Patent Application
20240051992
The present invention relates to thiophosphorylated peptide, which are capable of enzymatically induced self-assembly to form larger structures, and are capable of targeted delivery to the Golgi apparatus.
Golgi apparatus (GA), a stack of flattened membrane-enclosed disks that are dynamically regulated during cell cycles in mammalian cells, is considered as the “heart” of intracellular transportation (Kulkarni-Gosavi et al., “Form and Function of the Golgi Apparatus: Scaffolds, Cytoskeleton and Signalling,” FEBS. Lett. 593:2289-2305 (2019); Lee et al., “Bi-directional Protein Transport Between the ER and Golgi,” Annu. Rev. Cell. Dev. Biol. 20:87-123 (2004)). Increasing numbers of studies have revealed that Golgi is a hub for different signaling pathways that drive the survival and migration of cancer cells (Bivona et al., “Phospholipase Cy Activates Ras on the Golgi Apparatus by Means of RasGRP1,” Nature 424:694-698 (2003); Farber-Katz et al., “DNA Damage Triggers Golgi Dispersal Via DNA-PK and GOLPH3,” Cell 156:413-427 (2014)). Although Golgi is emerging as an important target for cancer therapy, there are, however, few approaches for targeting Golgi (Armstrong et al., “Manno-epi-cyclophellitols Enable Activity-Based Protein Profiling of Human α-Mannosidases and Discovery of New Golgi Mannosidase II Inhibitors,” J. Am. Chem. Soc. 142:13021-13029 (2020); Van Den Elsen et al., “Structure of Golgi a-mannosidase II: A Target for Inhibition of Growth and Metastasis of Cancer Cells,” Embo. J. 20:3008-3017 (2001)). While Golgi mannosidase II inhibitors are able to inhibit cancer cells, the selectivity (Dennis et al., “Growth Inhibition of Human Melanoma Tumor Xenografts in Athymic Nude Mice by Swainsonine,” Cancer Res. 50:1867-72 (1990)) or efficacy (Armstrong et al., “Manno-epi-cyclophellitols Enable Activity-Based Protein Profiling of Human α-Mannosidases and Discovery of New Golgi Mannosidase II Inhibitors,” J. Am. Chem. Soc. 142:13021-13029 (2020)) of the inhibitors remains to be improved. In addition, several studies reported the imaging of Golgi, including the commercial dyes for staining Golgi (van Echten-Deckert, et al., “1-Methylthiodihydroceramide, A Novel Analog of Dihydroceramide, Stimulates Sphinganine Degradation Resulting in Decreased De Novo Sphingolipid Biosynthesis,” J. Biol. Chem. 273:1184-91 (1998)), a smart “off—on” fluorescence probe for imaging the Golgi in cancer cells (Zhang et al., “An Off-on COX-2-specific Fluorescent Probe: Targeting the Golgi Apparatus of Cancer Cells,” J. Am. Chem. Soc. 135″11663-11669 (2013)), and carbon quantum dots localizing at Golgi (Li et al., “Chiral Nanoprobes for Targeting and Long-term Imaging of the Golgi Apparatus. Chem. Sci. 8:6829-6835 (2017)). These imaging agents, however, require 30 minutes (Zhang et al., “An Off-on COX-2-specific Fluorescent Probe: Targeting the Golgi Apparatus of Cancer Cells,” J. Am. Chem. Soc. 135″11663-11669 (2013)) or longer incubation time (Zhang et al., “An Off-on COX-2-specific Fluorescent Probe: Targeting the Golgi Apparatus of Cancer Cells,” J. Am. Chem. Soc. 135″11663-11669 (2013)) or pretreatment (van Echten-Deckert, et al., “1-Methylthiodihydroceramide, A Novel Analog of Dihydroceramide, Stimulates Sphinganine Degradation Resulting in Decreased De Novo Sphingolipid Biosynthesis,” J. Biol. Chem. 273:1184-91 (1998)) and they have yet to lead to an approach for selectively inhibiting the cancer cells. Thus, there is an unmet need of targeting Golgi, particularly to inhibit cancer cells.
The present invention is directed to overcoming these and other deficiencies in the art.
A first aspect of the invention relates to a peptide having the structure according to formula (Ia) or (Ib):
wherein,
A second aspect of the invention relates to a peptide having the structure according to formula (II):
wherein,
A third aspect of the invention relates to a pharmaceutical composition that includes a peptide according to the first or second aspect in an aqueous medium. In certain embodiments, the peptides present in the pharmaceutical composition form micelle structures and a therapeutic agent is encapsulated within the micelle structures.
A fourth aspect relates to dephosphorylated peptides according to the first aspect or deesterified peptides according to the second aspect, which may take one or more forms.
According to one embodiment, a nanofiber is formed in an aqueous medium and includes self-assembled, dephosphorylated forms of the peptide according to the first aspect or deesterified peptides according to the second aspect.
According to another embodiment, a supramolecular hydrogel is formed in an aqueous medium and includes a self-assembled, dephosphorylated form of the peptide according to the first aspect or a self-assembled, deesterified form of the peptide according to the second aspect.
A fifth aspect of the invention relates to a method of delivering a therapeutic agent into the Golgi apparatus. This method includes the steps of encapsulating a therapeutic agent within a micelle structure of a pharmaceutical composition according to the third aspect; and contacting a cell with the pharmaceutical composition, whereby micelle structures are taken up by the cell and targeted to the Golgi apparatus within the cell.
A sixth aspect of the invention relates to a method of delivering a drug moiety into the Golgi apparatus. This method includes the steps of providing a peptide according to the first or second aspect, wherein Z1 or Z2 is a drug moiety (e.g., a therapeutic agent), or a composition according to the third aspect; and contacting a cell with the peptide or the composition, whereby the peptide, or micelle structures formed by the peptide, is taken up by the cell and targeted to the Golgi apparatus within the cell.
A seventh aspect of the invention relates to a method of treating a patient having a cancerous condition. This method includes the step of administering a pharmaceutical composition according to the third aspect to a patient having a cancerous condition, wherein said administering is effective to inhibit cancer cell survival.
An eighth aspect of the invention relates to a method of treating a patient having Alzheimer's or Parkinson's disease. This method includes the step of administering a pharmaceutical composition according to the third aspect to a patient having a Alzheimer's or Parkinson's disease, wherein said administering is effective to treat symptoms of disease.
A ninth aspect of the invention relates to a method of imaging a cell. This method includes the steps of providing a peptide according to the first or second aspect, wherein Z1 or Z2 is a fluorophore, or a composition comprising the peptide; contacting a cell with the peptide or the composition, whereby the peptide, or micelle structures formed by the peptide, are taken up by the cell and targeted to the Golgi apparatus within the cell; and obtaining an image of the cell, whereby the Golgi apparatus is identified by fluorescence from the fluorophore.
The accompanying examples of enzymatic noncovalent synthesis (“ENS”) (He et al., “Enzymatic Noncovalent Synthesis,” Chem. Rev. 120:9994-10078 (2020), which is hereby incorporated by reference in its entirety) were facilitated by an oxygen atom of the phosphoester bond in a phosphopeptide (pO1) being changed to a sulfur atom to make pS1 for fast enzymatic self-assembly. These studies presented here show that pS1 undergoes rapid dephosphorylation catalyzed by alkaline phosphatase (“ALP”) to form S1 that assembles. Unexpectedly, treating HeLa cells with pS1 shows that S1 instantly accumulates at Golgi of the HeLa cells at the concentration as low as 500 nM. Such an enzymatic accumulation of Golgi (
As discussed in the accompanying examples, the invention offers a number of advantages. First, unlike previously reported agents that require at least 30 minutes incubation time or even longer time or pretreatment, peptides of the invention only takes minutes to target and image Golgi, which is over one order of magnitude more efficient than the reported agents. Second, peptides of the invention can make a connection with proteins inside the Golgi, which means it can manipulate specific proteins or even cells, and it will have much broader applications compared with previously reported agents. Third, the design of the peptides of formula (I) is quite straightforward compared with previously reported agents, which means it is more economically beneficial. This work illustrates an efficient and effective approach to image Golgi in live cells. Fourth, the thiophosphate peptides enable delivery of inhibitors into cells. Additional examples demonstrate that similar results can be achieved using the thioesterified peptide (AcS1) according to formula (II).
One aspect of the invention relates to a peptide comprising the structure according to formula (Ia) or (lb):
wherein,
Another aspect of the invention relates to a peptide having the structure according to formula (II):
wherein,
As discussed more fully below, the peptides are capable of forming micelle structures in an aqueous medium, capable of cellular uptake and targeting to the Golgi apparatus upon enzymatic dephosphorylation (i.e., hydrolytic phosphatase cleavage of thiophosphate group to form a thiol group) or deesterification (i.e., hydrolytic cleavage of the thioester group to form a thiol group). Following enzymatic cleavage, the resulting peptide is capable of self-assembling to form nanofibers and, eventually, a hydrogel in an aqueous medium.
The term “amino acid” further includes analogues, derivatives, and congeners of any specific amino acid referred to herein, as well as C-terminal or N-terminal protected amino acid derivatives (e.g., modified with an N-terminal or C-terminal protecting group). Furthermore, the term “amino acid” includes both D- and L-amino acids. Hence, an amino acid which is identified herein by its name, three letter or one letter symbol and is not identified specifically as having the D or L configuration, is understood to assume any one of the D or L configurations. For example, 2-Nal or 2-nal refer to the L and D configurations, respectively, of the analogue 3-(2-naphthyl)-alanine.
Naturally occurring amino acids are identified throughout by the conventional three-letter and/or one-letter abbreviations, corresponding to the trivial name of the amino acid, in accordance with the following list: Alanine (Ala), Arginine (Arg), Asparagine (Asn), Aspartic acid (Asp), Cysteine (Cys), Glutamic acid (Glu), Glutamine (Gln), Glycine (Gly), Histidine (His), Isoleucine (Ile), Leucine (Leu), Lysine (Lys), Methionine (Met), Phenylalanine (Phe), Proline (Pro), Serine (Ser), Threonine (Thr), Tryptophan (Trp), Tyrosine (Tyr), and Valine (Val). The abbreviations are accepted in the peptide art and are recommended by the IUPAC-IUB commission in biochemical nomenclature.
As used herein, the term “about” when used in connection with a numerical value denotes an interval of accuracy that is ±10% in certain embodiments, ±5% in other embodiments, ±2.5% in still further embodiments, and ±1% in yet another embodiment.
As used herein and in the appended claims, the singular “a”, “an” and “the” include the plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a “peptide” includes a plurality of such peptides.
In some embodiments of the invention, the peptide, following phosphatase cleavage of the thiophosphate group is capable of self-assembling to form nanofibers containing the cleavage product and a hydrogel composed of such nanofibers, when present in an aqueous medium. A hydrogel may be defined as a three-dimensional, hydrophilic or amphiphilic polymeric network capable of taking up a quantity of water, typically a large quantity of water. The networks are composed of homopolymers or copolymers, are insoluble due to the presence of covalent chemical or physical (ionic, hydrophobic interactions, entanglements) crosslinks. The crosslinks provide the network structure and physical integrity. Hydrogels exhibit a thermodynamic compatibility with water that allows them to swell in aqueous media. The chains of the network are connected in such a fashion that pores exist and that a substantial fraction of these pores are of dimensions between 1 nm and 1000 nm.
As used herein and is well-known in the art, the term “hydrogel” refers to a material that comprises fibrous networks formed of water-soluble natural or synthetic polymer chains, typically (though not exclusively) containing more than 95% water, often more than 96%, 97%, 98%, or 99% water.
The term “gelling” or “gelation” means a thickening of the medium that may result in a gelatinous consistency and even in a solid, rigid consistency that does not flow under its own weight.
A “gelator” is defined herein to include a non-polymeric organic compound whose molecules can establish, between themselves, at least one physical interaction leading to a self-assembly of the molecules in a carrier fluid to form a gel. The gel may result from the formation of a network of molecular nanofibers due to the stacking or aggregation of gelator molecules. The gelator is the product of enzymatic cleavage of the peptide.
The peptide chain can have any length that is sufficient to allow for self-assembly after enzymatic cleavage. This includes peptides up to about 70 amino acids, up to about 65 amino acids, up to about 60 amino acids, up to about 55 amino acids, up to about 50 amino acids, up to about 45 amino acids, up to about 40 amino acids, up to about 35 amino acids, up to about 30 amino acids, up to about 25 amino acids, up to about 20 amino acids, up to about 15 amino acids, or up to about 10 amino acids. In certain embodiments, the peptide chain is preferably less than about 20 amino acids in length (e.g., from 2 to 20 amino acids), such as from 3 to 5 amino acids, from 5 to 7 amino acids, from 8 to 10 amino acids, from 11 to 13 amino acids, from 14 to 17 amino acids, or from 18 to 20 amino acids.
In certain embodiments of the invention, the peptide chain contains only D-amino acids. In an alternative embodiment, the peptide chain contains only L-amino acids, or a mixture of L- and D-amino acids.
To promote self-assembly, the peptide chain preferably includes aromatic amino acids, including one or more of phenylalanine, tyrosine, and tryptophan, or any derivatives thereof.
In the peptide chain, the amino acid residue to which the Z2 moiety is covalently linked is one that has (or had, prior to such covalent linkage) a reactive sidechain. This amino acid having the reactive sidechain can be (i) one having a basic sidechain with a reactive amino group, such as Arg or Lys; or (ii) one having a nucleophilic sidechain with a reactive hydroxyl group or thiol group, such as Ser or Thr or Cys, but preferably Ser or Cys; or (iii) one having a basic sidechain with a reactive imidazole group, such as His. The amino acids having a reactive amino group will typically form a —NH—C(O)— covalent bond with the Z2 moiety as described herein. The amino acids having a reactive hydroxyl group will typically form a —O—C(O)— covalent bond with the Z2 moiety (see Ono et al., Bull. Chem. Soc. Japan 51(8):2401-2 404 (1978), which is hereby incorporated by reference in its entirety). The amino acids having a reactive thiol group will typically form a —S—C(O)— covalent bond with the Z2 moiety (see Ingenito et al., JACS 121:11369-74 (1999), which is hereby incorporated by reference in its entirety).
In one embodiment, the peptide chain includes an aromatic group linked to the amino terminus of the peptide chain. The aromatic group can be any suitable single- or multi-ring aromatic moiety that facilitates self-assembly as discussed herein. Exemplary aromatic groups include, without limitation, phenylacetyl, naphthylacetyl, fluorenylacetyl, pyrenylacetyl, and cinnamoyl.
Exemplary peptides chains include, without limitation: FF, FFK, ff, ffk, FFKY (SEQ ID NO: 1), FFFKY (SEQ ID NO: 2), FFGKY (SEQ ID NO: 3), FFGK (SEQ ID NO: 4), FFGKF (SEQ ID NO: 5), ffky, fffky, ffgky, ffgk, ffgkf, FFK(Dmt) (SEQ ID NO: 6), FFFK(Dmt) (SEQ ID NO: 7), FFGK(Dmt) (SEQ ID NO: 8), ffk(dmt), fffk(dmt), ffgk(dmt), FFCY (SEQ ID NO: 9), FFFCY (SEQ ID NO: 10), FFGCY (SEQ ID NO: 11), FFGC (SEQ ID NO: 12), FFGCF (SEQ ID NO: 13), ffcy, fffcy, ffgcy, ffgc, ffgcf, FFC(Dmt) (SEQ ID NO: 14), FFFC(Dmt) (SEQ ID NO: 15), FFGC(Dmt) (SEQ ID NO: 16), ffc(dmt), fffc(dmt), ffgc(dmt), wherein Dmt is 2,6-dimethyl-L-tyrosine and dmt is 2,6-dimethyl-D-tyrosine.
The linker, J, which is present in Formula (Ib) and Formula (II) can be —(CH2)2—, as in Formula (Ia), or it can be another —(CH2)n— moiety where n is an integer from 3 to 10, such as 3, 4, 5, 6, 7, 8, 9, or 10; a —(CH2)m—NH—(CH2)m— moiety where each m is independently an integer from 2 to 10, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, as demonstrated in the examples, solid phase synthesis can be used to react S-(2-aminoethyl) thiophosphate (or cysteamine thiophosphate) with the C-terminal carboxylic acid to form —NH(CH2)2S—P(O)(OH)2. In this example, the linker, J, is —(CH2)2—. An alternative thiophosphate source is (3-aminopropyl)aminoethyl thiophosphate (or amifostine), which when reacted with the C-terminal carboxylic acid of the peptide will form —NH(CH2)3NH(CH2)2S—P(O)(OH)2. In this latter example, the linker, J, is —(CH2)3NH(CH2)2—. As an alternative, in Formula (II), an S-(aminoalkyl) alkylthioate can be reacted with the C-terminal carboxylic acid to form the thioester (—SC(O)(CH)nCH3) group. Exemplary S-(aminoalkyl) alkylthioates that are available include, without limitation, S-(2-aminoethyl) ethanethioate, S-(3-aminopropyl) ethanethioate, and S-(4-aminobutyl) ethanethioate, which in some embodiments are commercially available as HCl or HBr salts.
Exemplary peptides, having Z1 and/or Z2 groups as defined herein, include without limitation:
According to one approach, the peptides of the present invention can be synthesized by standard peptide synthesis operations. These include both FMOC (9-fluorenylmethyloxy-carbonyl) and tBoc (tert-butyloxy-carbonyl) synthesis protocols that can be carried out on automated solid phase peptide synthesis instruments including, without limitation, the Applied Biosystems 431 A, 433 A synthesizers and Peptide Technologies Symphony or large scale Sonata or CEM Liberty automated solid phase peptide synthesizers. The use of alternative peptide synthesis instruments is also contemplated. Peptides prepared using solid phase synthesis are recovered in a substantially pure form.
Coupling of the thiophosphate moiety or thioester moiety can be carried out by reacting the peptide (e.g., resulting from solid peptide synthesis) with a thiophosphate containing moiety or thioester containing moiety under suitable reaction conditions. By way of example, the peptide can be dissolved in DMF with HBTU, followed by the addition of amifostine (or salt thereof), cysteamine S-phosphate (or salt thereof), or an S-(2-aminoalkyl) alkylthioate. The resulting peptide product can be purified using HPLC.
In certain embodiments, the peptide chain further includes, as Z1 and/or Z2, a therapeutic agent covalently bonded to the N-terminal residue of peptide chain or a sidechain of an amino acid residue within the peptide chains, as noted above. Covalent attachment may be carried out directly to the N-terminus or the sidechain of an reactive sidechain. The therapeutic agent, Z1 and/or Z2, can be any type of therapeutic agent that is capable of being so-modified. Exemplary therapeutic agents include, without limitation, antioxidants, coenzymes, vitamins, metabolites, analgesics, anti-inflammatory agents, antihelminthics, anti-arrhythmic agents, anti-bacterial agents, anti-viral agents, anti-coagulants, anti-depressants, anti-diabetics, anti-epileptics, anti-fungal agents, anti-gout agents, anti-hypertensive agents, anti-thrombogenic agents, anti-claudication agents, anti-atherosclerotic drugs, vascular agents, anti-malarials, anti-migraine agents, anti-muscarinic agents, anti-neoplastic agents, erectile dysfunction improvement agents, immunosuppressants, anti-protozoal agents, anti-thyroid agents, anxiolytic agents, sedatives, hypnotics, neuroleptics, β-blockers, cardiac inotropic agents, corticosteroids, diuretics, anti-parkinsonian agents, gastro-intestinal agents, histamine receptor antagonists, keratolyptics, lipid regulating agents, anti-anginal agents, Cox-2 inhibitors, leukotriene inhibitors, macrolides, muscle relaxants, nutritional agents, opioid analgesics, protease inhibitors, sex hormones, stimulants, anti-osteoporosis agents, anti-obesity agents, cognition enhancers, anti-urinary incontinence agents, anti-benign prostate hypertrophy agents, essential fatty acids, non-essential fatty acids, cytokines, growth factors, antibodies, radioprotective agents, and cardioprotective agents.
Any of a variety of fluorophore molecules or fluorescent nanoparticles can be used as the Z1 or Z2 moiety. Exemplary fluorophores include, without limitation, 4-nitro-2,1,3-benzoxadiazolyl, 5-(dimethylamino)naphthalene-1-sulfonyl, 4-(N,N-dimethylaminosulfonyl)-2,1,3-benzoxadiazolyl, and 9-acridinyl. The accompanying examples demonstrate use of 4-nitro-2,1,3-benzoxadiazolyl. 5-(dimethylamino)naphthalene-1-sulfonyl chloride is a reagent that reacts with primary amino groups in both aliphatic and aromatic amines to produce stable blue- or blue-green-fluorescent sulfonamide adducts. 4-(N,N-Dimethyl aminosulfonyl)-7-isothiocyanato-2,1,3-benzoxadiazole can be used to react with primary amines to afford a stable and strong fluorophore (Matsunaga et al., Anal. Chem. 67(23):4276-82 (1995), which is hereby incorporated by reference in its entirety). 9-acridinyl carboxylic acid can be used to label primary amines in peptides (Carlson et al., Org. Lett. 2(10):1465-1468 (2000), which is hereby incorporated by reference in its entirety).
Several classes of nanoparticles are known in the prior art and the skilled person can select the appropriate type of nanoparticle according to the specific therapeutic or diagnostic requirements.
Examples of nanoparticles to be coupled with the peptide are quantum dots, Noble metal clusters, superparamagnetic iron oxide nanoparticles (IONPs), block-copolymer micelles, nanocells, dendrimers, nanotubes, polymersomes, XPclad™ nanoparticles, and nanoparticles consisting of amorphous silica surrounded by a crystalline luminescent calcium phosphate layer (e.g., ORMOBEAD™).
The ORMOBEAD particles can be suitably modified on the surface with polyethylene imine or TRIAMO yielding amine groups or 6-amino hexanoic acid (AHA) or with adipic acid yielding carboxyl groups. These groups can be used for the coupling to the peptides of the invention. The ORMOBEAD technology is disclosed by Dembski et al. (News Analytik, p. 1-3 (2013), which is hereby incorporated by reference in its entirety).
In one embodiment of the invention SiO2/Zn2SiO4:Mn2+ and SiO2/Ca10 (PO4)6OH:Eu3+ core-shell nanoparticles with diameters below 100 nm are used as nanoparticles for coupling with the peptides of the invention. These particles are disclosed by Dembski et al., (Optical Materials, 1106-1110 (2011); and GIT-Labor-Fachzeitschrift, p. 48-49 (2011), each of which is hereby incorporated by reference in its entirety).
In a further embodiment luminescent dye-labeled hybrid nanoparticles can be used. These nanoparticles consist of a SiO2-based particle matrix with covalently attached organic fluorophores. They combine the optical properties of organic dye molecules and the inorganic particle matrix properties. As a result they show an increased resistance to photobleaching and a decreased dye leakage. Respective nanoparticles are disclosed by Probst (Expert Review of Molecular Diagnostics 12(1): 49-64 (2012), which is hereby incorporated by reference in its entirety).
In another embodiment, cadmium-free quantum dots can be used. These nanoparticles show bright emission in the visible and near infra-red region of the spectrum. Respective nanoparticles are developed by Nanoco Technologies Ltd. (Manchester, UK) and are disclosed in WO07/020416, WO08/100276, WO10/52455, WO10/15824, WO10/10329 and WO13/93631, each of which is hereby incorporated by reference in its entirety.
In one embodiment of the invention (group II-alloyed) group semiconductor quantum dots, group III-V quantum dots or micronized semiconductor nanocrystal complexes as developed by Evident Technologies (Troy, N.Y., USA) can be used. These nanoparticles are disclosed in WO07/118118, WO08/94292, WO06/17125 or WO05/110916, respectively, each of which is hereby incorporated by reference in its entirety.
In another embodiment superparamagnetic iron oxide nanoparticles (IONPs), block-copolymer micelles, nanocells, dendrimers, nanotubes, polymersomes and XPclad® nanoparticles can be used. Respective nanoparticles are disclosed by Singh and Lillard (Exp Mol Pathol, 86(3):215-223 (2009), which is hereby incorporated by reference in its entirety) and Xie et al. (Adv Drug Deliv Rev 62(11):1064-1079 (2010), which is hereby incorporated by reference in its entirety).
In a further embodiment of the invention non-Cd-nanoparticles can be used, comprising a core area being covered by a shell area which represents an antireflective coating of the core area. Respective nanoparticles are disclosed by US 2008/0286826 A1, which is hereby incorporated by reference in its entirety.
In another embodiment of the invention, magnetic particles can be used, which are especially suited for targeted drug delivery. In a preferred embodiment said magnetic particles consist of superparamagnetic metal oxides and/or metals and are coated with the peptides of the invention and optionally with one or more additional drugs. Respective magnetic particles are disclosed by EP 1 267 843 B1, which is hereby incorporated by reference in its entirety.
Another aspect of the invention relates to a pharmaceutical composition that includes the peptide, as described herein, in an aqueous medium.
In certain embodiments the peptides in the pharmaceutical composition form micelle structures. Additionally, the pharmaceutical composition can further contain a therapeutic agent encapsulated within the micelle structures. These therapeutic agents can be hydrophobic.
Exemplary therapeutic agents include, but are not limited to, the following: analgesics, anti-inflammatory agents, antihelminthics, anti-arrhythmic agents, anti-bacterial agents, anti-viral agents, anti-coagulants, anti-depressants, anti-diabetics, anti-epileptics, anti-fungal agents, anti-gout agents, anti-hypertensive agents, anti-thrombogenic agents, anti-claudication agents, anti-atherosclerotic drugs, vascular agents, anti-malarials, anti-migraine agents, anti-muscarinic agents, anti-neoplastic agents (e.g., antiproliferative or chemotherapeutic agents), erectile dysfunction improvement agents, immunosuppressants, anti-protozoal agents, anti-thyroid agents, anxiolytic agents, sedatives, hypnotics, neuroleptics, β-blockers, cardiac inotropic agents, corticosteroids, diuretics, anti-parkinsonian agents, gastro-intestinal agents, histamine receptor antagonists, keratolyptics, lipid regulating agents, anti-anginal agents, Cox-2 inhibitors, leukotriene inhibitors, macrolides, muscle relaxants, nutritional agents, opioid analgesics, protease inhibitors, sex hormones, stimulants, anti-osteoporosis agents, anti-obesity agents, cognition enhancers, anti-urinary incontinence agents, anti-benign prostate hypertrophy agents, essential fatty acids, non-essential fatty acids, antioxidants, and mixtures thereof.
Further non-limiting examples of the therapeutic agents in the pharmaceutical composition include: acetretin, albendazole, albuterol, aminoglutethimide, amiodarone, amlodipine, amphetamine, amphotericin B, arginine, atorvastatin, atovaquone, azithromycin, baclofen, beclomethasone, benazepril, benzonatate, betamethasone, bicalutanide, budesonide, bupropion, busulfan, butenafine, calcifediol, calcipotriene, calcitriol, camptothecin, candesartan, capsaicin, captopril, carbamezepine, carotenes, celecoxib, cerivastatin, cetirizine, chlorpheniramine, cholecalciferol, cilazepril, cilostazol, cimetidine, cinnarizine, ciprofloxacin, cisapride, clarithromycin, clemastine, clomiphene, clomipramine, clonidine, clopidogrel, codeine, coenzyme Q10, cyclobenzaprine, cyclosporin, danazol, dantrolene, dexchlorpheniramine, diclofenac, dicoumarol, digoxin, dehydroepiandrosterone, dihydroergotamine, dihydrotachysterol, dirithromycin, donezepil, doxazosin, efavirenz, eprosartan, ergocalciferol, ergotamine, essential fatty acid sources, etodolac, etoposide, famotidine, fenofibrate, fentanyl, fexofenadine, finasteride, fluconazole, flurbiprofen, fluvastatin, fosphenyloin, frovatriptan, fuirazolidone, gabapentin, gemfibrozil, glibenclamide, glipizide, glyburide, glimepiride, griseofulvin, halofantrine, ibuprofen, irbesartan, irinotecan, isosorbide dinitrate, isotretinoin, itraconazole, ivermectin, ketenserin, ketoconazole, ketorolac, lamotrigine, lansoprazole, leflunomide, lisinopril, loperamide, loratadine, losartan, lovastatin, L-thryroxine, lutein, lycopene, medroxyprogesterone, mifepristone, mefloquine, megestrol acetate, methadone, methoxsalen, methyldopa, metronidazole, miconazole, midazolam, miglitol, minoxidil, mitoxantrone, montelukast, moxonidine, nabumetone, nalbuphine, naratriptan, nelfinavir, nifedipine, nil solidipine, nilutanide, nitrofurantoin, nitroglycerin, nizatidine, omeprazole, oprevelkin, oestradiol, oxaprozin, paclitaxel, paracalcitol, paroxetine, pentazocine, pioglitazone, pizofetin, prazosin, pravastatin, prednisolone, probucol, progesterone, pseudoephedrine, pyridostigmine, rabeprazole, raloxifene, rofecoxib, repaglinide, rifabutine, rifapentine, rimexolone, ritanovir, rizatriptan, rosiglitazone, saquinavir, sertraline, sibutramine, sildenafil citrate, simvastatin, sirolimus, spironolactone, sumatriptan, tacrine, tacrolimus, tamoxifen, tamsulosin, targretin, tazarotene, telmisartan, teniposide, terbinafine, terazosin, tetrahydrocannabinol, tiagabine, ticlopidine, tirofibran, tizanidine, topiramate, topotecan, toremitfene, tramadol, tretinoin, troglitazone, trovafloxacin, ubidecarenone, urapidil, valsartan, venlafaxine, verteporfin, vigabatrin, vitamin A, vitamin D, vitamin E, vitamin K, zafirlukast, zileuton, zolmitriptan, zolpidem, zopiclone, pharmaceutically acceptable salts, isomers, and derivatives thereof, and mixtures thereof.
In certain aspects of the invention, the therapeutic agent in the pharmaceutical composition is an antiproliferative or chemotherapeutic drug. Exemplary antiproliferative or chemotherapeutic drugs include, but are not limited to, Abarelix, aldesleukin, Aldesleukin, Alemtuzumab, Alitretinoin, Allopurinol, Altretamine, Amifostine, Anastrozole, Arsenic trioxide, Asparaginase, Azacitidine, β-lapachone, BCG Live, Bevacuzimab, Avastin, Fluorouracil, Bexarotene, Bleomycin, Bortezomib, Busulfan, Calusterone, Capecitabine, Camptothecin, Carboplatin, Carmustine, Celecoxib, Cetuximab, Chlorambucil, Cisplatin, Cladribine, Clofarabine, Cyclophosphamide, Cytarabine, Dactinomycin, Darbepoetin alfa, Daunorubicin, Denileukin, Dexrazoxane, Docetaxel, Doxorubicin (neutral), Doxorubicin hydrochloride, Dromostanolone Propionate, Epirubicin, Epoetin alfa, Erlotinib, Estramustine, Etoposide Phosphate, Etoposide, Exemestane, Filgrastim, floxuridine fludarabine, Fulvestrant, Gefitinib, Gemcitabine, Gemtuzumab, Goserelin Acetate, Histrelin Acetate, Hydroxyurea, Ibritumomab, Idarubicin, Ifosfamide, Imatinib Mesylate, Interferon Alfa-2a, Interferon Alfa-2b, Irinotecan, Lenalidomide, Letrozole, Leucovorin, Leuprolide Acetate, Levamisole, Lomustine, Megestrol Acetate, Melphalan, Mercaptopurine, 6-MP, Mesna, Methotrexate, Methoxsalen, Mitomycin C, Mitotane, Mitoxantrone, Nandrolone, Nelarabine, Nofetumomab, Oprelvekin, Oxaliplatin, Paclitaxel, Palifermin, Pamidronate, Pegademase, Pegaspargase, Pegfilgrastim, Pemetrexed Disodium, Pentostatin, Pipobroman, Plicamycin, Porfimer Sodium, Procarbazine, Quinacrine, Rasburicase, Rituximab, Sargramostim, Sorafenib, Streptozocin, Sunitinib Maleate, Talc, Tamoxifen, Temozolomide, Teniposide, VM-26, Testolactone, Thioguanine, 6-TG, Thiotepa, Topotecan, Toremifene, Tositumomab, Trastuzumab, Tretinoin, ATRA, Uracil Mustard, Valrubicin, Vinblastine, Vincristine, Vinorelbine, Zoledronate, and Zoledronic acid.
In certain embodiments the therapeutic agent of the pharmaceutical composition is a protein or polypeptide. The protein or polypeptide can be a cytokine or growth factor (such as VEGF, FGF, MCP-1, PIGF, KGF, PDGF), or an antibody or binding portion thereof.
In certain embodiments, the therapeutic agent of the pharmaceutical composition is a treatment for Alzheimer's Disease or Parkinson's Disease. Exemplary therapeutic agents for the treatment of these diseases include, without limitation, a cholinesterase inhibitor, an N-methyl D-aspartate (NMDA) antagonist, L-Dopa, a dopamine agonist, an MAO B inhibitor, a COMT inhibitor, an anticholinergic, or amantadine.
In a further embodiment, the therapeutic agent of the pharmaceutical composition is an antioxidant, a coenzyme, a vitamin, a metabolite, or a mineral.
In certain embodiments, the peptide of the pharmaceutical composition is present at a concentration of from about 100 nM to about 500 μM. Furthermore, the therapeutic agent can be present in an amount from about 1 nM to about 10 μM. Single doses of the pharmaceutical composition may contain from 0.1 μg to 0.1 g of the peptide, preferably 4 μg to 0.04 g or 400 μg to 0.4 g; and any effective amount of the therapeutic agent. Typically, single doses of the therapeutic agent range from 1 μg/kg·body weight to 1000 mg/kg·body weight (although lesser or greater dosages are also contemplated).
In some embodiments, the carrier is an aqueous medium. In one embodiment, the aqueous medium is a sterile isotonic aqueous buffer, which is typically well tolerated for administration to an individual. Additional exemplary aqueous media include, without limitation, normal saline (about 0.9% NaCl), phosphate buffered saline (“PBS”), sterile water/distilled autoclaved water (“DAW”), as well as cell growth medium (e.g., MEM, with or without serum), aqueous solutions of dimethyl sulfoxide (“DMSO”), polyethylene glycol (“PEG”), and/or dextran (less than 6% per by weight).
To improve patient tolerance to administration, the pharmaceutical composition may have a pH of about 4.5 to about 8.5. In some embodiments, sodium hydroxide or hydrochloric is added to the pharmaceutical composition to adjust the pH.
In other embodiments, the pharmaceutical composition includes a weak acid or salt as a buffering agent to maintain pH. Citric acid has the ability to chelate divalent cations and can thus also prevent oxidation, thereby serving two functions as both a buffering agent and an antioxidant stabilizing agent. Citric acid is typically used in the form of a sodium salt, typically 10-500 mM. Other weak acids or their salts can also be used.
The pharmaceutical composition may also include solubilizing agents, preservatives, stabilizers, emulsifiers, and the like. A local anesthetic (e.g., lidocaine, benzocaine, etc.) may also be included in the compositions, particularly for injectable forms, to ease pain at the site of the injection.
Another aspect of the present invention relates to enzymatic (e.g., phosphatase) cleavage products of the peptides, whereby cleavage of the thiophosphate group disrupts the ability of the (resulting cleaved) peptide to form micelle structures. Instead, given the loss of the phosphate group from the C-terminus of the peptide, the resulting product is capable of self-assembly to form nanofibers and possibly larger hydrogel assemblies containing those nanofibers.
Exemplary cleavage products include, without limitation, the following peptides:
Thus, in one embodiment, the invention relates to a nanofiber formed in an aqueous medium which includes self-assembled, enzymatically modified forms of the peptide as described herein. As used herein, the term “nanofiber” is defined as a fiber of material having any shape wherein at least one dimension, e.g. the diameter, width, thickness, and the like, is about 100 nm or less. Nanofiber diameters may be about 50 nm or less, about 40 nm or less, about 30 nm or less, about 20 nm or less, about 10 nm or less, about 5 nm or less, about 4 nm or less, about 3 nm or less, about 2 nm or less, or about 1 nm or less in diameter. Although the peptides of the present invention, upon self-assembly, as described herein, form nanofibers, persons of skill in the art should appreciate that such peptides may also form microfibrils that are larger than 100 nm thick.
In another embodiment, the invention relates to a supramolecular hydrogel formed in an aqueous medium that includes a self-assembled, enzymatically modified form of the peptide.
In these embodiments, the nanofiber or supramolecular hydrogel includes the enzymatically modified form of the peptide as described above, but the nanofiber or supramolecular hydrogel may optionally include an enzymatically unmodified form of the peptide, which may be incorporated into the same.
In addition, where combinations of peptides are present in the pharmaceutical composition, then nanofibers and supramolecular hydrogels formed by the enzymatically modified forms of the peptides may also include combinations thereof. These nanofibers and supramolecular hydrogels that contain combinations of enzymatically modified forms of the peptides may or may not contain a tethered therapeutic agent (Z).
Based on the various combinations of therapeutic agents, both tethered and micelle-containing, and combinations thereof, the peptides can be used to deliver the therapeutic agents in patients for the treatment of various disease conditions.
Therefore, one aspect of the invention relates to a method of delivering a therapeutic agent into the Golgi apparatus comprising encapsulating a therapeutic agent within a micelle structure of a pharmaceutical composition as described herein, and then contacting a cell with the pharmaceutical composition, whereby micelle structures are taken up by the cell and targeted to Golgi apparatus within the cell. As discussed hereinafter, the peptides of the invention form micelles, which turn into nanofibers after dephosphorylation or de-esterification. The formation of micelles of pS1 or AcS1 likely facilitates the cellular uptake by caveolin-mediated endocytosis, similar to the cellular uptake of peptide amphiphiles. In any event, when the micelle structure is altered by way of dephosphorylation or de-esterification of the component peptides, the micelle-delivered therapeutic agent is released into or adjacent the contacted cell.
Some embodiments of the method relate to the delivering of a therapeutic agent into Golgi apparatus wherein the cell is ex vivo or in vivo. For in vivo contact to occur, a pharmaceutical composition of the invention is administered to an individual administering is carried out orally, parenterally, subcutaneously, intravenously, intradermally, intramuscularly, intraperitoneally, by implantation, by intracavitary or intravesical instillation, intraarterially, intralesionally, intradermally, peritumorally, intratumorally, or by introduction into one or more lymph nodes. Other modes of administration that are effective to present the micelle to cells that are intentionally targeted (i.e., for delivery of the therapeutic agent) can also be used.
Administration of the pharmaceutical composition can be repeated on a daily schedule (i.e., once, twice, or thrice daily), or according to a periodic schedule (i.e., once weekly, bimonthly, once monthly).
Individuals that can be treated include both veterinary patients, typically but not exclusively mammals, as well as human patients.
A further aspect of the invention relates to a method of treating a patient having a cancerous condition. This method includes administering a pharmaceutical composition as described herein to a patient having a cancerous condition, where the administering is effective to inhibit cancer cell survival. Modes and frequency of administration, and patient groups include those identified above.
The cancerous conditions to be treated in accordance with this aspect can involve cancer cells present in a solid tumor, present as a metastatic cell, or present in a heterogenous population of cells that includes both cancerous and noncancerous cells. Exemplary cancer conditions include, without limitation, cancers or neoplastic disorders of the brain and CNS (glioma, malignant glioma, glioblastoma, astrocytoma, multiforme astrocytic gliomas, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma), pituitary gland, breast (Infiltrating, Pre-invasive, inflammatory cancers, Paget's Disease, Metastatic and Recurrent Breast Cancer), blood (Hodgkin's Disease, Leukemia, Multiple Myeloma, Lymphoma), lymph node cancer, lung (Adenocarcinoma, Oat Cell, Non-small Cell, Small Cell, Squamous Cell, Mesothelioma), skin (melanoma, basal cell, squamous cell, Kaposi's Sarcoma), bone cancer (Ewing's Sarcoma, Osteosarcoma, Chondrosarcoma), head and neck (laryngeal, pharyngeal, and esophageal cancers), oral (jaw, salivary gland, throat, thyroid, tongue, and tonsil cancers), eye, gynecological (Cervical, Endrometrial, Fallopian, Ovarian, Uterine, Vaginal, and Vulvar), genitourinary (Adrenal, bladder, kidney, penile, prostate, testicular, and urinary cancers), and gastrointestinal (appendix, bile duct (extrahepatic bile duct), colon, gallbladder, gastric, intestinal, liver, pancreatic, rectal, and stomach cancers).
In this aspect of the invention, the pharmaceutical composition contains the peptide in combination with a cancer therapeutic agent of the type described above, where the cancer therapeutic agent is tethered to the peptide chain, introduced separately to the pharmaceutical composition and encapsulated within the micelle structure, both are used together to deliver different forms of the same therapeutic agent (i.e., tethered and untethered forms of the same active agent), or both are used to deliver two different therapeutic agents in combination (i.e., one therapeutic agent tethered and the other untethered but encapsulated within the micelle structure).
While any class of antineoplastic agent, anticancer drug, or chemotherapeutic drug is contemplated for use in connection with the present invention, exemplary agents within these classes include alkylating agents, platinum drugs, antimetabolites, anthracycline and nonanthracycline antitumor antibiotics, topoisomerase inhibitors, mitotic inhibitors, corticosteroids and targeted cancer therapies (such as imatinib, Gleevec®; gefitinib, Iressa®; sunitinib, Sutent®; and bortezomib, Velcade®).
Yet another aspect of the invention relates to a method of treating a patient having Alzheimer's or Parkinson's disease. This method includes administering a pharmaceutical composition as described herein to a patient having a Alzheimer's or Parkinson's disease, wherein the administering is effective to treat symptoms of such disease. Modes and frequency of administration, and patient groups include those identified above.
In this aspect of the invention, the pharmaceutical composition contains the peptide in combination with a therapeutic agent suitable for treating Alzheimer's or Parkinson's disease, including those described above, where the therapeutic agent is tethered to the peptide chain, introduced separately to the pharmaceutical composition and encapsulated within the micelle structure, both are used together to deliver different forms of the same therapeutic agent (i.e., tethered and untethered forms of the same active agent), or both are used to deliver two different therapeutic agents in combination (i.e., one therapeutic agent tethered and the other untethered but encapsulated within the micelle structure).
Yet another aspect of the invention relates to a method of imaging a cell. The method includes providing a peptide or a composition as described herein, wherein Z1 or Z2 is a fluorophore, and then contacting a cell with the peptide or the composition, whereby the peptide, or micelle structures formed by the peptide, are taken up by the cell and targeted to the Golgi apparatus within the cell. Thereafter, an image of the cell can be obtained using any desired image capture equipment or techniques compatible with the fluorophore used as Z1 and/or Z2. In the image, the Golgi apparatus is identified by fluorescence from the fluorophore.
The examples below are intended to exemplify the practice of embodiments of the disclosure but are by no means intended to limit the scope thereof.
Reagents and Instruments: 2-Cl-trityl chloride resin (1.0 mmol/g), Fmoc protected amino acid, and HBTU were obtained from GL Biochem (Shanghai, China). N,N-diisopropylethylamine (DIEA) and solvents were obtained from Fisher Scientific. Alkaline phosphatase (“ALP”) was purchased from Biomatik (Cat. No. A1130, alkaline phosphatase [ALP], >1300 U/mg, in 50% glycerol.). Cysteamine S-phosphate, cysteamine hydrochloride and O-phosphorylethanolamine were all purchased from Sigma-Aldrich. All the chemical reagents and solvents were used as received from commercial sources without further purification. Minimum Essential Media (MEM), Dulbecco's Modified Eagle Medium (DMEM), McCoy's 5A Medium, and RPMI-1640 Medium were purchased from ATCC. Fetal bovine serum (FBS) and Penicillin-Streptomycin from Gibco by Life Technologies. All precursors and compounds were purified by a reverse phase HPLC (Agilent 1100 Series) equipped with an XTerra C18 RP column, and HPLC grade acetonitrile (0.1% TFA) and HPLC grade water (0.1% TFA) were used as the eluents. The LC-MS spectra were obtained with a Waters Acquity Ultra Performance LC with Waters MICROMASS detector. Transmission electron microscope (TEM) images were obtained on Morgagni 268 transmission electron microscope. 1H-NMR spectra of compounds were obtained using Varian Unity Inova 400 MHz. Fluorescence images were taken by ZEISS LSM 880 confocal laser scanning microscope.
Cell Lines: All cell lines used (HeLa, Saos-2, SJSA-1, OVSAHO, HCC1937, HepG2, OVCAR-4, SKOV-3, MCF-7, HEK293, HS-5) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). HeLa, HepG2, MCF-7 and HEK293 cells were cultured in MEM medium with 10% FBS and 1% P/S (100 U mL−1 penicillin and 100 μg mL−1 streptomycin, Invitrogen Life Technologies). Saos-2, SKOV-3 cells were cultured in McCoy's 5A medium with 15% FBS and 1% P/S. HS-5 cells were cultured in DMEM medium with 10% FBS and 1% P/S. SJSA-1, OVSAHO, HCC1937, OVCAR-4 cells were cultured in RPMI-1640 medium with 10% FBS and 1% P/S. All the cells were cultured at 37° C. in a humidified atmosphere of 5% CO2. To determine the cytotoxicity of the compounds, cells were seeded in 96-well cell plate at 1.0×104 cells/well for 24 h followed by culture medium removal, and then fresh culture medium containing compounds were added. For fluorescence imaging, cells were seeded in confocal dish at 1.5×105 cells/dish for 24 h followed by the addition of fresh medium containing compounds, and then the fluorescence of cells were taken by ZEISS LSM 880 confocal laser scanning microscope with the 63× oil lens.
Critical micelle concentration (CMC) determination: A series of solutions of compounds (pS1, pS2), from the concentration of 0.0625 μM to 400 μM, were prepared in deionized water and adjusted the pH to 7.4. The count rates of the solutions were measured and recorded by an ALV/DLS/SLS-5000 Light Scattering System, which were converted to the intensity of scattered light and then plotted against concentrations to determine the CMC values. Then, ALP (0.1 U/mL) was added to the series of solutions and after 24 h, the same procedures were proceeded to determine CMC values of dephosphorylated pS1 and pS2 (S1 and S2).
Transmission Electron Microscopy Experiments: The 400 mesh copper grids coated with carbon film was glowing discharged and sample solutions (5 μL of each) was placed onto the grids. After 30 seconds, the sample solution was removed, and the grids were stained by uranyl acetate (2% v/v) and allowed to dry in air. TEM images were obtained with Morgagni 268 transmission electron microscope at the HV of 80 kV with filament of 2.
Determination of Dephosphorylation Rates in vitro: To determine the dephosphorylation rates of thiophosphopeptides in vitro, pS1, pO1, pS2, pO2 solutions (PBS, pH 7.4) were prepared at concentration of 120 μM and ALP was added into the solutions to make the final concentration of ALP to 0.1 U/mL at 37° C. At the designated time, the same volume of methanol was added to eliminate the activity of ALP. LC/MS was used analyse the results.
Staining cells with FlAsH-EDT 2: Briefly, after incubating HeLa cell lines (1.5×105) in 3.5 cm confocal dish for 24 h at 37° C. in a humidified atmosphere of 5% CO2, we discarded the medium and added fresh medium containing pS2 (10 μM) and incubated them together for 4 h. After 4 h, medium was removed followed by three-time wash by cell imaging buffer to eliminate FBS. Then, we added FlAsH-EDT2 solution (cell imaging buffer, 1 μM) into the confocal dish and incubated the HeLa cells at 37° C. for 1 h. Meanwhile, we prepared 1,2-Ethanedithiol (EDT) solution (cell imaging buffer, 250 μM) and after the one-hour incubation of HeLa cells with FlAsH-EDT2, FlAsH-EDT2 was discarded and EDT solution was added to the confocal dish to eliminate the unspecific binding of FlAsH-EDT2 for 10 min. Ten minutes later, EDT solution was removed and cells were washed with cell imaging buffer three times and the fluorescence images were taken by ZEISS LSM 880 confocal laser scanning microscope.
The precursor NBD-β-Alanine was synthesized according to Scheme 1 below:
Briefly, to a 10 mL water solution of (3-Alanine (5.5 mmol, 490 mg) and potassium carbonate (16.5 mmol, 2.07g), NBD-Cl (5 mmol, 1g) in 60 mL of MeOH was added to the above solution, dropwise with stirring under nitrogen gas protection. After stirring at room temperature for 6 h, methanol was removed by a rotary evaporator and the residual solution was acidified to pH 3 by HCl (1 N). The acidic aqueous solution was then extracted by diethyl ether. The combined organic solution was dried over anhydrous sodium sulfate, and then concentrated by a rotary evaporator. The resulting dark-yellow powder (NBD-β-Alanine) was directly used for solid phase peptide synthesis.
The synthesis of all peptides was performed by solid phase synthesis. Standard Fmoc chemistry solid phase peptide synthesis was carried out using 2-chlorotrityl chloride resin and the corresponding Fmoc-protected amino acids with side chains properly protected. Briefly, the 2-Cl resin (1 g) was swelling in dry DCM for 30 min and then the first amino acid was loaded onto the resin. After loading the first amino acid to the resin, the capping regent (DCM:MeOH:DIEA=17:2:1) was used to cap all the active sites of the resin for additional 15 min. Fmoc group was then removed with 20% piperidine in DMF, the next Fmoc-protected amino acid was coupled to the free amino group using HBTU as the coupling reagent. The peptide chain was cleaved from the resin by 95% TFA (95% TFA, 2.5% TIPS, 2.5% H2O) for 1 h. After the solvent was removed by a rotary evaporator, 30 mL of dry diethyl ether was added to the residual solution and followed by centrifuging at 10000 rpm for 8 min. The resulting solid products were dried by a lyophilizer. NBD-containing peptides were obtained as a solid orange powder and naphthylalanine-capped peptides were obtained as solid white powder.
Synthesis of pS1 and pS2: Briefly, 0.1 mmol of synthesized peptide (NBD-ff or Nap-ff) was dissolved in 2 mL dry DMF and 0.12 mmol of HBTU was added directly into the solution. After stirring the mixture for 30 minutes, 0.12 mmol of Cysteamine S-Phosphate sodium salt was added into the mixture and DIEA was added dropwise to adjust pH around 8, then keep stirring for 8 h. After that, the solvent is air dried and the remained oily product was dissolved in methanol and purified by RP-HPLC to obtain NBD-ff and Nap-ff bearing the C-terminal aminoethyl-thiophsphate group.
LC/MS data was obtained for pS1 and pS2 to confirm their structure. The calculated molecular weight (Mw) of pS1 is 685.17, and the observed M/Z=684.32. The calculated molecular weight (Mw) of pS2 is 619.19, and the observed M/Z=618.45.
Synthesis of SI: Briefly, 0.1 mmol of synthesized peptide (NBD-ff) was dissolved in 2 mL dry DMF and 0.12 mmol of HBTU was added directly into the solution. After stirring the mixture for 30 minutes, 0.12 mmol of cysteamine hydrochloride was added into the mixture and DIEA was added dropwise to adjust pH around 8, and stirring was continued for 8 h. After that, the solvent was air dried and the remaining oily product was dissolved in methanol and purified by RP-HPLC to obtain the titled compound.
Synthesis of pO1 and pO2: Briefly, 0.1 mmol of synthesized peptide (NBD-ff or Nap-ff) was dissolved in 2 mL dry DMF and 0.12 mmol of HBTU was added directly into the solution. After stirring the mixture for 30 minutes, 0.12 mmol of O-Phosphorylethanolamine was added into the mixture and DIEA was added dropwise to adjust pH around 8, and stirring was continued for 8 h. After that, the solvent was air dried and the remaining oily product was dissolved in methanol and purified by RP-HPLC to obtain the titled compounds. LC/MS data was obtained for pO1 and pO2 to confirm their structure. The calculated molecular weight (Mw) of pO1 is 669.19, and the observed M/Z=668.46. The calculated molecular weight (Mw) of pO2 is 603.21, and the observed M/Z=602.46.
As shown in
Based on the results presented herein, it is contemplated that other peptide cores that enable self-assembly and resist proteolysis can be used in replacement of the di-D-phenylalanine core.
The design of pS1 ensures the fast dephosphorylation of the thiophosphopeptide by ALP (
HeLa cells were incubated with CellLight® Golgi-RFP (Herrera et al., “A Bispecific Antibody Promotes Aggregation of Ricin Toxin on Cell Surfaces and Alters Dynamics of Toxin Internalization and Trafficking,” PLoS One 11:(6):e0156893 (2016), which is hereby incorporated by reference in its entirety) for 24 hours to transfect RFP at the Golgi, and then the HeLa cells were incubated with pS1 (10 μM) for 8 minutes (
The concentration of pS1 is another important factor that determines the accumulation of S1 at Golgi. Golgi fluorescence was assessed by fixing the incubation time at 4 minutes and varying the concentration pS1 at 10, 5, and 2 μM (
Unlike pS1, S1, at the concentration of 10 μM and when incubated with HeLa cells for 8 minutes, hardly results in any fluorescence in the cells, indicating slower cell uptake of S1 than that of pS1 and indicating the importance of enzymatic dephosphorylation for targeting Golgi. As shown in
To further understand the mechanism of Golgi-accumulation of S1 assemblies resulting from the rapid enzymatic dephosphorylation of pS1, the rate of fluorescence increase in Golgi was examined over a 16 minute period in HeLa cells treated pS1 and several inhibitors. The fluorescence increase in the Golgi of HeLa cells incubated with pS1 was the only reference (
The exocytosis of accumulated S1 was also examined by incubating HeLa cells pretreated with pS1 in a fresh culture medium. The intensity of fluorescence at the Golgi of the HeLa cells drops only slightly over time, confirming that the enzymatically formed assemblies of Si are largely trapped in the Golgi. Inhibiting protein disulfide isomerases (PDIs) decrease disulfide bonds of cysteine rich proteins (CRPs) (Lyles and Gilbert, “Catalysis of the Oxidative Folding of Ribonuclease A By Protein Disulfide Isomerase: Pre-steady-state Kinetics and the Utilization of the Oxidizing Equivalents of the Isomerase,” Biochemistry 30:619-625 (1991), which is hereby incorporated by reference in its entirety) that are transported to Golgi, contributing to a slight decrease of the rate of Golgi accumulation of Si. This result implies the peptide assemblies likely form disulfide bonds with CRPs. This observation agrees with the formation of dimers of S1 (or S2) in the cell lysate treated with pS1 (or pS2), suggesting that a certain extent of covalent linkage between assemblies likely contributes to the retention of the assemblies in the Golgi. Moreover, the addition of N-ethylmaleimide almost completely eliminates the accumulation of S1 at Golgi (
To examine the applicability of the process illustrated in
Using the same solid-phase synthesis described in Example 1, a nonfluorescent analogue of pS1 (pS2) was synthesized using naphthyl group to replace NBD (
The cytotoxicity of pS2 was assessed against HeLa, HEK293, and HS-5 cells, and the IC50 values were 2.8 μM, >100 μM and >100 μM, respectively (
The preceding examples illustrate that rapid dephosphorylation of thiophosphopeptides enables instantly targeting of Golgi apparatus and selectively inhibiting cancer cell survival. These observations agree with several known facts:
The fluorophore-labeled precursor peptide, NBD-ff, was synthesized according to Example 1. 0.1 mmol of synthesized peptide was dissolved in 2 mL dry DMF and 0.12 mmol of HBTU was added directly into the solution. After stirring the mixture for 30 minutes, 0.12 mmol of S-(2-aminoethyl) ethanethioate hydrobromide was added into the mixture and DIEA was added dropwise to adjust pH around 8, with stirring for an additional 8 h. After that, the solvent was air dried and the remaining oily product was dissolved in methanol and purified by RP-HPLC to obtain NBD-ff bearing the C-terminal aminoethyl-thioester group, designated AcS1. The same procedure will be carried out using the intermediate peptide, Nap-ff, to generate AcS2.
The responsiveness of AcS1 to various enzymes was next assessed in a cell free assay by treating AcS1 with alkaline phosphatase (ALP, 0.1 U/mL), enterokinase (ENTK, 1 U/mL), trypsin (1 U/mL), and carboxylesterase (CES, 1 U/mL) for 24 h. Measurement of AcS1 or its de-esterified product, 51, was performed by mass spectroscopy. The results are shown in
HeLa cells were treated with AcS1 (10 μM) in accordance with the preceding Examples, and peptide uptake was measured over a period of time using CLSM. As shown in
Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions, and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.
This application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 63/148,412, filed Feb. 11, 2021, which is hereby incorporated by reference in its entirety.
This invention was made with government support under CA142746 and CA252364 awarded by National Institutes of Health, and DMR-2011846 awarded by the National Science Foundation. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/016151 | 2/11/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63148412 | Feb 2021 | US |
