3 by purified E. coli-derived Fc-L10-OSK1, also referred to as “Fc-L-OSK1”, is shown in Table 35 (in Example 50 herein below).


Example 41
Fc-L10-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1 [K7S,E16K,K20D] Expressed by Mammalian Cells

Fc-L10-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1 [K7S,E16K,K20D], inhibitors of Kv1.3, were expressed in mammalian cells. A DNA sequence coding for the Fc region of human IgG1 fused in-frame to a linker sequence and a monomer of the Kv1.3 inhibitor peptide OSK1, OSK1[K7S], OSK1[E16K,K20D], or OSK1[K7S,E16K,K20D] was constructed as described below. Methods for expressing and purifying the peptibody from mammalian cells (HEK 293 and Chinese Hamster Ovary cells) are disclosed herein.


For construction of Fc-L10-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] expression vectors, a PCR strategy was employed to generate the full length genes, OSK1, OSK1[K7S], OSK1[E16K,K20D], and OSK1[K7S,E16K,K20D], each linked to a four glycine and one serine amino acid linker with two stop codons and flanked by BamHI and NotI restriction sites as shown below.


Two oligos for each of OSK1, OSK1[K7S], OSK1[E16K,K20D], and [K7S,E16K,K20D]OSK1 with the sequence as depicted below were used in a PCR reaction with PfuTurbo HotStart DNA polymerase (Stratagene) at 95° C.-30 sec, 55° C.-30 sec, 75° C.-45 sec for 35 cycles; BamHI (ggatcc) and NotI (gcggccgc) restriction sites are underlined.










OSK1:



Forward primer:








(SEQ ID NO: 876)









cat gga tcc gga gga gga gga agc ggc gtg atc atc



aac gtg aag tgc aag atc agc cgc cag tgc ctg gag


ccc tgc aag aag gcc g;





Reverse primer:








(SEQ ID NO: 877)









cat gcg gcc gct tac tac ttg ggg gtg cag tgg cac



ttg ccg ttc atg cac ttg ccg aag cgc atg ccg gcc


ttc ttg cag ggc tcc a;





OSK1 [K7S]:


Forward primer:








(SEQ ID NO: 878)









cat gga tcc gga gga gga gga agc ggc gtg atc atc



aac gtg agc tgc aag atc agc cgc cag tgc ctg gag


ccc tgc aag aag gcc g;





Reverse primer:








(SEQ ID NO: 879)









cat gcg gcc gct tac tac ttg ggg gtg cag tgg cac



ttg ccg ttc atg cac ttg ccg aag cgc atg ccg gcc


ttc ttg cag ggc tcc a;





OSK1 [E16K,K20D]:


Forward primer:








(SEQ ID NO: 880)









cat gga tcc gga gga gga gga agc ggc gtg atc atc



aac gtg aag tgc aag atc agc cgc cag tgc ctg aag


ccc tgc aag gac gcc g;





Reverse primer:








(SEQ ID NO: 881)









cat gcg gcc gct tac tac ttg ggg gtg cag tgg cac



ttg ccg ttc atg cac ttg ccg aag cgc atg ccg gcg


tcc ttg cag ggc ttc a;





OSK1 [K7S,E16K,K20D]:


Forward primer:








(SEQ ID NO: 882)









cat gga tcc gga gga gga gga agc ggc gtg atc atc



aac gtg agc tgc aag atc agc cgc cag tgc ctg aag


ccc tgc aag gac gcc g;





Reverse primer:








(SEQ ID NO: 883)









cat gcg gcc gct tac tac ttg ggg gtg cag tgg cac



ttg ccg ttc atg cac ttg ccg aag cgc atg ccg gcg


tcc ttg cag ggc ttc a.






The resulting PCR products were resolved as the 155 bp bands on a four percent agarose gel. The 155 bp PCR product was purified using PCR Purification Kit (Qiagen), then digested with BamHI and NotI (Roche) restriction enzymes, and agarose gel was purified by Gel Extraction Kit (Qiagen). At the same time, the pcDNA3.1(+) CMVi-hFc-Shk[2-35] vector was digested with BamHI and NotI restriction enzymes and the large fragment was purified by Gel Extraction Kit. The gel purified PCR fragment was ligated to the purified large fragment and transformed into One Shot® Top10F′ (Invitrogen). DNAs from transformed bacterial colonies were isolated and digested with BamHI and NotI restriction enzymes and resolved on a two percent agarose gel. DNAs resulting in an expected pattern were submitted for sequencing. Although, analysis of several sequences of clones yielded a 100% percent match with the above sequences, only one clone from each gene was selected for large scaled plasmid purification. The DNA of Fc-L10-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] in pCMVi vector was resequenced to confirm the Fc and linker regions and the sequence was 100% identical to the above sequences. The sequences and pictorial representations of Fc-L10-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] are depicted in FIG. 42A-B, FIG. 43A-B, FIG. 44A-B and FIG. 45A-B, respectively.


HEK-293 cells used in transient transfection expression of Fc-L110-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] in pCMVi protein were cultured in growth medium containing DMEM High Glucose (Gibco), 10% fetal bovine serum (FBS from Gibco), 1× non-essential amino acid (NEAA from Gibco) and 1× Penicillin/Streptomycine/Glutamine (Pen/Strep/Glu from Gibco). 5.6 μg each of Fc-L10-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] in pCMVi plasmid that had been phenol/chloroform extracted was transfected into HEK-293 cells using FuGENE 6 (Roche). The cells were recovered for 24 hours, and then placed in DMEM High Glucose, 1×NEAA and 1× Pen/Strep/Glu medium for 48 hours. Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] were purified from medium conditioned by these transfected HEK-293 cells using a protocol described in Example 50 herein below.


Fifteen μl of conditioned medium was mixed with an in-house 4× Loading Buffer (without β-mercaptoethanol) and electrophoresed on a Novex 4-20% tris-glycine gel using a Novex Xcell II apparatus at 101V/46 mA for 2 hours in a 1× Gel Running solution (25 mM Tris Base, 192 mM Glycine, 3.5 mM SDS) along with 20 μl of BenchMark Pre-Stained Protein ladder (Invitrogen). The gel was then soaked in Electroblot buffer (25 mM Tris base, 192 mM glycine, 20% methanol,) for 5 minutes. -A nitrocellulose membrane from Invitrogen (Cat. No. LC200, 0.2 μm pores size) was soaked in Electroblot buffer. The pre-soaked gel was blotted to the nitrocellulose membrane using the Mini Trans-Blot Cell module according to the manufacturer instructions (Bio-Rad Laboratories) at 300 mA for 2 hours. The blot was rinsed in Tris buffered saline solution pH7.5 with 0.1% Tween20 (TBST). Then, the blot was first soaked in a 5% milk (Camation) in TBST for 1 hour at room temperature, followed by washing three times in TBST for 10 minutes per wash. Then, incubated with 1:1000 dilution of the HRP-conjugated Goat anti-human IgG, (Fcγ) antibody (Pierece Biotechnology Cat. no. 31413) in TBST with 5% milk buffer for 1 hour with shaking at room temperature. The blot was then washed three times in TBST for 15 minutes per wash at room temperature. The primary antibody was detected using Amersham Pharmacia Biotech's ECL western blotting detection reagents according to manufacturers instructions. Upon ECL detection, the western blot analysis displayed the expected size of 66 kDa under non-reducing gel conditions (FIG. 46).


Plasmids containing the Fc-L10-OSK1, Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K,K20D], and Fc-L10-OSK1[K7S,E16K,K20D] inserts in pCMVi vector were digested with XbaI and NotI (Roche) restriction enzymes and gel purified. The inserts were individually ligated into SpeI and NotI (Roche) digested pDSRα24 (Amgen Proprietary) expression vector. Integrity of the resulting constructs were confirmed by DNA sequencing. Although, analysis of several sequences of clones yielded a 100% percent match with the above sequence, only one clone was selected for large scaled plasmid purification.


AM1 CHOd- (Amgen Proprietary) cells used in the stable expression of Fc-L10-OSK1 protein were cultured in AM1 CHOd- growth medium containing DMEM High Glucose, 10% fetal bovine serum, 1× hypoxantine/thymidine (HT from Gibco), 1×NEAA and 1× Pen/Strep/Glu. 5.6 μg of pDSRα-24-Fc-L10-OSK1 plasmid was transfected into AM1 CHOd- cells using FuGene 6. Twenty-four hours post transfection, the cells were split 1:11 into DHFR selection medium (DMEM High Glucose plus 10% Dialyzed Fetal Bovine Serum (dFBS), 1×NEAA and 1× Pen/Strep/Glu) at 1:50 dilution for colony selection. The cells were selected in DHFR selection medium for thirteen days. The ten 10-cm2 pools of the resulting colonies were expanded to ten T-175 flasks, then were scaled up ten roller bottles and cultured under AM1 CHOd- production medium (DMEM/F12 (1:1), 1×NEAA, 1× Sodium Pyruvate (Na Pyruvate), 1× Pen/Strep/Glu and 1.5% DMSO). The conditioned medium was harvested and replaced at one-week intervals. The resulting six liters of conditioned medium were filtered through a 0.45 μm cellulose acetate filter (Corning, Acton, Mass.), and characterized by SDS-PAGE analysis as shown in FIG. 47. Then, transferred to Protein Chemistry for purification.


Twelve colonies were selected after 13 days on DHFR selection medium and picked into one 24-well plate. The plate was allowed to grow up for one week, and then was transferred to AM1 CHOd- production medium for 48-72 hours and the conditioned medium was harvested. The expression levels were evaluated by Western blotting similar to the transient Western blot analysis with detection by the same HRP-conjugated Goat anti-human IgG, (Fcγ) antibody to screen 5 μl of conditioned medium. All 12 stable clones exhibited expression at the expected size of 66 kDa. Two clones, A3 and C2 were selected and expanded to T175 flask for freezing with A3 as a backup to the primary clone C2 (FIG. 48).


The C2 clone was scaled up into fifty roller bottles (Corning) using selection medium and grown to confluency. Then, the medium was exchanged with a production medium, and let incubate for one week. The conditioned medium was harvested and replaced at the one-week interval. The resulting fifty liters of conditioned medium were filtered through a 0.45 μm cellulose acetate filter (Corning, Acton, Mass.), and characterized by SDS-PAGE analysis (data not shown). Further purification was accomplished as described in Example 42 herein below.


Example 42
Purification of Fc-L10-OSK1, Fc-L10-OSK1(K7S), Fc-L10-OSK1(E16K,K20D), and Fc-L10-OSK1(K7S,E16K,K20D) Expressed by Mammalian Cells

Purification of Fc-L10-OSK1. Approximately 6 L of CHO (AM1 CHOd-) cell-conditioned medium (see, Example 41 above) was loaded on to a 35 ml MAb Select column (GE Healthcare) at 10 ml/min 7° C., and the column was washed with several column volumes of Dulbecco's phosphate buffered saline without divalent cations (PBS) and sample was eluted with a step to 100 mM glycine pH 3.0. The MAb Select elution was directly loaded on to an inline 65 ml SP-HP column (GE Healthcare) in S-Buffer A (20 mM NaH2PO4, pH 7.0) at 10 ml/min 7° C. After disconnecting the MAb select column, the SP-HP column was then washed with several column volumes S-Buffer A, and then developed using a linear gradient from 5% to 60% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.0) at 10 ml/min followed by a step to 100% S-Buffer B at 7° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data. The pooled material was then concentrated to about 20 ml using a Pall Life Sciences Jumbosep 10K Omega centrifugal ultra-filtration device. The concentrated material was then buffer exchanged by diluting with 20 ml of 20 mM NaH2PO4, pH 7.0 and reconcentrated to 20 ml using the Jumbosep 10K Omega filter. The material was then diluted with 20 ml 20 mM NaH2PO4, 200 mM NaCl, pH 7.0 and then reconcentrated to 22 ml. The buffer exchanged material was then filtered though a Pall Life Sciences Acrodisc with a 0.22 μm, 25 mm Mustang E membrane at 1 ml/min room temperature. A spectral scan was then conducted on 50 μl of the filtered material diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 49A, black trace). The concentration of the filtered material was determined to be 4.96 mg/ml using a calculated molecular mass of 30,371 g/mol and extinction coefficient of 35,410 M−1 cm−1. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 49B). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 30-fold dilution of the sample in Charles Rivers Laboratories Endotoxin Specific Buffer yielding a result of 1.8 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 149 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 49C). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). One milliliter of the resultant solution was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from about 200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses. (FIG. 49D). The product was then stored at −80° C.


The yield for the mammalian Fc-L10-OSK1 prep was 115 mg from 6 L, the purity was >90% judging by SDS-PAGE; Fc-L10-OSK1 ran as the expected dimer judging by SEC-HPLC, and the mass is with the expected range judging by MS.


The activity of purified Fc-L10-OSK1 in blocking human Kv1.3 and human Kv1.1 is described in Example 43 herein below.


Purification of Fc-L10-OSK1(K7S). Fc-L10-OSK1(E16K,K20D), and Fc-L10-OSK1(K7S,E16K,K20D). Approximately 500 mL of medium conditioned by transfected HEK-293 (see, Example 41 above) was combined with a 65% slurry of MAb Select resin (1.5 ml) (GE Healthcare) and 500 μl 20% NaN3. The slurry was then gently agitated for 3 days at 4° C. followed by centrifugation at 1000 g for 5 minutes at 4° C. using no brake. The majority of the supernatant was then aspirated and the remaining slurry in the pellet was transferred to a 14 ml conical tube and combined with 12 ml of Dulbecco's phosphate buffered saline without divalent cations (PBS). The slurry was centrifuged at 2000 g for 1 minute at 4° C. using a low brake and the supernatant was aspirated. The PBS wash cycle was repeated an additional 3 times. The bound protein was then eluted by adding 1 ml of 100 mM glycine pH 3.0 and gently agitating for 5 min at room temperature. The slurry was then centrifuged at 2000 g for 1 minute at 4° C. using a low brake and the supernatant was aspirated as the first elution. The elution cycle was repeated 2 more times, and all 3 supernatants were combined into a single pool. Sodium acetate (37.5 μl of a 3 M solution) was added to the elution pool to raise the pH, which was then dialyzed against 10 mM acetic acid, 5% sorbitol, pH 5.0 for 2 hours at room temperature using a 10 kDa SlideAlyzer (Pierce). The dialysis buffer was changed, and the dialysis continued over night at 4° C. The dialyzed material was then filtered through a 0.22 μm cellulose acetate filter syringe filter. Then concentration of the filtered material was determined to be 1.27 mg/ml using a calculated molecular mass of 30,330 and extinction coefficient of 35,410 M−1 cm−1 (FIG. 50A). The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 50B). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 25-fold dilution of the sample in Charles Rivers Laboratories Endotoxin Specific Buffer yielding a result of <1 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 50 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 50C). The product was then subject to mass spectral analysis by diluting 1 μl of the sample into 10 μl of sinapinic acid (10 mg per ml in 0.05% trifluoroacetic acid, 50% acetonitrile). One milliter of the resultant solution was spotted onto a MALDI sample plate. The sample was allowed to dry before being analyzed using a Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse). The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots. External mass calibration was accomplished using purified proteins of known molecular masses. (FIG. 50D). The product was then stored at −80° C.



FIGS. 51A-D show results from the purification and analysis for Fc-L10-OsK1(E16K, K20D), which was conducted using the same protocol as that for the Fc-L110-OsK1 (K7S) molecule (described above) with the following exceptions: the concentration was found to be 1.59 mg/ml using a calculated molecular mass of 30,357 g/mol and a calculated extinction coefficient of 35,410; the pyrogen level was found to be <1 EU/mg using a 32-fold dilution.



FIGS. 52A-D show results from the purification and analysis for Fc-L10-OsK1(K7S,E16K, K20D), which was conducted using the same protocol as that for the Fc-L10-OsK1(K7S) molecule (described above) with the following exceptions: the concentration was found to be 0.81 mg/ml using a calculated molecular mass of 30,316 g/mol and a calculated extinction coefficient of 35,410; the pyrogen level was found to be <1 EU/mg using a 16-fold dilution.


The activity of purified Fc-L10-OSK1[K7S], Fc-L10-OSK1[E16K, K20D] and Fc-L10-OSK1[K7S, E16K, K20D] in blocking human Kv1.3 and human Kv1.1 is described in Example 43 herein below.


Example 43
Electrophysiology of OSK1 and OSK1 Peptibody Analogs

A 38-residue peptide toxin of the Asian scorpion Orthochirus scrobiculosus venom (OSK1) was synthesized (see, Examples 41) to evaluate its impact on the human Kv1.1 and Kv1.3 channels, subtypes of the potassium channel family. The potency and selectivity of synthetic OSK1 in inhibiting the human Kv1.1 and Kv1.3 channels was evaluated by the use of HEK293 cell expression system and electrophysiology (FIG. 53). Whole cell patch clamp recording of stably expressed Kv1.3 channels revealed that the synthetic OSK1 peptide is more potent in inhibiting human Kv1.3 when compared to Kv1.1 (Table 33).


Fusion of OSK1 peptide toxin to antibody to generate OSK1 peptibody. To improve plasma half-life and prevent OSK1 peptide toxin from penetrating the CNS, the OSK1 peptide toxin was fused to the Fc-fragment of a human antibody IgG1 via a linker chain length of 10 amino acid residues (Fc-L10-OSK1), as described in Example 41 herein. This fusion resulted in a decrease in the potency of Kv1.3 by 5-fold when compared to the synthetic OSK1 peptide. However, it significantly improved the selectivity of OSK1 against Kv1.1 by 210-fold when compared to that of the synthetic peptide alone (4-fold; Table 33 and FIG. 54).


Modification of OSK1-peptibody (Fc-L10-OSK1). OSK1 shares 60 to 80% sequence homology to other members of scorpion toxins, which are collectively termed α-KTx3. Sequence alignment of OSK1 and other members of α-KTx3 family revealed 4 distinct structural differences at positions 12, 16, 20, and 36. These structural differences of OSK1 have been postulated to play an important role in its wide range of activities against other potassium channels, which is not observed with other members of α-KTx3 family. Hence, two amino acid residues at position 16 and 20 were restored to the more conserved amino acid residues within the OSK1 sequence in order to evaluate their impact on selectivity against other potassium channels such as Kv1.1, which is predominantly found in the CNS as a heterotetromer with Kv1.2. By substituting for glutamic acid at position 16, and for lysine at position 20, the conserved lysine and aspartic acid residues, respectively (i.e., Fc-L10-OSK1[E16K, K20D]), we did not observe a significant change in potency when compared to that of Fc-L10-OSK1 (1.3-fold difference; FIG. 56 and Table 33). However, this double mutation removed the blocking activity against Kv1.1. The selectivity ratio of Kv1.1/Kv1.3 was 403-fold, which was a significant improvement over the selectivity ratio for Fc-L10-OSK1 (210-fold). A single amino acid mutation at position 7 from lysine to serine (Fc-L10-OSK1[K7S]) produced a slight change in potency and selectivity by 2- and 1.3-fold, respectively, when compared to those of Fc-L10-OSK1 (FIG. 55 and Table 33). There was a significant decrease in potency as well as selectivity when all three residues were mutated to generate Fc-L10-OSK1[K7S, E16K, K20D] (FIG. 57 and Table 33).


As demonstrated by the results in Table 33, we dramatically improved selectivity against Kv1.1 by fusing the OSK1 peptide toxin to the Fc-fragment of the human antibody IgG1, but reduced target potency against Kv1.3. The selectivity against Kv1.1 was further improved when 2 residues at two key positions were restored to the conserved residues found in other members of the α-KTx3 family.


Table 33 shows a summary of IC50 values for OSK1 and OSK1 analogs against hKv1.3 and hKv1.1 channels. All analogues are ranked based on their potency against hKv1.3. Also shown in the table is the selectively ratio of hKv1.1/hKv1.3 for all OSK1 analogs.
















hKv1.3: IC50
hKv1.1: IC50
hKv1.1/


Compound
[pM]
[pM]
hKv1.3


















Synthetic OSK1
39
160
4


Fc-L10-OSK1
198
41600
210


Fc-L10-OSK1[E16K, K20D]
248
100000
403


Fc-L10-OSK1[K7S]
372
100000
269


Fc-L10-OSK1[K7S, E16K, K20D]
812
10000
12









Example 44
Pharmacokinetic Study of PEG-ShK[1-35] Molecule in Rats

The intravenous (IV) pharmacokinetic profile was determined of a about 24-kDa 20K PEG-ShK[1-35] molecule and the about 4-kDa small native ShK peptide was determined in Spraque Dawley rats. The IV dose for the native ShK peptide and our novel 20K PEG-ShK[1-35] molecule was 1 mg/kg. This dose represented equal molar amounts of these two molecules. The average weight of the rats was about 0.3 kg and two rats were used for each dose & molecule. At various times following IV injection, blood was drawn and about 0.1 ml of serum was collected. Serum samples were stored frozen at −80° C. until analysis.


Assay Plate preparation for electrophysiology. Rat serum samples containing the 20K PEG-ShK[1-35] molecule or the native ShK peptide from pharmacokinetic studies were received frozen. Before experiments, each sample was thawed at room temperature and an aliquot (70 to 80 μl) was transferred to a well in a 96-well polypropylene plate. In order to prepare the Assay Plate, several dilutions were made from the pharmacokinetic serum samples to give rise to Test Solutions. Dilutions of serum samples from the pharmacokinetic study were into 10% Phosphate Buffered Saline (PBS, with Ca2, and Mg2+). For determination of the amount of our novel 20K PEG-ShK[1-35] molecule in serum samples from the pharmacokinetic study, the final serum concentrations in the Test Solutions were 90%, 30%, 10%, 3.3% and 1.1%. Purified 20K PEG-Shk[1-35] Standard inhibition curves were also prepared in the Assay Plate. To do this, 8-point serial dilutions of the purified 20K PEG-ShK[1-35] molecule (Standard) were prepared in either 90%, 30%, 10%, 3.3% or 1.1% rat serum and the final concentration of standard was 50, 16.7, 5.5, 1.85, 0.62, 0.21, 0.068 and 0.023 nM.


Cell preparation for electrophysiology. CHO cells stably expressing the voltage-activated K+ channel, KV1.3 were plated in T-175 tissue culture flasks (at a density of 5×106) 2 days before experimentation and allowed to grow to around 95% confluence. Immediately prior to the experiment, the cells were washed with PBS and then detached with a 2 ml mixture (1:1 volume ratio) of trypsin (0.25%) and versene (1:5000) at 37° C. (for 3 minutes). Subsequently, the cells were re-suspended in the flask in 10 ml of tissue culture medium (HAM's F-12 with Glutamax, Invitrogen, Cat#31765) with 10% FBS, 1×NEAA and 750 μg/ml of G418) and centrifuged at about 1000 rpm for 1½ minutes. The resultant cell pellet was re-suspended in PBS at 3-5×106 cells/ml.


IonWorks electrophysiology and data analysis. The ability of Test solutions or Standards in serum to inhibit K+ currents in the CHO-Kv1.3 cells was investigated using the automated electrophysiology system, IonWorks Quattro. Re-suspended cells, the Assay Plate, a Population Patch Clamp (PPC) PatchPlate as well as appropriate intracellular (90 mMK-Gluconate, 20 mMKF, 2 mM NaCl, 1 mM MgCl2, 10 mM EGTA, 10 mM HEPES, pH 7.35) and extracellular (PBS, with Ca2+ and Mg2+) buffers were positioned on IonWorks Quattro. Electrophysiology recordings were made from the CHO-Kv1.3 cells using an amphotericin-based perforated patch-clamp method. Using the voltage-clamp circuitry of the IonWorks Quattro, cells were held at a membrane potential of −80 mV and voltage-activated K+ currents were evoked by stepping the membrane potential to +30 mV for 400 ms. K+ currents were evoked under control conditions i.e., in the absence of inhibitor at the beginning of the experiment and after 10-minute incubation in the presence of the Test Solution or Standard. The mean K+ current amplitude was measured between 430 and 440 ms and the data were exported to a Microsoft Excel spreadsheet. The amplitude of the K+ current in the presence of each concentration of the Test Solution or Standard was expressed as a percentage of the K+ current in control conditions in the same well.


Standard inhibition curves were generated for each standard in various levels of rat serum and expressed as current percent of control (POC) versus log of nM concentration. Percent of control (POC) is inversely related to inhibition, where 100 POC is no inhibition and 0 POC is 100% inhibition. Linear regression over a selected region of the curve was used to derive an equation to enable calculation of drug concentrations within Test solutions. Only current values within the linear portion of the Standard curve were used to calculate the concentration of drug in Test solutions. The corresponding Standard curve in a given level of serum, was always compared to the same level of serum of Test solution when calculating drug level. The Standard curves for ShK and 20K PEG-ShK[1-35] are shown in FIG. 58A and FIG. 58B, respectively, and each figure contains linear regression equations for each Standard at a given percentage of serum. For the 20K PEG-ShK[1-35] standard curve the linear portion of the Standard curve was from 20 POC to 70 POC and only current values derived from the Test solution which fell within this range were used to calculate drug concentration within the Test solution.


The pharmacokinetic profile of our novel 20K PEG ShK[1-35] molecule after IV injection is shown in FIG. 59. The terminal half-life (t1/2 b) of this molecule is estimated from this curve to be between 6 to 12 hours long. Beyond 48 hours, the level of drug falls outside the linear range of the Standard curve and is not calculated. The calculated 6 to 12 hour half-life of our novel 20K PEG-ShK[1-35] molecule was substantially longer than the approximately 0.33 hour (or 20 min) half-life of the native ShK molecule reported earlier by C. Beeton et al. [C. Beeton et al. (2001) Proc. Natl. Acad. Sci. 98, 13942-13947], and is a desirable feature of a therapeutic molecule. A comparison of the relative levels of Kv1.3 inhibitor after an equal molar IV injection of ShK versus 20K PEG-ShK[1-35] is shown in FIG. 60. As can be seen from this figure examining 5% serum Test solutions, the 20K PEG-ShK[1-35] molecule showed significant suppression of Kv1.3 current (<70 POC) for more than 24 hours, whereas the native ShK peptide only showed a significant level of inhibition of Kv1.3 current for the first hour and beyond 1 hour showed no significant blockade. These data again demonstrate a desirable feature of the 20K PEG ShK[1-35] molecule as a therapeutic for treatment of autoimmune disease.


Example 45
PEGylated Toxin Peptide Suppressed Severe Autoimmune Encephalomyelitis in Animal Model

The 20KPEG-ShK inhibitor of Kv1.3 shows improved efficacy in suppressing severe autoimmune encephalomyelitis in rats. Using an adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE) model of multiple sclerosis described earlier [C. Beeton et al. (2001) J. Immunol. 166, 936], we examined the activity in vivo of our novel 20KPEG-ShK molecule and compared its efficacy to that of the ShK toxin peptide alone. The study design is illustrated in FIG. 61. The results from this in vivo study are provided in FIG. 62 and FIG. 63. The 20KPEG-ShK molecule delivered subcutaneously (SC) at 10 μg/kg daily from day −1 to day 3 significantly reduced disease severity and increased survival, whereas animals treated with an equal molar dose (10 μg/kg) of the small ShK peptide developed severe disease and died.


The 35-amino acid toxin peptide ShK (Stichodactyla helianthus neurotoxin) was purchased from Bachem Bioscience Inc and confirmed by electrophysiology to potently block Kv1.3 (see Example 36 herein). The synthesis, PEGylation and purification of the 20KPEG ShK molecule was as described herein above. The encephalomyelogenic CD4+ rat T cell line, PAS, specific for myelin-basic protein (MBP) originated from Dr. Evelyne Beraud. The maintenance of these cells in vitro and their use in the AT-EAE model has been described earlier [C. Beeton et al. (2001) PNAS 98, 13942]. PAS T cells were maintained in vitro by alternating rounds of antigen stimulation or activation with MBP and irradiated thymocytes (2 days), and propagation with T cell growth factors (5 days). Activation of PAS T cells (3×105/ml) involved incubating the cells for 2 days with 10 μg/ml MBP and 15×106/ml syngeneic irradiated (3500 rad) thymocytes. On day 2 after in vitro activation, 10-15×106 viable PAS T cells were injected into 6-12 week old female Lewis rats (Charles River Laboratories) by tail IV. Daily subcutaneous injections of vehicle (2% Lewis rat serum in PBS), 20KPEG-ShK or ShK were given from days −1 to 3 (FIG. 61), where day −1 represent 1 day prior to injection of PAS T cells (day 0). In vehicle treated rats, acute EAE developed 4 to 5 days after injection of PAS T cells (FIG. 62). Serum was collected by retro-orbital bleeding at day 4 and by cardiac puncture at day 8 (end of the study) for analysis of levels of inhibitor. Rats were weighed on days −1, 4, 6, and 8. Animals were scored blinded once a day from the day of cell transfer (day 0) to day 3, and twice a day from day 4 to day 8. Clinical signs were evaluated as the total score of the degree of paresis of each limb and tail. Clinical scoring: 0=No signs, 0.5=distal limp tail, 1.0=limp tail, 2.0=mild paraparesis, ataxia, 3.0=moderate paraparesis, 3.5=one hind leg paralysis, 4.0=complete hind leg paralysis, 5.0=complete hind leg paralysis and incontinence, 5.5=tetraplegia, 6.0=moribund state or death. Rats reaching a score of 5.5 were euthanized.


Treatment of rats with the Kv1.3 blocker PEG-ShK prior to the onset of EAE caused a lag in the onset of disease, inhibited the progression of disease, and prevented death in a dose-dependent manner (FIG. 62). Onset of disease in rats that were treated with the vehicle alone, 10 μg/kg ShK or 1 μg/kg of PEG-ShK was observed on day 4, compared to day 4.5 in rats treated with 10 μg/kg PEG-ShK or 100 μg/kg PEG-ShK. In addition, rats treated with vehicle alone, 10 μg/kg ShK or 1 μg/kg of PEG-ShK all developed severe disease by the end of the study with an EAE score of 5.5 or above. In contrast, rats treated with 10 μg/kg PEG-ShK or 100 μg/kg PEG-ShK, reached a peak clinical severity score average of <2, and all but one rat survived to the end of the study. Furthermore, we found that rat body weight correlated with disease severity (FIG. 63). Rats treated with vehicle alone, 10 μg/kg ShK or 1 μg/kg of PEG-ShK all lost an average of 31 g, 30 g, and 30 g, respectively, while rats treated with 10 μg/kg PEG-ShK or 100 μg/kg PEG-ShK lost 18 g and 11 g, respectively. Rats in the latter two groups also appeared to be gaining weight by the end of the study, a sign of recovery. It should be noted that rats treated with 10 μg/kg ShK and 10 μg/kg PEG-ShK received molar equivalents of the ShK peptide. The significantly greater efficacy of the PEG-ShK molecule relative to unconjugated ShK, is likely due to the PEG-ShK molecule's greater stability and prolonged half-life in vivo (see, Example 44).


Example 46
Compositions Including Kv1.3 Antagonist Peptides Block Inflammation in Human Whole Blood

Ex vivo assay to examine impact of Kv1.3 inhibitors on secretion of IL-2 and IFN-g. Human whole blood was obtained from healthy, non-medicated donors in a heparin vacutainer. DMEM complete media was Iscoves DMEM (with L-glutamine and 25 mM Hepes buffer) containing 0.1% human albumin (Bayer #68471), 55 μM 2-mercaptoethanol (Gibco), and 1× Pen-Strep-Gln (PSG, Gibco, Cat#10378-016). Thapsigargin was obtained from Alomone Labs (Israel). A 10 mM stock solution of thapsigargin in 100% DMSO was diluted with DMEM complete media to a 40 μM, 4× solution to provide the 4× thapsigargin stimulus for calcium mobilization. The Kv1.3 inhibitor peptide ShK (Stichodacytla helianthus toxin, Cat# H2358) and the BKCa1 inhibitor peptide IbTx (Iberiotoxin, Cat# H9940) were purchased from Bachem Biosciences, whereas the Kv1.1 inhibitor peptide DTX-k (Dendrotoxin-K) was from Alomone Labs (Israel). The CHO-derived Fc-L10-ShK[2-35] peptibody inhibitor of Kv1.3 was obtained as described herein at Example 4 and Example 39. The calcineurin inhibitor cyclosporin A was obtained from the Amgen sample bank, but is also available commercially from a variety of vendors. Ten 3-fold serial dilutions of inhibitors were prepared in DMEM complete media at 4× final concentration and 50 μl of each were added to wells of a 96-well Falcon 3075 flat-bottom microtiter plate. Whereas columns 1-5 and 7-11 of the microtiter plate contained inhibitors (each row with a separate inhibitor dilution series), 50 μl of DMEM complete media alone was added to the 8 wells in column 6 and 100 μl of DMEM complete media alone was added to the 8 wells in column 12. To initiate the experiment, 100 μl of whole blood was added to each well of the microtiter plate. The plate was then incubated at 37° C., 5% CO2 for one hour. After one hour, the plate was removed and 50 μl of the 4× thapsigargin stimulus (40 μM) was added to all wells of the plate, except the 8 wells in column 12. The plates were placed back at 37° C., 5% CO2 for 48 hours. To determine the amount of IL-2 and IFN-g secreted in whole blood, 100 μl of the supernatant (conditioned media) from each well of the 96-well plate was transferred to a storage plate. For MSD electrochemilluminesence analysis of cytokine production, 20 μl of the supernatants (conditioned media) were added to MSD Multi-Spot Custom Coated plates (www.meso-scale.com). The working electrodes on these plates were coated with four Capture Antibodies (hIL-5, hIL-2, hlFNg and hIL-4) in advance. After addition of 20 μl of conditioned media to the MSD plate, 150 μl of a cocktail of Detection Antibodies and P4 Buffer were added to each well. The 150 μl cocktail contained 20 ul of four Detection Antibodies (hIL-5, hIL-2, hIFNg and hIL-4) at 1 μg/ml each and 130 ul of 2×P4 Buffer. The plates were covered and placed on a shaking platform overnight (in the dark). The next morning the plates were read on the MSD Sector Imager. Since the 8 wells in column 6 of each plate received only the thapsigargin stimulus and no inhibitor, the average MSD response here was used to calculate the “High” value for a plate. The calculate “Low” value for the plate was derived from the average MSD response from the 8 wells in column 12 which contained no thapsigargin stimulus and no inhibitor. Percent of control (POC) is a measure of the response relative to the unstimulated versus stimulated controls, where 100 POC is equivalent to the average response of thapsigargin stimulus alone or the “High” value. Therefore, 100 POC represents 0% inhibition of the response. In contrast, 0 POC represents 100% inhibition of the response and would be equivalent to the response where no stimulus is given or the “Low” value. To calculate percent of control (POC), the following formula is used: [(MSD response of well)−(“Low”)]/[(“High”)−(“Low”)]×100. The potency of the molecules in whole blood was calculated after curve fitting from the inhibition curve (IC) and IC50 was derived using standard curve fitting software. Although we describe here measurement of cytokine production using a high throughput MSD electrochemillumenescence assay, one of skill in the art can readily envision lower throughput ELISA assays are equally applicable for measuring cytokine production.


Ex vivo assay demonstrating Kv1.3 inhibitors block cell surface activation of CD40L & IL-2R. Human whole blood was obtained from healthy, non-medicated donors in a heparin vacutainer. DMEM complete media was Iscoves DMEM (with L-glutamine and 25 mM Hepes buffer) containing 0.1% human albumin (Bayer #68471), 55 μM 2-mercaptoethanol (Gibco), and 1× Pen-Strep-Gln (PSG, Gibco, Cat#10378-016). Thapsigargin was obtained from Alomone Labs (Israel). A 10 mM stock solution of thapsigargin in 100% DMSO was diluted with DMEM complete media to a 40 μM, 4× solution to provide the 4× thapsigargin stimulus for calcium mobilization. The Kv1.3 inhibitor peptide ShK (Stichodacytla helianthus toxin, Cat# H2358) and the BKCa1 inhibitor peptide IbTx (Iberiotoxin, Cat# H9940) were purchased from Bachem Biosciences, whereas the Kv1.1 inhibitor peptide DTX-k (Dendrotoxin-K) was from Alomone Labs (Israel). The CHO-derived Fc-L110-ShK[2-35] peptibody inhibitor of Kv1.3 was obtained as described in Example 4 and Example 39. The calcineurin inhibitor cyclosporin A was obtained from the Amgen sample bank, but is also available commercially from a variety of vendors. The ion channel inhibitors ShK, IbTx or DTK-k were diluted into DMEM complete media to 4× of the final concentration desired (final=50 or 100 nM). The calcineurin inhibitor cyclosporin A was also diluted into DMEM complete media to 4× final concentration (final=10 μM). To appropriate wells of a 96-well Falcon 3075 flat-bottom microtiter plate, 50 μl of either DMEM complete media or the 4× inhibitor solutions were added. Then, 100 μl of human whole blood was added and the plate was incubated for 1 hour at 37° C., 5% CO2. After one hour, the plate was removed and 50 μl of the 4× thapsigargin stimulus (40 μM) was added to all wells of the plate containing inhibitor. To some wells containing no inhibitor but just DMEM complete media, thapsigargin was also added whereas others wells with just DMEM complete media had an additional 50 μl of DMEM complete media added. The wells with no inhibitor and no thapsigargin stimulus represented the untreated “Low” control. The wells with no inhibitor but which received thapsigargin stimulus represented the control for maximum stimulation or “High” control. Plates were placed back at 37° C., 5% CO2 for 24 hours. After 24 hours, plates were removed and wells were process for FACS analysis. Cells were removed from the wells and washed in staining buffer (phosphate buffered saline containing 2% heat-inactivated fetal calf serum). Red blood cells were lysed using BD FACS Lysing Solution containing 1.5% formaldehyde (BD Biosciences) as directed by the manufacturer. Cells were distributed at a concentration of 1 million cells per 100 microliters of staining buffer per tube. Cells were first stained with 1 microliter of biotin-labeled anti-human CD4, washed, then stained simultaneously 1 microliter each of streptavidin-APC, FITC-labeled anti-human CD45RA, and phycoerythrin (PE)-labeled anti-human CD25 (IL-2Ra) or PE-labeled anti-human CD40L. Cells were washed with staining buffer between antibody addition steps. All antibodies were obtained from BD Biosciences (San Diego, Calif.). Twenty to fifty thousand live events were collected for each sample on a Becton Dickinson FACSCaliber (Mountain View, Calif.) flow cytometer and analyzed using FlowJo software (Tree Star Inc., San Carlos, Calif.). Dead cells, monocytes, and granulocytes were excluded from the analysis on the basis of forward and side scatter properties.



FIG. 64 and FIG. 67 demonstrate that Kv1.3 inhibitors ShK and Fc-L10-ShK[2-35] potently blocked IL-2 secretion in human whole blood, in addition to suppressing activation of the IL-2R on CD4+ T cells. The Kv1.3 inhibitor Fc-L10-ShK[2-35] was more than 200 times more potent in blocking IL-2 production in human whole blood than cyclosporine A (FIG. 64) as reflected by the IC50. FIG. 65 shows that Kv1.3 inhibitors also potently blocked secretion of IFNg in human whole blood, and FIG. 66 demonstrates that upregulation of CD40L on T cells was additionally blocked. The data in FIGS. 64-67 show that the Fc-L10-ShK[2-35] molecule was stable in whole blood at 37° C. for up to 48 hours, providing potent blockade of inflammatory responses. Toxin peptide therapeutic agents that target Kv1.3 and have prolonged half-life, are sought to provide sustained blockade of these responses in vivo over time. In contrast, despite the fact the Kv1.3 inhibitor peptide ShK also showed potent blockade in whole blood, the ShK peptide has a short (˜20 min) half-life in vivo (C. Beeton et al. (2001) Proc. Natl. Acad. Sci. 98, 13942), and cannot, therefore, provide prolonged blockade. Whole blood represents a physiologically relevant assay to predict the response in animals. The whole blood assays described here can also be used as a pharmacodynamic (PD) assay to measure target coverage and drug exposure following dosing of patients. These human whole blood data support the therapeutic usefulness of the compositions of the present invention for treatment of a variety immune disorders, such as multiple sclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease, contact-mediated dermatitis, rheumatoid arthritis, psoriatic arthritis, asthma, allergy, restinosis, systemic sclerosis, fibrosis, scleroderma, glomerulonephritis, Sjogren syndrome, inflammatory bone resorption, transplant rejection, graft-versus-host disease, and lupus.


Example 47
PEGylated Peptibodies

By way of example, PEGylated peptibodies of the present invention were made by the following method. CHO-expressed FcL10-OsK1 (19.2 mg; MW 30,371 Da, 0.63 micromole) in 19.2 ml A5S, 20 mM NaBH3CN, pH 5, was treated with 38 mg PEG aldehyde (MW 20 kDa; 3×, Lot 104086). The sealed reaction mixture was stirred in a cold room overnight. The extent of the protein modification during the course of the reaction was monitored by SEC HPLC using a Superose 6 HR 10/30 column (Amersham Pharmacia Biotech) eluted with a 0.05 M phosphate buffer, 0.5 M NaCl, pH 7.0 at 0.4 ml/min. The reaction mixture was dialyzed with A5S, pH 5 overnight. The dialyzed material was then loaded onto an SP HP FPLC column (16/10) in A5S pH 5 and eluted with a 1 M NaCl gradient. The collected fractions were analyzed by SEC HPLC, pooled into 3 pools, exchanged into DPBS, concentrated and submitted for functional testing (Table 34).


In another example, FcL10-ShK1 (16.5 mg; MW 30,065 Da, 0.55 micro mole) in 16.5 ml A5S, 20 mM NaBH3CN, pH 5 was treated with 44 mg PEG aldehyde (MW 20 kDa; 4×, Lot 104086). The sealed reaction mixture was stirred in a cold room overnight. The extent of the protein modification during the course of the reaction was monitored by SEC HPLC using a Superose 6 HR 10/30 column (Amersham Pharmacia Biotech) eluted with a 0.05 M phosphate buffer, 0.5 M NaCl, pH 7.0 at 0.4 ml/min. The reaction mixture was dialyzed with A5S, pH 5 overnight. The dialyzed material was loaded onto an SP HP FPLC column (16/10) in A5S pH 5 and was eluted with a 1 M NaCl gradient. The collected fractions were analyzed by SEC HPLC, pooled into 3 pools, exchanged into DPBS, concentrated and submitted for functional testing (Table 34).


The data in Table 34 demonstrate potency of the PEGylated peptibody molecules as Kv1.3 inhibitors.


Table 34 shows determination of IC50 made by whole cell patch clamp electrophysiology with HEK 293 as described in Example 36 herein above. The sustained IC50 was derived from the current 400 msecs after voltage ramp from −80 mV to +30 mV. Pool #2 samples comprised di-PEGylated peptibodies and Pool #3 samples comprised mono-PEGylated peptibodies.

















PEGylated Peptibody
Pool #
IC50 Sustained (nM)









PEG-Fc-L10-SHK (2-35)
3
0.175 (n = 4)



PEG-Fc-L10-SHK (2-35)
2
0.158 (n = 4)



PEG-Fc-L10-OSK1
3
0.256 (n = 3)



PEG-Fc-L10-OSK1
2
0.332 (n = 3)










Example 48
PEGylated Toxin Peptides

Shk and Osk-1 PEGylation, purification and analysis. Synthetic Shk or OSK1-1 toxin peptides were selectively PEGylated by reductive alkylation at their N-termini. Conjugation was achieved, with either Shk or OSK-1 toxin peptides, at 2 mg/ml in 50 mM NaH2PO4, pH 4.5 reaction buffer containing 20 mM sodium cyanoborohydride and a 2 molar excess of 20 kDa monomethoxy-PEG-aldehyde (Nektar Therapeutics, Huntsville, Ala.). Conjugation reactions were stirred overnight at room temperature, and their progress was monitored by RP-HPLC. Completed reactions were quenched by 4-fold dilution with 20 mM NaOAc, pH 4, adjusted to pH 3.5 and chilled to 4° C. The PEG-peptides were then purified chromatographically at 4° C.; using SP Sepharose HP columns (GE Healthcare, Piscataway, N.J.) eluted with linear 0-1M NaCl gradients in 20 mM NaOAc, pH 4.0. (FIG. 68A and FIG. 68B) Eluted peak fractions were analyzed by SDS-PAGE and RP-HPLC and pooling determined by purity >97%. Principle contaminants observed were di-PEGylated toxin peptide and unmodified toxin peptide. Selected pools were concentrated to 2-5 mg/ml by centrifugal filtration against 3 kDa MWCO membranes and dialyzed into 10 mM NaOAc, pH 4 with 5% sorbitol. Dialyzed pools were then sterile filtered through 0.2 micron filters and purity determined to be >97% by SDS-PAGE and RP-HPLC (FIG. 69A and FIG. 69B). Reverse-phase HPLC was performed on an Agilent 1100 model HPLC running a Zorbax 5 μm 300SB-C8 4.6×50 mm column (Phenomenex) in 0.1% TFA/H20 at 1 ml/min and column temperature maintained at 40° C. Samples of PEG-peptide (20 μg) were injected and eluted in a linear 6-60% gradient while monitoring wavelengths 215 nm and 280 nm.


Electrophysiology performed by patch clamp on whole cells (see, Example 36) yielded a peak IC50 of 1.285 nM for PEG-OSK1 and 0.169 nM for PEG-ShK[1-35] (FIG. 74), in a concentration dependent block of the outward potassium current recorded from HEK293 cells stably expressing human Kv1.3 channel. The purified PEG-ShK[1-35] molecule, also referred to as “20K PEG-ShK[1-35]” and “PEG-ShK”, had a much longer half-life in vivo than the small ShK peptide (FIG. 59 and FIG. 60). PEG-ShK[1-35] suppressed severe autoimmune encephalomyelitis in rats (Example 45, FIGS. 61-63) and showed greater efficacy than the small native ShK peptide.


PEG conjugates of OSK1 peptide analogs were also generated and tested for activity in blocking T cell inflammation in the human whole blood assay (Example 46). As shown in Table 43, OSK1[Ala12], OSK1[Ala29], OSK1[Nal34] and OSK1[Ala29, 1Nal34] analogs containing an N-terminal 20K PEG conjugate, all provided potent blockade of the whole blood cytokine response in this assay. The 20K PEG-ShK was also highly active (Table 43).


Example 49
Fc Loop Insertions of ShK and OSK1 Toxin Peptides

As exemplified in FIG. 70, FIG. 71, FIG. 72, and FIG. 73, disulphide-constrained toxin peptides were inserted into the human IgG1 Fc-loop domain, defined as the sequence D137E138T139T140K141, according to the method published in Example 1 in Gegg et al., Modified Fc molecules, WO 2006/036834 A2 [PCT/US2005/034273]). Exemplary FcLoop-L2-OsK1-L2, FcLoop-L2-ShK-L2, FcLoop-L2-ShK-L4, and FcLoop-L4-OsK1-L2 were made having three linked domains. These were collected, purified and submitted for functional testing.


The peptide insertion for these examples was between Fc residues Leu139 and Thr140 and included 2-4 Gly residues as linkers flanking either side of the inserted peptide. However, alternate insertion sites for the human IgG1 Fc sequence, or different linkers, are also useful in the practice of the present invention, as is known in the art, e.g., as described in Example 13 of Gegg et al., Modified Fc molecules, WO 2006/036834 A2 [PCT/US2005/034273]).


Purified FcLoop OSK1 and FcLoop ShK1 molecules were tested in the whole blood assay of inflammation (see, Example 46). FcLoop-L2-OsK1-L2, FcLoop-L4-OsK1-L4 and FcLoop-L2-ShK-L2 toxin conjugates all provided potent blockade of the whole blood cytokine response in this assay, with IC50 values in the pM range (Table 43).


Example 50
Purification of ShK(2-35)-L-Fc from E. coli

Frozen, E. coli paste (117 g), obtained as described in Example 16 herein above, was combined with 1200 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 7.5 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 17,700 g for 30 min at 4° C. The pellet was then resuspended in 1200 ml 1% deoxycholic acid using a tissue grinder and then centrifuged at 17,700 g for 30 min at 4° C. The pellet was then resuspended in 1200 ml water using a tissue grinder and then centrifuged at 17,700 g for 30 min at 4° C. 6.4 g of the pellet (total 14.2 g) was then dissolved in 128 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. 120 ml of the pellet solution was then incubated with 0.67 ml of 1 M DTT for 60 min at 37° C. The reduced material was transferred to 5500 ml of the refolding buffer (3 M urea, 50 mM tris, 160 mM arginine HCl, 2.5 mM EDTA, 2.5 mM cystamine HCl, 4 mM cysteine, pH 9.5) at 2 ml/min, 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for 3 days at 4° C.


The refold was diluted with 5.5 L of water, and the pH was adjusted to 8.0 using acetic acid, then the solution was filtered through a 0.22 μm cellulose acetate filter and loaded on to a 35 ml Amersham Q Sepharose-FF (2.6 cm I.D.) column at 10 ml/min in Q-Buffer A (20 mM Tris, pH 8.5) at 8° C. with an inline 35 ml Amersham Mab Select column (2.6 cm I.D.). After loading, the Q Sepharose column was removed from the circuit, and the remaining chromatography was carried out on the Mab Select column. The column was washed with several column volumes of Q-Buffer A, followed by elution using a step to 100 mM glycine pH 3.0. The fractions containing the desired product immediately loaded on to a 5.0 ml Amersham SP-Sepharose HP column at 5.0 ml/min in S-Buffer A (10 mM NaH2PO4, pH 7.0) at 8° C. The column was then washed with several column volumes of S-Buffer A followed by a linear gradient from 5% to 60% S-Buffer B (10 mM NaH2PO4, 1 M NaCl, pH 7.0) followed by a step to 100% S-Buffer B. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE. The fractions containing the bulk of the desired product were pooled and then applied to a 50 ml MEP Hypercel column (2.6 cm I.D.) at 10 ml/min in MEP Buffer A (20 mM tris, 200 mM NaCl, pH 8.0) at 8° C. Column was eluted with a linear gradient from 5% to 50% MEP Buffer B (50 mM sodium citrate pH 4.0) followed by a step to 100% MEP Buffer B. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the bulk of the desired product were pooled.


The MEP-pool was then concentrated to about 10 ml using a Pall Jumbo-Sep with a 10 kDa membrane. A spectral scan was then conducted on 50 μl of the combined pool diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 76A). Then concentration of the material was determined to be 3.7 mg/ml using a calculated molecular mass of 30,253 and extinction coefficient of 36,900 M−1 cm−1. The purity of the material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 76B). The macromolecular state of the product was then determined using size exclusion chromatography on 70 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 76C). The product was then subject to mass spectral analysis by chromatographing approximately 4 μg of the sample through a RP-HPLC column (Vydac C4, 1×150 mm). Solvent A was 0.1% trifluoroacetic acid in water and solvent B was 0.1% trifluoroacetic acid in 90% acetonitrile, 10% water. The column was pre-equilibrated in 10% solvent B at a flow rate of 80 μl per min. The protein was eluted using a linear gradient of 10% to 90% solvent B over 30 min. Part of the effluent was directed into a LCQ ion trap mass spectrometer. The mass spectrum was deconvoluted using the Bioworks software provided by the mass spectrometer manufacturer. (FIG. 76D). The product was filtered through a 0.22 μm cellulose acetate filter and then stored at −80° C.


In Table 35, IC50 data for the purified E. coli-derived ShK[2-35]-L-Fc are compared to some other embodiments of the inventive composition of matter.









TABLE 35








E. coli-derived recombinant Fc-L-ShK[1-35], Fc-L-ShK[2-35], Fc-L-



OSK1, Shk[1-35]-L-Fc and ShK[2-35]-L-Fc peptibodies containing Fc at


either the N-terminus or C-terminus show potent blockade of human


Kv1.3. The activity of the CHO-derived Fc-L10-ShK[1-35] R1Q mutant is


also shown. Whole cell patch clamp electrophysiology (WCVC), by


methods described in Example 36, was performed using HEK293/Kv1.3


cells and the IC50 shown is the average from dose-response curves from


3 or more cells. IonWorks ™ (IWQ) planar patch clamp electrophysiology


by methods described in Example 44 was on CHO/Kv1.3 cells and the


average IC50 is shown. The inventive molecules were obtained by


methods as described in the indicated Example: E. coli-derived Fc-L-


ShK[1-35] (Example 3 and Example 38), E. coli-derived Fc-L-ShK[2-35]


(Example 4 and Example 39), E. coli Fc-L-OSK1 (Example 10 and


Example 40), ShK[1-35]-L-Fc (Example 15 and Example 51), and


ShK[2-35]-L-Fc (Example 16 and this Example 50). CHO-derived


Fc-L10-ShK[1-35] R1Q molecule was generated using methods similar to


those described for CHO-derived Fc-L10-ShK[1-35].










Kv1.3 IC50
Kv1.3 IC50


Molecule
by WCVC (nM)
by IWQ (nM)






E. coli-derived Fc-L-ShK[1-35]

1.4




E. coli-derived Fc-L-ShK[2-35]

1.3
2.8



E. coli-derived Fc-L-OSK1

3.2



E. coli-derived Shk[1-35]-L-Fc


2.4



E. coli-derived ShK[2-35]-L-Fc


4.9


CHO-derived Fc-L10-ShK[1-35]

2.2


R1Q









Example 51
Purification of Met-ShK(1-35)-Fc from E. coli

Frozen, E. coli paste (65 g), obtained as described in Example 15 herein above was combined with 660 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 7.5 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 17,700 g for 30 min at 4° C. The pellet was then resuspended in 660 ml 1% deoxycholic acid using a tissue grinder and then centrifuged at 17,700 g for 30 min at 4° C. The pellet was then resuspended in 660 ml water using a tissue grinder and then centrifuged at 17,700 g for 30 min at 4° C. 13 g of the pellet was then dissolved in 130 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. 10 ml of the pellet solution was then incubated with 0.1 ml of 1 M DTT for 60 min at 37° C. The reduced material was transferred to 1000 ml of the refolding buffer (2 M urea, 50 mM tris, 160 mM arginine HCl, 2.5 mM EDTA, 1.2 mM cystamine HCl, 4 mM cysteine, pH 8.5) at 2 ml/min, 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for 3 days at 4° C.


The refold was diluted with 1 L of water, and filtered through a 0.22 μm cellulose acetate filter then loaded on to a 35 ml Amersham Q Sepharose-FF (2.6 cm I.D.) column at 10 ml/min in Q-Buffer A (20 mM Tris, pH 8.5) at 8° C. with an inline 35 ml Amersham Mab Select column (2.6 cm I.D.). After loading, the Q Sepharose column was removed from the circuit, and the remaining chromatography was carried out on the Mab Select column. The column was washed with several column volumes of Q-Buffer A, followed by elution using a step to 100 mM glycine pH 3.0. The fractions containing the desired product immediately loaded on to a 5.0 ml Amersham SP-Sepharose HP column at 5.0 ml/min in S-Buffer A (20 mM NaH2PO4, pH 7.0) at 8° C. The column was then washed with several column volumes of S-Buffer A followed by a linear gradient from 5% to 60% S-Buffer B (20 mM NaH2PO4, 1 M NaCl, pH 7.0) followed by a step to 100% S-Buffer B. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE. The fractions containing the bulk of the desired product were pooled.


The S-pool was then concentrated to about 10 ml using a Pall Jumbo-Sep with a 10 kDa membrane. A spectral scan was then conducted on 20 μl of the combined pool diluted in 700 μl PBS using a Hewlett Packard 8453 spectrophotometer (FIG. 77A). Then concentration of the material was determined to be 3.1 mg/ml using a calculated molecular mass of 30,409 and extinction coefficient of 36,900 M−1 cm−1. The purity of the material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 77B). The macromolecular state of the product was then determined using size exclusion chromatography on 93 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 77C). The product was then subject to mass spectral analysis by MALDI mass spectrometry.


An aliquot of the sample was spotted with the MALDI matrix sinapinic acid on sample plate. A Voyager DE-RP time-of-flight mass spectrometer equipped with a nitrogen laser (337 nm, 3 ns pulse) was used to collect spectra. The positive ion/linear mode was used, with an accelerating voltage of 25 kV. Each spectrum was produced by accumulating data from ˜200 laser shots (FIG. 77D). External mass calibration was accomplished using purified proteins of known molecular masses.


The IC50 for blockade of human Kv1.3 by purified E. coli-derived Met-ShK(1-35)-Fc, also referred to as “ShK[1-35]-L-Fc”, is shown in Table 35 herein above.


Example 52
Bacterial Expression of OsK1-L-Fc Inhibitor of Kv1.3

The methods to clone and express the peptibody in bacteria were as described in Example 3. The vector used was pAMG21amgR-pep-Fc and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of OsK1-L-Fc. Oligos used to form duplex are shown below:










SEQ ID NO: 1347









GGGTGTTATCATCAACGTTAAATGCAAAATCTCCCGTCAGTGCCTGGAAC



CGTGCAAAAAAGCTGGTATGCGT //;











SEQ ID NO: 1348









TTCGGTAAATGCATGAACGGTAAATGCCACTGCACCCCGAAATCTGGTGG



TGGTGCTTCT //;











SEQ ID NO: 1349









CACCACAACCACCACCACCACCAGATTTCGGGGTGCAGTGGCATTTACCG



TTCATGCATTTACCGAAACGCAT //;











SEQ ID NO: 1310









ACCAGCTTTTTTGCACGGTTCCAGGCACTGACGGGAGATTTTGCATTTAA



CGTTGATGATAAC //;






The oligos shown above were used to form the duplex shown below:














GGGTGTTATCATCAACGTTAAATGCAAAATCTCCCGTCAGTGCCTGGAACCGTGCAAAAA

SEQ ID NO: 1350



1
---------+---------+---------+---------+---------+---------+
60




    CAATAGTAGTTGCAATTTACGTTTTAGAGGGCAGTCACGGACCTTGGCACGTTTTT

SEQ ID NO: 1352















 G  V  I  I  N  V  K  C  K  I  S  R  Q  C  L  E  P  C  K  K
-
SEQ ID NO: 1351







AGCTGGTATGCGTTTCGGTAAATGCATGAACGGTAAATGCCACTGCACCCCGAAATCTGG


61
---------+---------+---------+---------+---------+---------+
120




TCGACCATACGCAAAGCCATTTACGTACTTGCCATTTACGGTGACGTGGGGCTTTAGACC














 A  G  M  R  F  G  K  C  M  N  G  K  C  H  C  T  P  K  S  G
-







TGGTGGTGGTTCT //


121
---------+-------
137



ACCACCACCAAGACCAC //






 G  G  G  S  G //
-







Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.


Example 53
Bacterial Expression of Gly-ShK(1-35)-L-Fc Inhibitor of Kv1.3

The methods to clone and express the peptibody in bacteria were as described in Example 3. The vector used was pAMG21amgR-pep-Fc and the oligos listed below were used to generate a duplex (see below) for cloning and expression in bacteria of Gly-ShK(1-35)-L-Fc. Oligos used to form duplex are shown below:










SEQ ID NO: 1313









GGGTCGTTCTTGTATTGATACTATTCCAAAATCTCGTTGTACTGCTTTTC



AATGTAAACATTCTATGAAATATCGTCTTTCTT //;











SEQ ID NO: 1314









TTTGTCGTAAAACTTGTGGTACTTGTTCTGGTGGTGGTGGTTCT //;












SEQ ID NO: 1353









CACCAGAACCACCACCACCAGAACAAGTACCACAAGTTTTACGACAAAAA



GAAAGACGATATTTCATAGAATGTTTACATTGA //;











SEQ ID NO: 1354









AAAGCAGTACAACGAGATTTTGGAATAGTATCAATACAAGAACG //;







The oligos shown above were used to form the duplex shown below:














GGGTCGTTCTTGTATTGATACTATTCCAAAATCTCGTTGTACTGCTTTTCAATGTAAACA

SEQ ID NO: 1355



1
---------+---------+---------+---------+---------+---------+
60




    GCAAGAACATAACTATGATAAGGTTTTAGAGCAACATGACGAAAAGTTACATTTGT

SEQ ID NO: 1357















 G  R  S  C  I  D  T  I  P  K  S  R  C  T  A  F  Q  C  K  H
-
SEQ ID NO: 1356







TTCTATGAAATATCGTCTTTCTTTTTGTCGTAAAACTTGTGGTACTTGTTCTGGTGGTGG


61
---------+---------+---------+---------+---------+---------+
120




AAGATACTTTATAGCAGAAAGAAAAACAGCATTTTGAACACCATGAACAAGACCACCACC














 S  M  K  Y  R  L  S  F  C  R  K  T  C  G  T  C  S  G  G  G
-







TGGTTCT //


121
---------+-
131




ACCAAGACCAC //






 G  S  G //
-







Bacterial expression of the peptibody was as described in Example 3 and paste was stored frozen.


Example 54
Bacterial Expression of CH2-OSK1 Inhibitor of Kv1.3

The methods to clone and express the fusion of a CH2 domain of an Fc with OSK1 in bacteria were generally as described in Example 3. The vector used was pAMG21.G2.H6.G3.CH2.(G4S)2.OSK. Briefly, the pAMG21 vector was modified to remove the multi-cloning site's BamHI. This allowed the BamHI in front of the OSK as a site to swap out different sequences for fusion with the OSK. The sequence upstream of the OSK1 coding sequence was ligated between the NdeI and BamHI sites.


The sequence of the entire vector, including the insert was the following:










SEQ ID NO: 4914









gtcgtcaacgaccccccattcaagaacagcaagcagcattgagaactttg






gaatccagtccctcttccacctgctgaccggatcagcagtccccggaaca





tcgtagctgacgccttcgcgttgctcagttgtccaaccccggaaacggga





aaaagcaagttttccccgctcccggcgtttcaataactgaaaaccatact





atttcacagtttaaatcacattaaacgacagtaatccccgttgatttgtg





cgccaacacagatcttcgtcacaattctcaagtcgctgatttcaaaaaac





tgtagtatcctctgcgaaacgatccctgtttgagtattgaggaggcgaga





tgtcgcagacagaaaatgcagtgacttcctcattgagtcaaaagcggttt





gtgcgcagaggtaagcctatgactgactctgagaaacaaatggccgttgt





tgcaagaaaacgtcttacacacaaagagataaaagtttttgtcaaaaatc





ctctgaaggatctcatggttgagtactgcgagagagaggggataacacag





gctcagttcgttgagaaaatcatcaaagatgaactgcaaagactggatat





actaaagtaaagactttactttgtggcgtagcatgctagattactgatcg





tttaaggaattttgtggctggccacgccgtaaggtggcaaggaactggtt





ctgatgtggatttacaggagccagaaaagcaaaaaccccgataatcttct





tcaacttttgcgagtacgaaaagattaccggggcccacttaaaccgtata





gccaacaattcagctatgcggggagtatagttatatgcccggaaaagttc





aagacttctttctgtgctcgctccttctgcgcattgtaagtgcaggatgg





tgtgactgatcttcaccaaacgtattaccgccaggtaaagaacccgaatc





cggtgtttacaccccgtgaaggtgcaggaacgctgaagttctgcgaaaaa





ctgatggaaaaggcggtgggcttcacttcccgttttgatttcgccattca





tgtggcgcacgcccgttcgcgtgatctgcgtcgccgtatgccaccagtgc





tgcgtcgtcgggctattgatgcgctcttgcaggggctgtgtttccactat





gacccgctggccaaccgcgtccagtgctccatcaccacgctggccattga





gtgcggactggcgacggagtctgctgccggaaaactctccatcacccgtg





ccacccgtgccctgacgttcctgtcagagctgggactgattacctaccag





acggaatatgacccgcttatcgggtgctacattccgaccgatatcacgtt





cacatctgcactgtttgctgccctcgatgtatcagaggaggcagtggccg





ccgcgcgccgcagccgtgtggtatgggaaaacaaacaacgcaaaaagcag





gggctggataccctgggcatggatgaactgatagcgaaagcctggcgttt





tgttcgtgagcgttttcgcagttatcagacagagcttaagtcccgtggaa





taaagcgtgcccgtgcgcgtcgtgatgcggacagggaacgtcaggatatt





gtcaccctggtgaaacggcagctgacgcgcgaaatcgcggaagggcgctt





cactgccaatcgtgaggcggtaaaacgcgaagttgagcgtcgtgtgaagg





agcgcatgattctgtcacgtaaccgtaattacagccggctggccacagct





tccccctgaaagtgacctcctctgaataatccggcctgcgccggaggctt





ccgcacgtctgaagcccgacagcgcacaaaaaatcagcaccacatacaaa





aaacaacctcatcatccagcttctggtgcatccggccccccctgttttcg





atacaaaacacgcctcacagacggggaattttgcttatccacattaaact





gcaagggacttccccataaggttacaaccgttcatgtcataaagcgccat





ccgccagcgttacagggtgcaatgtatcttttaaacacctgtttatatct





cctttaaactacttaattacattcatttaaaaagaaaacctattcactgc





ctgtccttggacagacagatatgcacctcccaccgcaagcggcgggcccc





taccggagccgctttagttacaacactcagacacaaccaccagaaaaacc





ccggtccagcgcagaactgaaaccacaaagcccctccctcataactgaaa





agcggccccgccccggtccgaagggccggaacagagtcgcttttaattat





gaatgttgtaactacttcatcatcgctgtcagtcttctcgctggaagttc





tcagtacacgctcgtaagcggccctgacggcccgctaacgcggagatacg





ccccgacttcgggtaaaccctcgtcgggaccactccgaccgcgcacagaa





gctctctcatggctgaaagcgggtatggtctggcagggctggggatgggt





aaggtgaaatctatcaatcagtaccggcttacgccgggcttcggcggttt





tactcctgtttcatatatgaaacaacaggtcaccgccttccatgccgctg





atgcggcatatcctggtaacgatatctgaattgttatacatgtgtatata





cgtggtaatgacaaaaataggacaagttaaaaatttacaggcgatgcaat





gattcaaacacgtaatcaatatcgggggtgggcgaagaactccagcatga





gatccccgcgctggaggatcatccagccggcgtcccggaaaacgattccg





aagcccaacctttcatagaaggcggcggtggaatcgaaatctcgtgatgg





caggttgggcgtcgcttggtcggtcatttcgaaccccagagtcccgctca





gaagaactcgtcaagaaggcgatagaaggcgatgcgctgcgaatcgggag





cggcgataccgtaaagcacgaggaagcggtcagcccattcgccgccaagc





tcttcagcaatatcacgggtagccaacgctatgtcctgatagcggtccgc





cacacccagccggccacagtcgatgaatccagaaaagcggccattttcca





ccatgatattcggcaagcaggcatcgccatgagtcacgacgagatcctcg





ccgtcgggcatgcgcgccttgagcctggcgaacagttcggctggcgcgag





cccctgatgctcttcgtccagatcatcctgatcgacaagaccggcttcca





tccgagtacgtgctcgctcgatgcgatgtttcgcttggtggtcgaatggg





caggtagccggatcaagcgtatgcagccgccgcattgcatcagccatgat





ggatactttctcggcaggagcaaggtgagatgacaggagatcctgccccg





gcacttcgcccaatagcagccagtcccttcccgcttcagtgacaacgtcg





agcacagctgcgcaaggaacgcccgtcgtggccagccacgatagccgcgc





tgcctcgtcctgcaattcattcaggacaccggacaggtcggtcttgacaa





aaagaaccgggcgcccctgcgctgacagccggaacacggcggcatcagag





cagccgattgtctgttgtgcccagtcatagccgaatagcctctccaccca





agcggccggagaacctgcgtgcaatccatcttgttcaatcatgcgaaacg





atcctcatcctgtctcttgatctgatcttgatcccctgcgccatcagatc





cttggcggcaagaaagccatccagtttactttgcagggcttcccaacctt





accagagggcgccccagctggcaattccggttcgcttgctgtccataaaa





ccgcccagtctagctatcgccatgtaagcccactgcaagctacctgcttt





ctctttgcgcttgcgttttcccttgtccagatagcccagtagctgacatt





catccggggtcagcaccgtttctgcggactggctttctacgtgttccgct





tcctttagcagcccttgcgccctgagtgcttgcggcagcgtgaagctaca





tatatgtgatccgggcaaatcgctgaatattccttttgtctccgaccatc





aggcacctgagtcgctgtctttttcgtgacattcagttcgctgcgctcac





ggctctggcagtgaatgggggtaaatggcactacaggcgccttttatgga





ttcatgcaaggaaactacccataatacaagaaaagcccgtcacgggcttc





tcagggcgttttatggcgggtctgctatgtggtgctatctgactttttgc





tgttcagcagttcctgccctctgattttccagtctgaccacttcggatta





tcccgtgacaggtcattcagactggctaatgcacccagtaaggcagcggt





atcatcaacaggcttacccgtcttactgtcgaagacgtgcgtaacgtatg





catggtctccccatgcgagagtagggaactgccaggcatcaaataaaacg





aaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcgg





tgaacgctctcctgagtaggacaaatccgccgggagcggatttgaacgtt





gcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgc





caggcatcaaattaagcagaaggccatcctgacggatggcctttttgcgt





ttctacaaactcttttgtttatttttctaaatacattcaaatatggacgt





cgtacttaacttttaaagtatgggcaatcaattgctcctgttaaaattgc





tttagaaatactttggcagcggtttgttgtattgagtttcatttgcgcat





tggttaaatggaaagtgaccgtgcgcttactacagcctaatatttttgaa





atatcccaagagctttttccttcgcatgcccacgctaaacattctttttc





tcttttggttaaatcgttgtttgatttattatttgctatatttatttttc





gataattatcaactagagaaggaacaattaatggtatgttcatacacgca





tgtaaaaataaactatctatatagttgtctttctctgaatgtgcaaaact





aagcattccgaagccattattagcagtatgaatagggaaactaaacccag





tgataagacctgatgatttcgcttctttaattacatttggagatttttta





tttacagcattgttttcaaatatattccaattaatcggtgaatgattgga





gttagaataatctactataggatcatattttattaaattagcgtcatcat





aatattgcctccattttttagggtaattatccagaattgaaatatcagat





ttaaccatagaatgaggataaatgatcgcgagtaaataatattcacaatg





taccattttagtcatatcagataagcattgattaatatcattattgcttc





tacaggctttaattttattaattattctgtaagtgtcgtcggcatttatg





tctttcatacccatctctttatccttacctattgtttgtcgcaagttttg





cgtgttatatatcattaaaacggtaatagattgacatttgattctaataa





attggatttttgtcacactattatatcgcttgaaatacaattgtttaaca





taagtacctgtaggatcgtacaggtttacgcaagaaaatggtttgttata





gtcgattaatcgatttgattctagatttgttttaactaattaaaggagga





ataacatatgggcggccatcatcatcatcatcatggcgggggaccgtcag





ttttcctcttccccccaaaacccaaggacaccctcatgatctcccggacc





cctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggt





caagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaa





agccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctc





accgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggt





ctccaacaaagccctcccagcccccatcgagaaaaccatctccggcggcg





gcggcagcggcggcggcggatccggtgttatcatcaacgttaaatgcaaa





atctcccgtcagtgcctggaaccgtgcaaaaaagctggtatgcgtttcgg





taaatgcatgaacggtaaatgccactgcaccccgaaataatgaattcgag





ctcactagtgtcgacctgcagggtaccatggaagcttactcgaagatccg





cggaaagaagaagaagaagaagaaagcccgaaaggaagctgagttggctg





ctgccaccgctgagcaataactagcataaccccttggggcctctaaacgg





gtcttgaggggttttttgctgaaaggaggaaccgctcttcacgctcttca





cgcggataaataagtaacgatccggtccagtaatgacctcagaactccat





ctggatttgttcagaacgctcggttgccgccgggcgttttttattggtga





gaatcgcagcaacttgtcgcgccaatcgagccatgtc//.






The insert DNA sequence was the following:










SEQ ID NO: 4915









atgggcggccatcatcatcatcatcatggcgggggaccgtcagttttcct






cttccccccaaaacccaaggacaccctcatgatctcccggacccctgagg





tcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc





aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcg





ggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcc





tgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaac





aaagccctcccagcccccatcgagaaaaccatctccggcggcggcggcag





cggcggcggcggatccggtgttatcatcaacgttaaatgcaaaatctccc





gtcagtgcctggaaccgtgcaaaaaagctggtatgcgtttcggtaaatgc





atgaacggtaaatgccactgcaccccgaaa//.






The amino acid sequence of the CH2-OSK1 fusion protein product was the following:










SEQ ID NO: 4917









MGGHHHHHHGGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF



NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN


KALPAPIEKTISGGGGSGGGGSGVIINVKCKISRQCLEPCKKAGMRFGKC


MNGKCHCTPK//.





SEQ ID NO:4917 includes the OSK1 sequence











(SEQ ID NO: 25)









GVIINVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK.







Purification and refolding of CH2-OSK1 expressed in bacteria. Frozen, E. coli paste (13.8 g) was combined with 180 ml of room temperature 50 mM tris HCl, 5 mM EDTA, pH 8.0 and was brought to about 0.1 mg/ml hen egg white lysozyme. The suspended paste was passed through a chilled microfluidizer twice at 12,000 PSI. The cell lysate was then centrifuged at 17,700 g for 50 min at 4° C. The pellet was then resuspended in 90 ml 1% deoxycholate using a tissue grinder and then centrifuged at 15,300 g for 40 min at 4° C. The pellet was then resuspended in 90 ml water using a tissue grinder and then centrifuged at 15,300 g for 40 min at 4° C. The pellet (3.2 g) was then dissolved in 64 ml 8 M guanidine HCl, 50 mM tris HCl, pH 8.0. The suspension was then incubated at room temperature (about 23° C.) for 30 min with gentle agitation followed by centrifugation at 15,300 g for 30 min at 4° C. The supernatant (22 ml) was then reduced by adding 220 μl 1 M dithiothreitol and incubating at 37° C. for 30 minutes. The reduced suspension (20 ml) was transferred to 2000 ml of the refolding buffer (1 M urea, 50 mM ethanolamine, 160 mM arginine HCl, 0.02% NaN3, 1.2 mM cystamine HCl, 4 mM cysteine, pH 9.8) at 4° C. with vigorous stirring. The stirring rate was then slowed and the incubation was continued for approximately 2.5 days at 4° C.


Ten milliliters of 500 mM imidazole was added to the refolding solution and the pH was adjusted to pH to 8.0 with 5 M acetic acid. The refold was then filtered through a 0.45 μm cellulose acetate filter with two pre-filters. This material was then loaded on to a 50 ml Qiagen Ni-NTA Superflow column (2.6 cm ID) in Ni-Buffer A (50 mM NaH2PO4, 300 mM NaCl, pH 7.5) at 15 ml/min 13° C. The column was then washed with 10 column volumes of Ni-Buffer A followed by 8% Ni-Buffer B (250 mM imidazole, 50 mM NaH2PO4, 300 mM NaCl, pH 7.5) at 25 ml/min. The column was then eluted with 60% Ni-Buffer B followed by 100% Ni-Buffer B at 10 ml/min. The peak fractions were collected and dialyzed against S-Buffer A (10 mM NaH2PO4, pH 7.1)


The dialyzed sample was then loaded on to a 5 ml Amersham SP-HP HiTrap column at 5 ml/min in S-Buffer A at 13° C. The column was then washed with several column volumes of S-Buffer A, followed by elution with a linear gradient from 0% to 60% S-Buffer B (10 mM NaH2PO4, 1 M NaCl, pH 7.1) followed by a step to 100% S-Buffer B at 1.5 ml/min 13° C. Fractions were then analyzed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE, and the fractions containing the desired product were pooled based on these data. The pool was then concentrated to about 1.6 ml using a Pall Macrosep with a 10 kDa membrane at 4° C. The concentrated sample was then filtered through a 0.22 μm cellulose acetate centrifugal filter.


A spectral scan was then conducted on 10 μl of the combined pool diluted in 150 μl water using a Hewlett Packard 8453 spectrophotometer (FIG. 78). The concentration of the filtered material was determined to be 3.35 mg/ml using a calculated molecular mass of 17,373 g/mol and extinction coefficient of 17,460 M−1 cm−1. The purity of the filtered material was then assessed using a Coomassie brilliant blue stained tris-glycine 4-20% SDS-PAGE (FIG. 79). The endotoxin level was then determined using a Charles River Laboratories Endosafe-PTS system (0.05-5 EU/ml sensitivity) using a 67-fold dilution of the sample in Charles Rivers Endotoxin Specific Buffer BG120 yielding a result of <1 EU/mg protein. The macromolecular state of the product was then determined using size exclusion chromatography on 50 μg of the product injected on to a Phenomenex BioSep SEC 3000 column (7.8×300 mm) in 50 mM NaH2PO4, 250 mM NaCl, pH 6.9 at 1 ml/min observing the absorbance at 280 nm (FIG. 80). The product was then subject to mass spectral analysis by chromatographing approximately 4 μg of the sample through a RP-HPLC column (Vydac C4, 1×150 mm). Solvent A was 0.1% trifluoroacetic acid in water and solvent B was 0.1% trifluoroacetic acid in 90% acetonitrile, 10% water. The column was pre-equilibrated in 10% solvent B at a flow rate of 80 μl per min. The protein was eluted using a linear gradient of 10% to 90% solvent B over 30 min. Part of the effluent was directed into a LCQ ion trap mass spectrometer. The mass spectrum was deconvoluted using the Bioworks software provided by the mass spectrometer manufacturer. (FIG. 81). The product was then stored at −80° C.


PEGylation of CH2-OSK1. The CH2-OSK1 fusion protein was diluted to 2 mg/ml in 50 mM sodium acetate, 10 mM sodium cyanoborohydride, pH 4.8 with a 4-fold molar excess of 20 kD methoxy-PEG-aldehyde (Nektar Therapeutics, Huntsville, Ala.). The reaction was allowed to proceed overnight (˜18 hrs) at 4° C. Upon completion, reaction was quenched with 4 volumes of 10 mM sodium acetate, 50 mM NaCl, pH 5, then loaded at 0.7 mg protein/ml resin to an SP Sepharose HP column (GE Healthcare, Piscataway, N.J.) equilibrated in 10 mM sodium acetate, 50 mM NaCl, pH 5. The mono-PEGylated CH2-Osk fusion was eluted with a linear 50 mM-1 M NaCl gradient (FIG. 82). Peak fractions were evaluated by SDS-PAGE and the mono-PEG-CH2-OSK1 fractions pooled, concentrated and dialyzed into Dulbecco's Phosphate Buffered Saline. The final product was analyzed by SDS-PAGE (FIG. 83).


As shown in Table 43, the purified CH2-OSK1 (“L2-6H-L3-CH2-L10-OsK1(1-38)”) and PEGylated CH2-OSK1 (“20 k PEG-L2-6H-L3-CH2-L10-OSK1(1-38)”) molecules were active in blocking inflammation in the human whole blood assay (See, Example 46).


Example 55
Bioactivity of OSK1 Peptide Analogs

The activity of OSK1 peptide analogs in blocking human Kv1.3 versus human Kv1.1 current is shown in FIGS. 84 through 86 and Tables 37-41. Three electrophysiology techniques were used (See, e.g., Example 36 and Example 44). Whole cell patch clamp (FIGS. 84 & 85 and Table 41) represents a low throughput technique which is well established in the field and has been available for many years. We also used two new planar patch clamp techniques, PatchXpress and IonWorks Quattro, with improved throughput which facilitate assessment of potency and selectivity of novel OSK1 analogs described in this application. The PatchXpress technique is of moderate throughput and the novel OSK1[Ala-12] analog (SEQ ID No:1410) had similar Kv1.3 potency and selectivity over Kv1.1 to that observed by whole cell patch clamp (FIG. 84 and Table 41). IonWorks Quattro represents a 384-well planar patch clamp electrophysiology system of high throughput. Using this IonWorks system, the novel OSK1[Ala-29] analog (SEQ ID No:1424) showed potent inhibition of the Kv1.3 current and improved selectivity over Kv1.1 (FIG. 86). The OSK1[Ala-29] analog (SEQ ID No:1424) showed similar Kv1.3 activity and selectivity over Kv1.1 by whole cell patch clamp electrophysiology (FIG. 85) to that observed by IonWorks (FIG. 86). The Kv1.3 and Kv1.1 activities of Alanine, Arginine, Glutamic acid and 1-Naphthylalanine analogs of OSK1 were determined by IonWorks electrophysiology and is reported in Tables 37-40. OSK1 peptide analogs identified by IonWorks to have good potency or Kv1.3 selectivity, were tested further in whole cell patch clamp studies (see, Table 41). The Kv1.3 IC50 of the His34Ala analog of OSK1 (SEQ ID No:1428) was 797 fold lower than its IC50 against Kv1.1 (Table 41), demonstrating that this analog is a highly selective Kv1.3 inhibitor. In this same assay, native OSK1 (SEQ ID NO: 25) showed only slight Kv1.3 selectivity, with the Kv1.1 IC50 being only 5 fold higher than Kv1.3.


The novel OSK1 peptide analogs described in this application which inhibit Kv1.3 are useful in the treatment of autoimmune disease and inflammation. Kv1.3 is expressed on T cells and Kv1.3 inhibitors suppress inflammation by these cells. As one measure of inflammation mediated by T cells, we examined the impact of OSK1 analogs on IL-2 and IFN-g production in human whole blood following addition of a pro-inflammatory stimulus (Tables 36-40 and Table 42). “WB/IL-2” in these tables refers to the assay measuring IL-2 response of whole blood (see, Example 46), whereas “WB/IFNg” refers to the assay measuring IFNg response of whole blood (see, Example 46). The IC50 values listed in Tables 36-40 and 42, represent the average IC50 value determined from experiments done with two or more blood donors. The whole blood assay (see Example 46) allows for a combined measurement of the potency of the analogs in blocking inflammation and Kv1.3, as well as an assessment of the stability of the molecules in a complex biological fluid. Using this assay, several OSK1 analogs were examined and found to potently suppress inflammation (FIGS. 90C & 90D, Tables 36-40). Some of these analogs showed reduced activity in this whole blood assay, which may indicate that these residues play an important role in binding Kv1.3. Relative to the immunosuppressive agent cyclosporin A, Kv1.3 peptide inhibitors ShK-Ala22, OSK1-Ala29, and OSK1-Ala12 were several orders of magnitude more potent in blocking the cytokine response in human whole blood (FIG. 90).


The solution NMR structure of OSK1 has been solved and is provided as pdb accession number “1SCO” in Entrez's Molecular Modeling Database (MMDB) [J. Chen et al. (2003) Nucleic Acids Res. 31, 474-7]. FIG. 89 shows space filling (FIG. 89A, 89B, 89D) and worm (FIG. 89C) Cn3D rendering of the OSK1 structure. Light colored OSK1 amino acid residues Phe25, Gly26, Lys27, Met29 and Asn30 are shown in FIG. 89B. Some analogs of these residues were found to significantly reduce Kv1.3 activity (Tables 37-40), implying that these residues may make important contacts with the Kv1.3 channel. The molecular structure shown in FIG. 89A indicates these amino acids reside on a common surface of the OSK1 three-dimensional (3D) structure. FIG. 89D shows OSK1 residues (light shading) Ser11, Met29 and His34. These residues when converted to some amino acid analogs, provide improved Kv1.3 selectivity over Kv1.1 (Table 41). Although about 23 amino acids are between residues Ser11 and His34 in the contiguous polypeptide chain, the structure shown in FIG. 89D illustrates that in the 3D structure of the folded molecule these residues are relatively close to one another. Upon comparing FIGS. 89D and 89B, one can see that residues His34 and Ser11 (FIG. 89D) are on the left and right side, respectively, and adjacent to the major Kv1.3 contact surface displayed in FIG. 89B. It is envisioned that molecular modeling can be used to identify OSK1 analogs with improved Kv1.3 activity and selectivity, upon considering the Kv1.3 and Kv1.1 bioactivity information provided in Tables 37 through 42 and the solution NMR structure of OSK1 described above. FIG. 89C shows a worm rendering of the OSK1 structure with secondary structure elements (beta strands and alpha helices) depicted. The primary amino acid sequence of OSK1 is provided in FIG. 89E and amino acid residues comprising the beta strands & alpha helix are underlined. Wiggly lines in FIG. 89C indicate amino acid residues between or beyond these secondary structure elements, whereas straight lines depict the three disulfide bridges in OSK1. The first beta strand (β1) shown in FIGS. 89C and 89E contains no disulfide bridges to link it covalently to other secondary structure elements of the OSK1 molecule, unlike beta strand 3 (β3) that has two disulfide bridges with the alpha helix (α1). As shown in Table 42, OSK1 analogs without beta strand 1 (labeled “des 1-7”) still retain activity in blocking inflammation (see SEQ ID No: 4989 of Table 42) suggesting that this region of OSK1 is not essential for the molecules Kv1.3 bioactivity.


OSK1 analogs containing multiple amino acid changes were generated and their activity in the human whole blood assay of inflammation is provided in Table 42. Several analogs retain high potency in this assay despite as many as 12 amino acid changes. Based on the improved Kv1.3 selectivity of analogs with single amino acid changes, anologs with multiple amino acid changes may result in additional improvements in selectivity. It is also envisioned that analogs with multiple amino acid changes may have improved activity or stability in vivo, alone or in the context of a peptide conjugate to a half-life prolonging moiety.


Kv1.3 peptide toxins conjugated to half-life prolonging moieties are provided within this application. The bioactivity of several toxin conjugates is described in Table 43. OSK1 analogs with a N-terminal half-life prolonging 20K PEG moiety (see Example 48) were found to provide potent suppression of the whole blood IL-2 (“WB/IL-2”) and IFNg (“WB/IFNg”) response (Table 43). The 20K PEG-ShK conjugate, shown earlier to have prolonged half-life in vivo (see Examples 44 and 48), was also highly active in this whole blood assay. The FcLoop-OSK1 conjugates (see Example 49) were highly active in blocking inflammation (Table 43), and the CH2-OSK1 or PEG-CH2-OSK1 conjugates (see Example 54) provided modest blockade of the whole blood cytokine response (Table 43). The IL-2 and IFNg cytokine response measured in this whole blood assay results from T cell activation. Since this cytokine response is Kv1.3 dependent and potently blocked by the Kv1.3 peptide and peptide-conjugate inhibitors described herein, these whole blood studies illustrate the therapeutic utility of these molecules in treatment of immune disorders.









TABLE 36







Activity of OSK1 analogs in blocking thapsigargin-induced IL-2 and IFNg


production in 50% human whole blood as described in Example 46.









Thapsigargin Induced IL-2 & IFNg in



Human Whole Blood

















Analog IC50



Average
Std Dev
Average
Std Dev
Divided by



IC50 (nM)
IC50 (nM)
IC50 (nM)
IC50 (nM)
Ala-1 IC50













OSK1 Analog
IL-2
IL-2
IFNg
IFNg
IL2
IFNg
















Ala-1 (SEQ ID No: 1400)
0.1220
0.0791
0.1194
0.0802
1.00
1.00


Ala-2 (SEQ ID No: 1401)
0.0884
0.0733
0.1035
0.0776
0.72
0.87


Ala-3 (SEQ ID No: 1402)
0.0883
0.0558
0.0992
0.1007
0.72
0.83


Ala-4 (SEQ ID No: 1403)
0.1109
0.1098
0.0873
0.0993
0.91
0.73


Ala-5 (SEQ ID No: 1404)
0.0679
0.0566
0.0670
0.0446
0.56
0.56


Ala-6 (SEQ ID No: 1405)
0.0733
0.0477
0.0805
0.0696
0.60
0.67


Ala-7 (SEQ ID No: 1406)
0.0675
0.0383
0.0591
0.0260
0.55
0.49


Ala-9 (SEQ ID No: 1407)
0.0796
0.0761
0.0711
0.0627
0.65
0.60


Ala-10 (SEQ ID No: 1408)
0.0500
0.0425
0.0296
0.0084
0.41
0.25


Ala-12 (SEQ ID No: 1410)
0.1235
0.0823
0.1551
0.0666
1.01
1.30


Ala-13 (SEQ ID No: 1411)
0.1481
0.0040
0.1328
0.0153
1.21
1.11


Ala-15 (SEQ ID No: 1412)
0.1075

0.1075

0.88
0.90


Ala-16 (SEQ ID No: 1413)
0.1009

0.1009

0.83
0.84


Ala-17 (SEQ ID No: 1414)
0.1730

0.1730

1.42
1.45


Ala-19 (SEQ ID No: 1415)
0.1625

0.1625

1.33
1.36


Ala-20 (SEQ ID No: 1416)
0.3790

0.3790

3.11
3.17


Ala-22 (SEQ ID No: 1418)
7.0860

7.0860

58.07
59.33


Ala-23 (SEQ ID No: 1419)
0.2747

0.2747

2.25
2.30


Ala-25 (SEQ ID No: 1421)
3.0800

3.0800

25.24
25.79


Ala-27 (SEQ ID No: 1423)
3.4510
2.5781
1.5792
1.9217
28.28
13.22


Ala-29 (SEQ ID No: 1424)
0.4469
0.1727
0.2919
0.2422
3.66
2.44


Ala-30 (SEQ ID No: 1425)
0.9710
0.7538
0.6370
0.2674
7.96
5.33


Ala-34 (SEQ ID No: 1428)
0.0725
0.0275
0.0573
0.0341
0.59
0.48


Pro-12, Lys-16, Asp-20, Ile-
0.5138
0.4064
0.5127
0.1597
4.21
4.29


23, Ile-29, Ala-34 (SEQ ID


No: 1393)
















TABLE 37







OSK1 Alanine Analogs.









Analogue Activity (IC50, pM)












SEQ ID NO:
Analogue
Kv1.3
Kv1.1
WB/IL-2
WB/IFNg















1400
G1A
41.11
13.89
122.035
119.425


1401
V2A
81.78
9.94
88.395
103.515


1402
I3A
96.59
10.64
88.255
99.16


1403
I4A
195.30
16.92
110.865
87.255


1404
N5A
159.98
14.01
67.91
66.985


1405
V6A
173.75
12.84
73.26
80.465


1406
K7A
181.04
21.88
67.5
59.075


1407
K9A
166.27
40.59
79.58
71.065


1408
I10A
91.23
4.46
49.97
29.63


1409
S11A
40.79
113.15
90
110


1410
R12A
389.90
55.89
123.49
155.1


1411
Q13A
249.46
21.65
148.05
132.75


1412
L15A
43.07
15.04
107.5
107.5


1413
E16A
21.55
6.87
100.9
100.9


1414
P17A
33.89
9.08
173
173


1415
K19A
210.48
16.85
162.5
162.5


1416
K20A
1036.08
185.01
379
379


1417


1418
G22A
>3000
>3000
7086
7086


1419
M23A
71.39
38.63
274.7
274.7


1420
R24A
>3000
1890.78


1421
F25A
1486.97
47.30
3080
3080


1422
G26A
710.98
733.36
12075
10730


1423
K27A
232.44
>3000
1232
1579.15


1424
M29A
59.47
>3333
446.9
291.85


1425
N30A
692.54
>3000
971
637


1426
G31A
70.17
61.78


1427
K32A


41.3
34


1428
H34A
19.36
368.41
72.54
57.29


1429
T36A


728.4
723.5


1430
P37A


956
849.7


1431
K38A


221
343
















TABLE 38







OSK1 Arginine Analogs.









SEQ

Analogue Activity (IC50, pM)












ID NO:
Analogue
Kv1.3
Kv1.1
WB/IL-2
WB/IFNg















1432
G1R
68.75
9.91
554
991


1433
V2R
133.34
25.79
775
986


1434
I3R
19.90
2.47
148
180


1435
I4R
10.41
1.92
168
175


1436
N5R
13.62
2.15
95
120


1437
V6R
8.65
2.40
84
115


1438
K7R
13.17
<1.52401
78
71


1439
K9R
11.99
2.01
107
77


1440
I10R
11.68
1.73
307
474


1441
S11R
16.72
210.05
2118
4070


1442
Q13R
15.34
<1.52401
160
172


1443
L15R
13.73
2.16
93
116


1444
E16R
10.36
<1.52401
556
454


1445
P17R
10.42
<1.52401
202
355


1446
K19R
12.57
2.41
44
62


1447
K20R
9.85
<1.52401
67
83


1448
A21R
14.92
2.61
90
149


1449
G22R
23.74
3.49
292
349


1450
M23R
12.34
2.01
182
148


1451
F25R
>3333
817.42
25027
30963


1452
G26R
>3333
>3333
100000
100000


1453
K27R
1492.94
>3333
15088
10659


1454
M29R
200.39
1872.11
11680
7677


1455
N30R
18.90
45.71
405
445


1456
G31R
22.16
1.59
314
343


1457
K32R
30.83
7.24
28
34


1458
H34R
13.57
4.49
92
108


1459
T36R
1308.07
26.55
9697
10050


1460
P37R
13.32
2.01
229
253


1461
K38R
14.99
1.84
39
40
















TABLE 39







OSK1 Glutamic Acid Analogs.









SEQ

Analogue Activity (IC50, pM)












ID NO:
Analogue
Kv1.3
Kv1.1
WB/IL-2
WB/IFNg















1462
G1E
185.78
50.97
1217
1252


1463
V2E
36.23
35.01
97
184


1464
I3E
22.00
42.99
120
160


1465
I4E
15.65
3.19
218
191


1466
N5E
23.38
4.44
100
65


1467
V6E
17.73
2.43
48
68


1468
K7E
14.16
<1.52401
58
68


1469
K9E
31.76
110.67
179
171


1470
I10E
120.35
33.50
2573
2736


1471
S11E
>3333
>3333
39878
16927


1472
R12E
89.71
193.25
1787
2001


1473
Q13E
45.87
6.28
1063
799


1474
L15E
47.48
436.05
785
1059


1475
P17E
14.47
1.81
520
947


1476
K19E
23.51
13.71


1477
K20E
25.45
5.76


1478
A21E
7.37
<1.52401
117
138


1479
G22E
13.88
2.56
109
164


1480
M23E
24.28
10.44
606
666


1481
R24E


7161
9543


1482
F25E
>3333
>3333
100000
100000


1483
G26E
>3333
>3333
100000
100000


1484
K27E
>3333
548.55
5548
7144


1485
M29E
>3333
>3333
27099
24646


1486
N30E


14024
24372


1487
G31E
12.01
2.37
95
111


1488
K32E
15.56
17.31
62
63


1489
H34E
330.15
1689.82
1618
2378


1490
T36E
161.06
>3333
1742
2420


1491
P37E
62.67
622.18
239
1604


1492
K38E
25.76
34.33
526
713
















TABLE 40







OSK1 Naphthylalanine Analogs.









SEQ

Analogue Activity (IC50, pM)












ID NO:
Analogue
Kv1.3
Kv1.1
WB/IL-2
WB/IFNg















1493
G1Nal
20.66
33.93
2793
2565


1494
V2Nal
11.55
2.46
750
524


1495
I3Nal
10.31
2.34
907
739


1496
I4Nal
15.03
<1.52401
1094
1014


1497
N5Nal
21.78
<1.52401
760
431


1498
V6Nal
20.97
<1.52401
1776
2465


1499
K7Nal
23.61
<1.52401
222
246


1500
K9Nal
65.82
2.92
1070
1217


1501
I10Nal
45.44
<1.52401
184
257


1502
S11Nal
95.87
>3333
23915
17939


1503
R12Nal
37.66
24.99
460
387


1504
Q13Nal
13.44
<1.52401
140
198


1505
L15Nal
17.84
<1.52401
358
370


1506
E16Nal
9.58
<1.52401
1025
1511


1507
P17Nal
16.19
<1.52401
193
357


1508
K19Nal
17.22
<1.52401
58
99


1509
K20Nal
13.53
<1.52401
74
125


1510
A21Nal
26.10
<1.52401
315
434


1511
G22Nal
>3333
426.27
10328
10627


1512
M23Nal
35.96
64.37
581
1113


1513
R24Nal
45.26
2.85
293
818


1514
F25Nal
28.63
51.75
1733
1686


1515
G26Nal
>3333
1573.75
9898
10651


1516
K27Nal
>3333
1042.84
14971
27025


1517
M29Nal
93.46
46.88
100000
100000


1518
N30Nal
>3333
1283.88
100000
37043


1519
G31Nal
33.76
<1.52401
331
467


1520
K23Nal
26.13
1.91
134
196


1521
H34Nal
60.31
>3333
3323
6186


1522
T36Nal


100000
37811


1523
P37Nal
70.80
6.74
1762
3037


1524
K38Nal


308
409
















TABLE 41







OSK1 Analogues with Improved Selectivity at Kv1.3 over Kv1.1


(whole cell patch clamp ePhys).











SEQ ID

Kv1.3
Kv1.1
Kv1.3 Selectivity


NO:
Analogue
(IC50, pM)
(IC50, pM)
(=Kv1.1/Kv.3 IC50)














25
wild-type
39
 202
5


1441
S11R
40
 9130
228


1502
S11Nal
1490
85324
57


1410
R12A
25
 440
17


1474
L15E
190
65014
342


1423
K27A
289
 10085*
35


1424
M29A
33
 3472
105


1454
M29R
760
23028
30


1425
N30A
766
10168
14


1428
H34A
16
12754
797


1521
H34Nal
215
29178
136


1489
H34E
1322
39352
30


1490
T36E
1921
83914
44


1491
P37E
241
15699
65





*PatchXpress data













TABLE 42







OSK1 Analogues with Multiple Amino Acid Substitutions.












# Amino






Acid

WB/IL-2
WB/IFNg


SEQ ID NO:
Changes
Amino Acid Changes
(IC50, nM)
(IC50, nM)














4988
2
M29A, H34Nal
0.087
0.102


296
2
E16K, K20D
1.579
1.32


4986
2
I4K(Gly), H34A
0.470
0.451


4987
2
H34A, K38K(Gly)
1.249
2.380


4985
2
K19K(Gly), H34A
1.514
1.633


1392
3
R12A, E16K, K20D
0.041
0.092


4990
3
E16K, K20D, H34A
65.462
26.629


298
3
E16K, K20D, T36Y
0.639
0.923


4991
4
S11Nal, R12A, M29A, H34Nal
12.665
14.151


1396
5
E16K, K20D, des36-38
3.941
6.988


1395
5
GGGGS-Osk1
1.357
2.204


1274
5
E16K, K20D, T36G, P37G, K38G
2.636
3.639


4992
5
S11Nal, R12A, M29A, H34Nal, P37E
3.511
5.728


4994
5
S11Nal, R12A, M23F, M29A, H34Nal
8.136
22.727


1398
5
R12P, E16K, K20D, T37Y, K38Nle
0.527
0.736


1397
5
R12P, E16Om, K20E, T37Y, K38Nle
6.611
18.454


4995
6
S11Nal, R12A, M23Nle, M29A, H34Nal, P37E
14.32
68.158


4916
7
des1, V2G, R12A, E16K, K19R, K20D, H34A
1.499
2.244


4993
7
S11Nal, R12A, L15E, M29A, H34Nal, T36E, P37E
>100
>100


4989
12
des1-7, E16K, K20D, des36-38
8.179
8.341
















TABLE 43







Bioactivity of OSK1 and OSK1 peptide analog conjugates with half-


life-extending moieties as indicated. Fcloop structures G2-OSK1-G2


(SEQ ID NO: 976), G4-OSK1-G2 (SEQ ID NO: 979), and G2-ShK-G2


(SEQ ID NO: 977) are described in Example 49, and CH2-L10-


OSK1(1-38) SEQ ID NO: 4917 is described in Example 54.










F1 (and

WB/IL-2



F2, if

(IC50,
WB/IFNg


present)
Short-hand Designation
nM)
(IC50, nM)













PEG
20k PEG-OSK1[Ala12]
0.270
0.137


PEG
20k PEG-OSK1[Ala29]
5.756
5.577


PEG
20k PEG-OSK1[Ala29, 1Nal34]
0.049
0.081


PEG
20k PEG-OSK1[1Nal11]
0.019
0.027


PEG
20k PEG-ShK
0.046
0.065


FcLoop
FcLoop-G2-OSK-G2
0.028
0.056


FcLoop
FcLoop-G4-OSK-G2
0.150
0.195


FcLoop
FcLoop-G2-ShK-G2
0.109
0.119


PEG-
20k PEG-L2-6H-L3-CH2-L10-
8.325
50.144


CH2
OsK1 (1-38)


CH2
L2-6H-L3-CH2-L10-OsK1 (1-38)
38.491
55.162









Example 56
Design and Expression of Monovalent Fc-Fusion Molecules

There may be pharmacokinetic or other reasons, in some cases, to prefer a monovalent dimeric Fc-toxin peptide fusion (as represented schematically in FIG. 2B) to a (“bivalent”) dimer (as represented schematically in FIG. 2C). However, conventional Fc fusion constructs typically result in a mixture containing predominantly dimeric molecules, both monovalent and bivalent. Monovalent dimeric Fc-toxin peptide fusions (or “peptibodies”), including monovalent dimeric Fc-OSK1 peptide analog fusions and Fc-ShK peptide analog fusions, can be isolated from conditioned media which also contains bivalent dimeric Fc-toxin peptide, and dimeric Fc lacking the toxin peptide fusion. Separation of all three species can be accomplished using ion exchange chromatography, for example, as described in Examples 1, 2, and 41 herein.


A number of other exemplary ways that a monovalent dimeric Fc-toxin peptide fusion can be produced with greater efficiency are provided here, including for the production of monovalent dimeric Fc-OSK1 peptide analog fusions:


(1) Co-expressing equal amounts of Fc and Fc-toxin peptide in the same cells (e.g. mammalian cells). With the appropriate design, a mixture of bivalent dimeric Fc-toxin peptide fusion, monovalent dimeric Fc-toxin peptide fusion and dimeric Fc will be produced and released into the conditioned media. The monovalent dimeric Fc-toxin peptide can be purified from the mixture using conventional purification methods, for example, methods described in Examples 1, 2, and 41 herein.


(2) Engineering and recombinantly expressing in mammalian cells a single polypeptide construct represented by the following schematic:





Signal peptide-Fc-furin cleavage site-linker-furin cleavage site-Fc-toxin peptide


Furin cleavage occurs as the molecule travels through the endoplasmic reticulum and the intra-molecular Fc pairing (resulting in monovalent dimeric Fc-toxin peptide fusion) can occur preferentially to intermolecular Fc pairing (resulting in dimeric Fc-toxin peptide being expressed into conditioned medium; FIG. 87A-B).


By way of example of method (2) above, a DNA construct was produced for recombinant expression in mammalian cells of the following schematic polypeptide construct:





Signal peptide-Fc-furin cleavage site-linker-furin cleavage site-Fc-ShK(2-35)


The DNA construct had the following nucleotide coding sequence:










SEQ ID NO: 5007









atggaatggagctgggtctttctcttcttcctgtcagtaacgactggtgt






ccactccgacaaaactcacacatgcccaccgtgcccagcacctgaactcc





tggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctc





atgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagcca





cgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgc





ataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgt





gtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaagga





gtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaa





ccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctg





cccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcct





ggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatg





ggcagccggagaacaactacaagaccacgcctcccgtgctggactccgac





ggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggca





gcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaacc





actacacgcagaagagcctctccctgtctccgggtaaacgaggcaagagg





gctgtggggggcggtgggagcggcggcgggggctcaggtggcgggggaag





tggcgggggagggagtggagggggagggagtggaggcgggggatccggcg





gggggggtagcaagcgtcgcgagaagcgggataagacccatacctgcccc





ccctgtcccgcgcccgagttgctcgggggccccagcgtgtttttgtttcc





tcccaagcctaaagatacattgatgattagtagaacacccgaagtgacct





gtgtcgtcgtcgatgtctctcatgaggatcccgaagtgaaattcaattgg





tatgtcgatggggtcgaagtccacaacgctaaaaccaaacccagagaaga





acagtataattctacctatagggtcgtgtctgtgttgacagtgctccatc





aagattggctcaacgggaaagaatacaaatgtaaagtgagtaataaggct





ttgcccgctcctattgaaaagacaattagtaaggctaagggccaacctag





ggagccccaagtctatacactccctcccagtagagacgagctgaccaaga





accaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatc





gccgtggagtgggagagcaatgggcagccggagaacaactacaagaccac





gcctcccgtgctggactccgacggctccttcttcctctacagcaagctca





ccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtg





atgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtc





tccgggtaaaggaggaggaggatccggaggaggaggaagcagctgcatcg





acaccatccccaagagccgctgcaccgccttccagtgcaagcacagcatg





aagtaccgcctgagcttctgccgcaagacctgcggcacctgctaa //.






The resulting expressed polypeptide (from vector pTT5-Fc-Fc-L10-Shk(2-35)) had the following amino acid sequence before furin cleavage (the first 19 residues are a signal peptide sequence; furin cleavage sites are underlined):










SEQ ID NO: 5008









mewswvflfflsvttgvhsdkthtcppcpapellggpsvflfppkpkdtl






misrtpevtcvvvdvshedpevkfnwyvdgvevhnaktkpreeqynstyr





vvsvltvlhqdwlngkeykckvsnkalpapiektiskakgqprepqvytl





ppsrdeltknqvsltclvkgfypsdiavewesngqpennykttppvldsd





gsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgkrgkr





avggggsggggsggggsggggsggggsggggsggggskrrekrdkthtcp





pcpapellggpsvflfppkpkdtlmisrtpevtcvvvdvshedpevkfnw





yvdgvevhnaktkpreeqynstyrvvsvltvlhqdwlngkeykckvsnka





lpapiektiskakgqprepqvytlppsrdeltknqvsltclvkgfypsdi





avewesngqpennykttppvldsdgsfflyskltvdksrwqqgnvfscsv





mhealhnhytqkslslspgkggggsggggsscidtipksrctafqckhsm





kyrlsfcrktcgtc //.







FIG. 87A-B demonstrates recombinant expression of a monovalent dimeric Fc-L-ShK(2-35) molecule product expressed by and released into the conditioned media from transiently transfected mammalian cells. FIG. 88 shows results from a pharmacokinetic study on the monovalent dimeric Fc-ShK(1-35) in SD rats. Serum samples were added to microtiter plates coated with an anti-human Fc antibody to enable affinity capture. Plates were then washed, captured samples were released by SDS and run on a polyacrylamide gel. Samples were then visualized by western blot using an anti-human Fc-specific antibody and secondary-HRP conjugate. The MW of bands from serum samples is roughly identical to the original purified material, suggesting little, if any, degradation of the protein occurred in vivo over a pro-longed half-life, in spite of the presence of Arg at position 1 of the ShK(1-35) sequence.


(3) Similar to (2) above, a Fc-toxin peptide fusion monomer can be conjugated with an immunoglobulin light chain and heavy chain resulting in a monovalent chimeric immunoglobulin-Fc-toxin peptide molecule. We have termed an immunoglobulin (light chain+heavy chain)-Fc construct a “hemibody”; such “hemibodies” containing a dimeric Fc portion can provide the long half-life typical of a dimeric antibody. The schematic representation in FIG. 92A-C illustrates an embodiment of a hemibody-toxin peptide fusion protein and its recombinant expression by mammalian cells.


If the antibody chosen is a target specific antibody (e.g., an anti-Kv1.3 or anti-IKCa1 antibody), the chimeric molecule may also enhance the targeting efficiency of the toxin peptide. FIG. 91A-B demonstrates that such chimeric molecules, in this example Fc-L10-ShK(2-35) dimerized with human IgG1 or human IgG2 light and heavy chains, can be expressed and released into the conditioned media from transfected mammalian cells.


Example 57
Osk1 PEGylated at Residue 4 by Oxime Formation

[Dp(AOA)-PEG)4]Osk1 Peptide Synthesis of reduced [Dpr(AOA)4]Osk1. [Dpr(AOA)4]Osk1, having the sequence:










(SEQ ID NO: 5009)









CVI[Dpr(AOA)]NVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK








can be synthesized in a stepwise manner on a Symphony™ multi-peptide synthesizer by solid-phase peptide synthesis (SPPS) using 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)/N-methyl morpholine (NMM)/N,N-dimethyl-formamide (DMF) coupling chemistry at 0.1 mmol equivalent resin scale on Fmoc-Lys(Boc)-Wang resin (Novabiochem). N-alpha-(9-fluorenylmethyloxycarbonyl)- and side-chain protected amino acids can be purchased from Novabiochem. The following side-chain protection strategy can be employed: Asp(OtBu), Arg(Pbf), Cys(Trt), Gln(Trt), His(Trt), Lys(Nε-Boc), Ser(OtBu), Thr(OtBu) and Tyr(OtBu). Dpr(AOA), i.e., N-α-Fmoc-N-b-(N-t.-Boc-amino-oxyacetyl)-L-diaminopropionic acid, can be purchased from Novabiochem (Cat. No. 04-12-1185). The protected amino acid derivatives (20 mmol) can be dissolved in 100 ml 20% dimethyl sulfoxide (DMSO) in DMF (v/v). Protected amino acids can be activated with 200 mM HBTU, 400 mM NMM in 20% DMSO in DMF, and coupling can be carried out using two treatments with 0.5 mmol protected amino acid, 0.5 mmol HBTU, 1 mmol NMM in 20% DMF/DMSO for 25 minutes and then 40 minutes. Fmoc deprotection reactions can be carried out with two treatments using a 20% piperidine in DMF (v/v) solution for 10 minutes then 15 minutes. Following synthesis and removal of the N-terminal Fmoc group, the resin can be then drained, and washed with DCM, DMF, DCM, and then dried in vacuo. The peptide-resin can be deprotected and released from the resin by treatment with a TFA/amionooxyacetic acid/TIS/EDT/H2O (90:2.5:2.5:2.5:2.5) solution at room temperature for 1 hour. The volatiles can be then removed with a stream of nitrogen gas, the crude peptide precipitated twice with cold diethyl ether and collected by centrifugation. The [Dpr(AOA)4]Osk1 peptide can be then analyzed on a Waters 2795 analytical RP-HPLC system using a linear gradient (0-60% buffer B in 12 minutes, A: 0.1% TFA in water also containing 0.1% aminooxyacetic acid, B: 0.1% TFA in acetonitrile) on a Jupiter 4 μm Proteo™ 90 Å column.


Reversed-Phase HPLC Purification. Preparative Reversed-phase high-performance liquid chromatography can be performed on C18, 5 μm, 2.2 cm×25 cm) column. The [Dpr(AOA)4]Osk1 peptide is dissolved in 50% aqueous acetronitrile containing acetic acid and amionooxyacetic acid and loaded onto a preparative HPLC column. Chromatographic separations can be achieved using linear gradients of buffer B in A (A=0.1% aqueous TFA; B=90% aq. ACN containing 0.09% TFA), typically 5-95% over 90 minutes at 15 mL/min. Preparative HPLC fractions can be characterized by ESMS and photodiode array (PDA) HPLC, combined and lyophilized.


Osk1 peptide analog PEGylated at residue 4 by oxime formation: [Dpr(AOA)-PEG)4]Osk1 (i.e., GVI[Dpr(AOA-PEG)]NVKCKISRQCLEPCKKAGMRFGKCMNGKCHCTPK//SEQ ID NO:5010) can be made as follows. Lyophilized [Dpr(AOA)4]Osk1 peptide can be dissolved in 50% HPLC buffer A/B (5 mg/mL) and added to a two-fold molar excess of MeO-PEG-aldehyde, CH3O—[CH2CH2O]n—CH2CH2CHO (average molecular weight 20 kDa). The aminoxyacetyl group within the peptide at residue 4 reacts with the aldehyde group of the PEG to form a covalent oxime linkage. The reaction can be left for 24 hours, and can be analyzed on an Agilent™ 1100 RP-HPLC system using Zorbax™ 300SB-C8 5 μm column at 40° C. with a linear gradient (6-60% B in 16 minutes, A: 0.1% TFA in water, B: 0.1% TFA/90% ACN in water). Mono PEGylated [Dpr(AOA)-PEG)4]Osk1 peptide can be then isolated using a HiTrap™ 5 mL SP HP cation exchange column on AKTA FPLC system at 4° C. at 1 mL/min using a gradient of 0-50% B in 25 column volumes (Buffers: A=20 mM sodium acetate pH 4.0, B=1 M NaCl, 20 mM sodium acetate, pH 4.0). The fractions can be analyzed using a 4-20 tris-Gly SDS-PAGE gel and RP-HPLC. SDS-PAGE gels can be run for 1.5 hours at 125 V, 35 mA, 5 W. Pooled product, mono-PEGylated [Dpr(AOA)-PEG)4]Osk1 peptide, can be then dialyzed at 4° C. in 3 changes of 1 L of A4S buffer (10 mM NaOAc, 5% sorbitol, pH 4.0). The dialyzed product can be then concentrated in 10 K microcentrifuge filter to 2 mL volume and sterile-filtered using 0.2 μM syringe filter to give the final product.


Folding of [Dpr(AOA)-PEG)4]Osk1 (Disulphide bond formation). The mono-PEGylated [Dpr(AOA)-PEG)4]Osk1 peptide can be dissolved in 20% AcOH in water (v/v) and can be then diluted with water to a concentration of approximately 0.15 mg peptide mL, the pH adjusted to about 8.0 using NH4OH (28-30%), and gently stirred at room temperature for 36 hours. Folding process can be monitored by LC-MS analysis. Following this, folded mono-PEGylated [Dpr(AOA)-PEG)4]Osk1 can be purified using by reversed phase HPLC using a 1″ Luna 5 μm C18 100 Å Proteo™ column with a linear gradient 0-40% buffer B in 120 min (A=0.1% TFA in water, B=0.1% TFA in acetonitrile). Mono-PEGylated (oximated) [Dpr(AOA)-PEG)4]Osk1 peptide disulfide connectivity can be C1-C4, C2-C5, and C3-C6.


Abbreviations

Abbreviations used throughout this specification are as defined below, unless otherwise defined in specific circumstances.


Ac acetyl (used to refer to acetylated residues)


AcBpa acetylated p-benzoyl-L-phenylalanine


ADCC antibody-dependent cellular cytotoxicity


Aib aminoisobutyric acid


bA beta-alanine


Bpa p-benzoyl-L-phenylalanine


BrAc bromoacetyl (BrCH2C(O)


BSA Bovine serum albumin


Bzl Benzyl


Cap Caproic acid


COPD Chronic obstructive pulmonary disease


CTL Cytotoxic T lymphocytes


DCC Dicylcohexylcarbodiimide


Dde 1-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)ethyl


ESI-MS Electron spray ionization mass spectrometry


Fmoc fluorenylmethoxycarbonyl


HOBt 1-Hydroxybenzotriazole


HPLC high performance liquid chromatography


HSL homoserine lactone


IB inclusion bodies


KCa calcium-activated potassium channel (including IKCa, BKCa, SKCa)


Kv voltage-gated potassium channel


Lau Lauric acid


LPS lipopolysaccharide


LYMPH lymphocytes


MALDI-MS Matrix-assisted laser desorption ionization mass spectrometry


Me methyl


MeO methoxy


MHC major histocompatibility complex


MMP matrix metalloproteinase


1-Nap 1-napthylalanine


NEUT neutrophils


Nle norleucine


NMP N-methyl-2-pyrrolidinone


PAGE polyacrylamide gel electrophoresis


PBMC peripheral blood mononuclear cell


PBS Phosphate-buffered saline


Pbf 2,2,4,6,7-pendamethyldihydrobenzofuran-5-sulfonyl


PCR polymerase chain reaction


Pec pipecolic acid


PEG Poly(ethylene glycol)


pGlu pyroglutamic acid


Pic picolinic acid


pY phosphotyrosine


RBS ribosome binding site


RT room temperature (25° C.)


Sar sarcosine


SDS sodium dodecyl sulfate


STK serine-threonine kinases


t-Boc tert-Butoxycarbonyl


tBu tert-Butyl


THF thymic humoral factor


Trt trityl

Claims
  • 1. An OSK1 peptide analog, or a pharmaceutically acceptable salt thereof, wherein the OSK1 peptide analog comprises an amino acid sequence of the formula:
  • 2. The OSK1 peptide analog of claim 1 wherein Xaa11, Xaa27, and Xaa32 are each independently selected from Ser, Thr, Ala, Gly, Leu, Ile, Val, Met, Cit, Homocitrulline, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Guf, and 4-Amino-Phe, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Lys, His, Trp, Arg, Nα Methyl-Arg; homoarginine, 1-Nal, 2-NaI, Orn, D-Orn, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine.
  • 3. The OSK1 peptide analog of claim 1 wherein Xaa2, Xaa5, Xaa29, Xaa30, and Xaa34 are each independently selected from Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Tyr, Ser, Thr, Asn, Gln, Glu, Asp, α-aminoadipic acid, and para-carboxyl-phenylalanine.
  • 4. The OSK1 peptide analog of claim 1 wherein Xaa16, Xaa19 and Xaa20 are each independently selected from Lys, His, Arg, Trp, Arg, Na Methyl-Arg; homoarginine, 1-Nal, 2-Nal, Orn, D-Orn, Cit, Na-Methyl-Cit, Homocitrulline, His, Ala, Gly, Leu, Ile, Val, Met, Oic, Pro, Hyp, Tic, D-Tic, D-Pro, Thz, Aib, Sar, Pip, Bip, Phe, Ser, Thr, Guf, and 4-Amino-Phe.
  • 5. The OSK1 peptide analog of claim 1 wherein Xaa36 and Xaa31, if present, are each independently selected from Pro, Ala, Gly, Leu, Ile, Val, Met, Oic, Hyp, Tic, D-Tic, D-Pro, Thz, Noα-Methyl-Cit, Homocitrulline, Aib, Sar, Pip, Tyr, Thr, Ser, Phe, Trp, 1-Nal, 2-Nal, and Bip.
  • 6. The OSK1 peptide analog of claim 1, wherein Xaa38, if present, is selected from Lys, His, Orn, Trp, D-Orn, Arg, Nα Methyl-Arg; homoarginine, Cit, Nα-Methyl-Cit, Homocitrulline, His, Guf, and 4-Amino-Phe.
  • 7. The OSK1 peptide analog of claim 1, wherein the OSK1 peptide analog comprises one or more additional amino acid residues at its N-terminal or C-terminal, or both.
  • 8. An OSK1 peptide analog that comprises an amino acid sequence selected from SEQ ID NOS:1391 through 4912, 4916, 4920 through 5006, 5009, 5010, and 5012 through 5015 as set forth in Tables 7A-J, or a pharmaceutically acceptable salt of the OSK1 peptide analog.
  • 9. The OSK1 peptide analog of claim 1 or claim 8, wherein the C-terminal carboxylic acid moiety is replaced with a moiety selected from (A)-COOR, where R is independently (C1-C8)alkyl, haloalkyl, aryl or heteroaryl;(B)-C(═O)NRR, where R is independently hydrogen, (C1-C8)alkyl, haloalkyl, aryl or heteroaryl; and(C)—CH2OR where R is hydrogen, (C1-C8) alkyl; aryl or heteroaryl.
  • 10. A pharmaceutical composition, comprising the composition of claim 1 and a pharmaceutically acceptable carrier.
  • 11. A pharmaceutical composition, comprising the composition of claim 8 and a pharmaceutically acceptable carrier.
Parent Case Info

This application claims priority from U.S. Provisional Application No. 60/854,674, filed Oct. 25, 2006, and U.S. Application No. 60/995,370, filed Sep. 25, 2007, both of which are hereby incorporated by reference. This application is related to U.S. Non-provisional application Ser. No. ______, filed Oct. 25, 2007, U.S. Non-provisional application Ser. No. ______, filed Oct. 25, 2007, U.S. Non-provisional application Ser. No. ______, filed Oct. 25, 2007, U.S. Non-provisional application Ser. No. ______, filed Oct. 25, 2007, and U.S. Non-provisional application Ser. No. ______, filed Oct. 25, 2007, all which applications are hereby incorporated by reference. This application is also related to U.S. Non-provisional application Ser. No. 11/584,177, filed Oct. 19, 2006, which claims priority from U.S. Provisional Application No. 60/729,083, filed Oct. 21, 2005, both of which applications are hereby incorporated by reference. The instant application contains a “lengthy” Sequence Listing which has been submitted via CD-R in lieu of a printed paper copy in accordance with 37 C.F.R. Section 1.821, and is hereby incorporated by reference in its entirety. Three copies of said CD-R, recorded on Oct. 18, 2007 are labeled CRF, “Copy 1” and “Copy 2”, respectively, and each contains only one identical 2.60 Mb file (A-1186-US-NP5 SeqList.txt). Throughout this application various publications are referenced within parentheses or brackets. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.

Provisional Applications (2)
Number Date Country
60854674 Oct 2006 US
60995370 Sep 2007 US