Patent Application
20190030114
The invention relates to products, and methods of identifying products, that inhibit calpain-1 or calpain-2 function, and methods for specifically inhibiting calpain-1 or calpain-2 activation or activity, or for activating calpain-1, and to methods of treating and preventing diseases that are susceptible to treatment with molecules that, interfere with calpain-1 or calpain-2 function, or activate or augment calpain-1 activity.
Generic calpain inhibitors and their use to treat diseases have been unsuccessful as therapeutics (Donkor, 2011, incorporated by reference). Herein, evidence is provided for the particular use of calpain-2 selective inhibitors, or separately calpain-1 inhibitors, or calpain-2 selective inhibitors and/or calpain-1 activators. The literature describes a few examples of inhibitors exhibiting higher selectivity for one calpain versus another (Li et al, 1996; Li et al, 1993, both incorporated by reference), but these disclosures acknowledge that the usefulness of a calpain-1 or calpain-2-selective inhibitor was unknown and required additional experimentation to determine if these compounds actually had therapeutic value.
Indeed, despite the lack of distinction between calpain-1 and calpain-2, the art has recognized a generalized need to develop selective inhibitors of calpain. Although generic calpain inhibitors (which includes more than 10 variants) have been used successfully as treatments in animal models of various diseases, none have progressed to clinical trials, in part due to lack of selectivity. Thus, there is a long-felt, but poorly understood need for more selective calpain inhibitors, and for a better understanding of the functions of calpain-1 and calpain-2. The evidence that compels such a conclusion is based on a few other distinctions among calpains that bear no insight into calpain-1 and calpain-2 distinguishability. For instance, calpain-10 gene (CAPN10) polymorphisms are associated with type 2 diabetes mellitus (T2DM); calpain-1 (μ-calpain), calpain-2 (m-calpain), calpain-3, and calpain-5 have also been linked to T2DM-associated metabolic pathways (Donkor, 2011).
Until this disclosure, it was not recognized that calpain-1 and calpain-2 are differentially linked to LTP, learning and memory, neurodegeneration, diseases of synaptic dysfunction, cell protective signaling cascades (calpain-1) and cell death cascades (calpain-2). Calpain-1 activation is linked to synaptic NMDA receptor stimulation, which accounts for its necessary role in LTP induction. It is also involved in neuroprotection elicited by prolonged synaptic NMDA receptor stimulation (see
Significant improvements in selectivity have been made in calpains versus cysteine proteinases such as cathepsins (Cuerrier et al, 2007, incorporated by reference) or other proteinases (Sorimachi et al, 2012, incorporated by reference). While the literature has described inhibitors with some degree of selectivity for calpain-2 and not calpain-1, or vice versa, none have been created with the benefit of a specific substrate until now. Calpain inhibitor IV (carboxybenzyl-Leu-Leu-Tyr-CH2-F) shows some selectivity for calpain-1 but the effect appears to vary with cell-type (Powers et al, 2002, incorporated by reference). While a complete rendering of the structural elements contributing to PTEN specificity may be difficult to capture in a small molecule, a full accounting of the specificity will provide a solid structural basis for designing a highly specific inhibitor of calpain-2.
Described herein, are compositions and methods related to isoform-selective calpain inhibitors. Isoform-selective inhibitors are directed at either calpain-1 or calpain-2, and thus, selectively reduce the activity of one isoform in comparison to the other. Selective inhibitors of calpain-1 or calpain-2 may inhibit catalytic activity, reduce expression, selectively degrade, inhibit or hasten chemical modification, or affect protein interactions between calpain-1 or calpain-2 and one or more of its interacting proteins. Selective inhibitors of calpain isoforms may also be conjugated to agents affecting the targeting, stability, mobility, penetrance, bioavailability, or concentration of an inhibitor.
Selective calpain inhibitors may exist in a multitude of different forms, including nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Various selective inhibitors of calpain-2 according to the invention may be derived from the calpain-2 polypeptide substrate, PTEN (SEQ ID NO: 1).
Also described herein are findings that calpain-1 and calpain-2 are differentially linked—by both differential substrate specificity and differential subcellular scaffolding—to discrete cellular pathways. In particular, applicants provide evidence that administration of calpain-2 inhibitors described herein is useful for inhibiting cell death, and enhancing cognition. Indeed, calpain-1 and calpain-2 are differentially linked to the induction of Long-term Potentiation (LTP), the physiological substrate of learning and memory, in that calpain-1 is directly linked to the induction of LTP. Therefore, in aspects of the invention related to the treatment of neurological disorders, calpain-1 activation functions positively in the induction of LTP. Whereas calpain-2 activation during the same process acts like a brake in the consolidation of LTP, and thus creates a threshold for LTP, and limits the extent of LTP during the consolidation period. The particular and differential functions of calpain-1 and calpain-2 in cell protection and cell death are also disclosed herein.
In various aspects, the invention also provides methods of identifying inhibitors selective for calpain-2. These inhibitors are useful to I) inhibit cellular activity related to cell death and pathology, II) lower the threshold for sustaining LTP, III) increase LTP, IV) enhance neuronal synaptic plasticity, learning, memory, and cognition, and/or V) treat certain neurodegenerative diseases. In the methods of the invention small molecule inhibitors, proteins, peptides, polypeptides, modified peptides and polypeptides, and nucleic acids that selectively inhibit calpain-2 may be administered alone, or in combination with other inhibitors of calpain-2 function, or activators of calpain-1 function, as applicants have discovered that calpain-1 is involved specifically in neuroprotection and in the induction of synaptic plasticity, as compared to calpain-2. In another aspect, the invention relates to products and compositions such as small molecules inhibitors, polypeptides, peptides, modified peptides, and nucleic acids that selectively inhibit calpain-2 alone or in combination with other molecules that selectively inhibit calpain-2 and have diminished effect, little effect, or no measurable effect on calpain-1: In yet another aspect, the invention relates to products and compositions of matter such as small molecules inhibitors, polypeptides, peptides, modified polypeptides, and nucleic acids that selectively activate or inhibit calpain-1 function alone or in combination with other activators or selective inhibitors. Overall, the invention provides for methods of treating diseases that are susceptible to being inhibited, ameliorated, retarded, reversed, or prevented by calpain-2-selective inhibitors, or calpain-1 selective activators, or combination thereof.
Columns represent mean±S.E.M. n=4-6. *p<0.05; **p<0.01; ***p<0.001 versus NMDA plus vehicle-injected group. One-way ANOVA followed by Bonferroni test.
As stated above, compositions and methods related to isoform-selective calpain inhibitors are described herein. In various embodiments of the invention, isoform-selective inhibitors are directed at either calpain-1, (which is also referred to by its alternative moneclature, μ-calpain) or calpain-2, (which is also referred to by its alternative moneclature, m-calpain). In particular, calpain-1 and calpain-2 refer to mammalian forms that include the calpain-1 examples of SEQ ID NOs: (2-6) and the calpain-2 examples of SEQ ID NO: (69-73) or catalytic fragment thereof. Calpains are known in the art as calcium activated neutral proteases and include a family of related molecules (Baudry et al, 2013). A catalytic fragment includes equal to or more than the first 300 amino acids of calpain-1 or calpain-2.
Generally, the term ‘inhibitor’ relates to a small molecule, peptide, polypeptide, protein, nucleic acid or modifications thereof. For example, a selective inhibitor of calpain-2, reduces its activity and has diminished, less inhibitory effect on calpain 1. On the other hand, a selective inhibitor of calpain-1 reduces its activity and has diminished inhibitory effect on calpain-2. Inhibitors of calpain-1 or calpain-2 include molecules that selectively inhibit catalytic activity competitively, non-competitively, selectively reduce their expression, selectively hasten their degradation, inhibit or hasten their chemical modification, or selectively affect protein interactions between calpain-1 or calpain-2 and one or more of its interacting proteins. Inhibitors may also comprise conjugates, compositions, or formulations that affect the targeting, stability, mobility, penetrance, bioavailability, or concentration of an inhibitor reaching the effective functional space of calpain-1 or-2.
In particular, a ‘calpain-2 selective inhibitor’ or a ‘selective calpain-2 inhibitor’ is a small molecule, peptide, polypeptide, protein, or modification thereof with a calpain-2 inhibition constant (Ki) equal to or more than 10-fold lower than its Ki for calpain-1. For instance, a Ki for calpain-2 of 0.1 μM or lower and a Ki for calpain-1 of 1 μM or higher would meet the definition of ‘calpain-2-selective inhibitor’. A ‘highly selective calpain-2 inhibitor’ would preferably exhibit at least a 20-fold lower Ki for calpain-2 than calpain-1. A calpain-1 selective inhibitor is a small molecule, peptide, polypeptide, protein, or modification thereof with a calpain-1 inhibition constant (Ki) equal to or more than 7-fold lower than its Ki for calpain-2. A highly selective calpain-1 inhibitor is a small molecule, peptide, polypeptide, protein, or modification thereof with a calpain-1 inhibition constant (Ki) equal to or more than 20-fold lower than the Ki for calpain-2. At least four highly selective calpain-2 inhibitors are disclosed in the art. Li, et al, 1996, disclose molecules 18, 19, and 56, and z-Leu-Phe-CONH-nPr is disclosed as the most highly-selective calpain-2 inhibitor with Ki for calpain-1 of 15 μM and Ki for calpain-2 as 0.05 μM in U.S. Pat. No. 6,235,929, column 14, Table I. However, the literature also describes the Ki of z-Leu-Phe-CONH-nPr for calpain-2 as being 0.35 μM and the Ki for calpain-1 as 0.05 μM (U.S. Pat. No. 6,235,929, and Li et al, 1996). Thus, the structural information disclosed in the literature for highly selective calpain-2 inhibitors is somewhat unpredictable, with one of four known highly selective calpain-2 inhibitors being of indefinite selectivity.
In various embodiments of the invention, a calpain inhibitor is a “small molecule.” A “small molecule” refers to a composition that has a molecular weight of less than 5 kD and more preferably less than about 4 kD, and most preferably less than 1 kD. Small molecules can be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic molecules.
The terms “Polypeptide”, “Peptide”, and “Protein”, as used herein, are interchangeable and are defined to mean a biomolecule composed of amino acids linked by peptide bonds. This includes linked or individual compounds with an amine or amide bond on one end, an alpha carbon variably having two hydrogen atoms or a hydrogen atom and an R group or two R groups, a carboxylic acid group linked to the alpha-carbon on the other end, or an additional amide bond linking said amino acid with another amino acid. Polypeptides of the invention accommodate R groups of naturally occurring amino acids, including, glycine, alanine, cysteine, methionine, serine, threonine, leucine, isoleucine, valine, glutamate, aspartate, histamine, arginine, lysine, phenylalanine, tyrosine, proline, tryptophan, glutamine, and asparagine.
In various protein embodiments of the invention, a selective calpain inhibitor is an antibody that inhibits calpain-2. For example, the invention accomodates antibodies that inhibit substrate binding to calpain-2, which block access of calpain-2 to substrates, or which inhibit phosphorylation of calpain-2 at serine 50 (Shiraha et al, 2002; Zadran et al, 2010, incorporated by reference) by steric masking or allosteric modulation of calpain-2, or that bind calpain-2 such that it is inhibited. Antibodies can be polyclonal, monoclonal, single chain, anti-idiotypic, chimeric, or humanized versions of such antibodies or fragments thereof. Antibodies may be from any species in which an immune response can be raised.
Antibodies, peptides, polypeptides, and modified peptides that selectively block phosphorylation of Serine 50 of m-calpain are also contemplated, such as those described in USPPN 2003/0148264, which is incorporated by reference. These can be made using techniques defined in the art, such as phage display, a technique to generate highly variant peptide libraries as fusion proteins on the protein coat displayed on the surface of bacteriophage particles (Clackson et al, 1991; Cwirla et al, 1990, both incorporated by reference). Fusion proteins identified from a phage display library can be screened against a peptide target, such as unphosphorylated serine 50 of calpain-2. Such polypeptides can be used to selectively block phosphorylation of serine 50 by binding to, and sterically masking the phosphorylation site and thereby treat the diseases of LTP impairment disclosed herein.
‘Modifications’ refer to changes to peptides and polypeptides that are not present in peptides or polypeptides with the linked or unlinked features of any or all of naturally occuring amino acids. Modifications to the amino terminal, or carboxyl terminal, or R groups are made to increase or decrease the affinity of a peptide or polypeptide for calpain-1 or calpain-2, or increase or decrease the half-life, or increase the bioavailability, or to increase their concentration in the calpain-1 or calpain-2 target space.
Variants include polypeptides that differ in amino acid sequence due to mutagenesis. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, retaining activity. In some embodiments, the variants have improved activity relative to the native protein. In further respect to the notion of ‘variants’, it is recognized that DNA sequences of a protein may be altered by various methods, and that these alterations may result in DNA sequences encoding proteins with amino acid sequences different than that encoded by a protein of the present invention.
Indeed, in various embodiments of the invention, polypeptide inhibitors may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions of one or more amino acids. For example, in certain embodiments, the polypeptides of SEQ ID NOs: 1-6 and 69-73, may include up to 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, and about 35 or more amino acid substitutions, deletions, truncations, orminsertions. In yet another aspect, a sequence described herein contains 1, about 2, about 3, about 4, about 5, about 10, or about 20 additions or truncations to the C and/or N terminus region on a sequence described herein. In a non-limiting aspect, the disclosure provides for a sequence containing 1, about 2, about 3, about 4, about 5, about 10, or about 20 additions or deletions to the C and/or N terminus region of one or more of SEQ ID NOs: 1-6 and 69-73. The skilled artisan will further appreciate that changes can be introduced by mutation of the nucleotide sequences of the invention thereby leading to changes in the amino acid sequence of the encoded proteins, without altering the biological activity of the proteins. Thus, variant isolated nucleic acid molecules can be created by introducing one or more nucleotide substitutions, additions, or deletions into the corresponding nucleotide sequence disclosed herein, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleotide sequences are also encompassed by the present invention.
Conservative amino acid substitutions may be made at one or more, predicted, nonessential amino acid residues of a polypeptide calpain inhibitor according to the invention. A ‘nonessential’ amino acid residue is a residue that can be altered from the wild-type sequence of a protein without altering the biological activity, whereas an ‘essential’ amino acid residue is required for biological activity. A ‘conservative amino acid substitution’ is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
In an aspect, amino acid substitutions may be made in nonconserved regions that retain function. In general, such substitutions would not be made for conserved amino acid residues, or for amino acid residues residing within a conserved motif, where such residues are essential for protein activity. Examples of residues that are conserved and that may be essential for protein activity include, for example, residues that are identical between all proteins contained in an alignment of similar or related toxins to the sequences of the invention. Examples of residues that are conserved but that may allow conservative amino acid substitutions and still retain activity include, for example, residues that have only conservative substitutions between all proteins contained in an alignment of similar or related toxins to the sequences of the invention (e.g., residues that have only conservative substitutions between all proteins contained in the alignment homologous proteins). However, one of skill in the art would understand that functional variants may have minor conserved or nonconserved alterations in the conserved residues.
In an aspect, the disclosure provides for a protein or polypeptide having an amino acid sequence that is at least about 60%, 65%, about 70%, 75%, about 80%, 85%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the aminomacid sequence of any of the sequences described herein. In another aspect, the disclosure provides for a protein or polypeptide having an amino acid sequence that is at least about 60%, 65%, about 70%, 75%, about 80%, 85%, about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of any of the sequences described SEQ ID NOs: 1-194 and 201-22.
In another aspect, variants can include polypeptides encoded by a nucleic acid molecule that hybridizes to the nucleic acid molecules described herein, or complement thereof, under stringent conditions. Variants include polypeptides that differ in amino acid sequence due to mutagenesis. Variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein, that is, retaining activity. In some embodiments, the variants have improved activity relative to the native protein.
It is recognized that DNA sequences of a protein may be altered by various methods, and that these alterations may result in DNA sequences encoding proteins with amino acid sequences different than that encoded by a protein of the present invention. This protein may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions of one or more amino acids of SEQ ID NO: SEQ ID NO: 1-6 and 69-73, including up to 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 15, about 20, about 25, about 30, and about 35 or more amino acid substitutions, deletions, truncations, or insertions. In yet another aspect, a sequence described herein contains 1, about 2, about 3, about 4, about 5, about 10, or about 20 additions or truncations to the C and/or N terminus region on a sequence described herein. In a non-limiting aspect, the disclosure provides for a sequence containing 1, about 2, about 3, about 4, about 5, about 10, or about 20 additions or deletions to the C and/or N terminus region of one or more of SEQ ID NOs: 1-6 and 69-73.
One aspect of the invention pertains to isolated or recombinant nucleic acid molecules comprising nucleotide sequences encoding proteins or polypeptides or biologically active portions thereof, as well as nucleic acid molecules sufficient for use as hybridization probes to identify nucleic acid molecules encoding proteins with regions of sequence homology. As used herein, the term “nucleic acid molecule” is intended to include DNA molecules (e.g., recombinant DNA, cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. An “isolated” or “recombinant” nucleic acid sequence (or DNA) is used herein to refer to a nucleic acid sequence (or DNA) that is no longer in its natural environment, for example in an in vitro or in a recombinant bacterial or plant host cell. In some embodiments, an isolated or recombinant nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For purposes of the invention, ‘isolated’ when used to refer to nucleic acid molecules excludes isolated chromosomes.
The calpain-2 substrate, Stargazin
Stargazin gamma-2, a protein involved in α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking, is another calpain-2 specific substrate. In particular, the cytoplasmic C-terminus, shown in
The calpain-2 substrate, PTEN
The human tumor suppressor protein PTEN (SEQ ID NO: 1) is disclosed herein as having a calpain-2 specific cleavage site. Hence, PTEN is a calpain-2 specific substrate. While the literature discloses some examples of calpain-1 or calpain-2 specific substrates (i.e., Ki greater than 10 fold different), it has generally been taught that there are no differences in calpain-1 and calpain-2 substrates, in spite of the fact that calpains require, or ‘read’, a large area of their substrates and do not cleave small peptides lacking structural information (Goll et al, 2003, incorporated by reference). PTEN is the first calpain substrate with sites of significant differential sensitivity to calpain-2 versus calpain-1. Potential PTEN calpain-2 cleavage sites are underlined and in bold in
Until this disclosure, target protein/substrate specificity between calpain-1 versus calpain-2 was generally unrecognized. Crystal structures of calpain-1 (Pal et al, 2003, incorporated by, reference) and calpain-2 (Horfield et al, 1999, incorporated by reference) have provided for design of calpain inhibitors, but disclosure of calpain-1 or calpain-2 selective inhibitors using said crystal structures cocrystallized with inhibitors has not taught selectivity (Cuerrier et al, 2006, incorporated by reference). Polypeptide cleavage sites sensitive to calpain-2 but insensitive to calpain-1 both in vivo and in vitro are disclosed herein. In other embodiments PTEN, or polypeptide fragments of PTEN, or modified polypeptide fragments of PTEN are calpain-2 selective inhibitors. Polypeptide fragments of PTEN, as recited in SEQ ID NO: 1, or SEQ ID NOs: 146-194, alternatively are embodiments of the invention.
Preferred embodiments of the polypeptide invention comprise polypeptides with at least about 80%, about 90%, about 95%, about 98%, about 99%, or 100% sequence identity to the peptides of SEQ ID NO: SEQ ID NO: 1, 146-194. The peptides can comprise additional modifications or domains such as those that increase targeting across the BBB, or increase the half-life in vivo or in vitro, or increase the bioavailability, or combination of modifications or other polypeptide domains thereof; for example, a transferrin polypeptide fragment, an insulin fragment, an LDL binding protein fragment, a rabies virus glycoprotein fragment. It is expected that peptides or polypeptide fragments, or modified polypeptide fragments of the calpain-2 selective cleavage sites of PTEN will inhibit calpain-2, as measured by Ki, more than calpain-1, as measured by Ki.
In various other embodiments, a polypeptide calpain inhibitor contains at least 350 consecutive amino acids of calpain-1 or calpain-2 wherein the fragment is less than the full-length native forms and has proteolytic activity. In various other embodiments, a polypeptide calpain inhibitor contains at least four consecutive amino acids of calpain-1 or calpain-2.
In various embodiments of polypeptide inhibitors of the invention, the polypeptides are substituted with groups R1 and R2, in which R1 and R2 are linked by a covalent bond. In various other embodiments, R1 is a polypeptide fragment or modified polypeptide fragment that is a selective inhibitor of calpain-1 or calpain-2, or highly selective inhibitor of calpain-1 or calpain-2, or a molecule that improves absorption, bioavailability, half-life, or targeting such as (a transferrin polypeptide fragment, an insulin fragment, an LDL binding protein fragment, a rabies virus glycoprotein fragment); and R2 is a polypeptide or modified polypeptide that is a selective inhibitor of calpain-1 or calpain-2, or highly selective inhibitor of calpain-1 or calpain-2, or a polypeptide fragment or modified polypeptide fragment that improves absorption, bioavailability, half-life, or targeting, such as (a transferrin polypeptide fragment, an insulin fragment, an LDL binding protein fragment, a rabies virus glycoprotein fragment).
In various embodiments, a selective inhibitor of calpain-2 according to the invention is a molecule based on the following formula:
wherein: M1 is —O, —N, —S, or —C substituted to covalently link a blocking group selected from Y1—PhCH2—, Y1—Ph(CH2)2—, PhCH2—Y1, or Ph(CH2)2—Y1—, wherein Y1 is a polypeptide, or modified polypeptide covalently linked to a molecule that improves absorption, bioavailability, half-life, or targeting such as (a transferrin polypeptide fragment, an insulin fragment, an LDL binding protein fragment, a rabies virus glycoprotein fragment); or wherein Y1 is —H, a substitution for linking small molecule, polypeptide, or modified polypeptide moieties for improving half-life, bioavailability or targeting, such as (a transferrin polypeptide fragment, an insulin fragment, an LDL binding protein fragment, a rabies virus glycoprotein fragment); or Y1 is an —O, —N, —S, or —C substitution to link polypeptides that improve membrane permeability or blood brain barrier passage selected from (SEQ ID NOs: 195-200), a molecule that improves absorption, bioavailability, half-life, or targeting such as (a transferrin polypeptide fragment, an insulin fragment, an LDL binding protein fragment, a rabies virus glycoprotein fragment), or M1 is —O, —N, —S, or —C substituted to covalently link a small molecule, polypeptide, or modified polypeptide that improves membrane permeability or blood brain barrier passage, a polypeptide selected from SEQ ID NOs: (195-200), a molecule that improves absorption, bioavailability, half-life, or targeting such as (a transferrin polypeptide fragment, an insulin fragment, an LDL binding protein fragment, a rabies virus glycoprotein fragment); R1 is a functional group covalently bonded to the alpha-carbon having an L orientation, and having an amino acid side chain of leucine, phenylalanine, tyrosine, valine, isoleucine, methionine, alanine, or a modified amino acid side chain; and R2 is —CH3, —CH2CH3, —(CH2)2CH3, —CH(CH3)2, —CH2CH(CH3)2, —CH(CH3)CH2CH3, —C6H4(4-OH), C6H4(3-OH), C6H4(2-OH), C6H4(2-CH3), C6H4(3-CH3), C6H4(4-CH3), C6H4(2-OCH3), C6H4(3-OCH3), C6H4(4-OCH3), C6H4(2-NH2), C6H4(3-NH2), C6H4(4-NH2), C6H4(2-NHCH3), C6H4(3-NHCH3), C6H4(4-NHCH3), C6H4(2-N(CH3)2), C6H4(3-N(CH3)2), or C6H4(4-N(CH3)2); R3 is —H, —OCH3, ═NH, —NH2, —SH, ═O, ═S, —OCH2CH3, —O(CH2)2CH3, —OCH(CH3)2, —SCH3, —SCH2CH3, —S(CH2)2CH3, —SCH(CH3)2, —OH, —CH3, —F, —Cl, —Br, —I; X1 is —C6H3(3,5-R4,R5), —CHR6—C6H3-(3,5-R4,R5), -2-pyridyl, -2-pyridyl(3,5, R4,R5), —CHR6-2- pyridyl(3,5, R4,R5), -3-pyridyl(3,5, R4, R5), —CHR6-3-pyridyl(3,5,R4,R5), -4-pyridyl(3,5, R4, R5), or —CHR6-4-pyridyl(3,5,R4,R5); wherein R4 is —H, —OCH3, —OCH2CH3, —O(CH2)2CH3, —OCH(CH3)2, —SCH3, SCH2CH3, S(CH2)2CH3, —SCH(CH3)2, —OH, —CH3, —CH2CH3, —CN, —CHNH, —NH2, —NHCH3, —N(CH3)2, —F, —Cl, —Br, or —I; R5 is —H, —OCH3, —OCH2CH3, —O(CH2)2CH3, —OCH(CH3)2, —SCH3, SCH2CH3, —S(CH2)2CH3, —SCH(CH3)2, —OH, —CH3, —CH2CH3, —CN, —CHNH, —NH2, —NHCH3, —N(CH3)2, —F, —Cl, —Br, or —I; and R6 is —H, —OCH3, —OCH2CH3, —O(CH2)2CH3, —OCH(CH3)2, —SCH3, —SCH2CH3, —S(CH2)2CH3, —SCH(CH3)2, —OH, —CH3, —CH2CH3, —CN, —CHNH, —NH2, —NHCH3, —N(CH3)2, —F, —Cl, —Br, or —I.
In various other embodiments, a selective of calpain-2 according to the invention is a molecule based on the following formula:
wherein: R1 is X1—PhCH2—, or X1—Ph(CH2)2—; wherein X1 is —H, or a substitution for linking a molecule that improves absorption, bioavailability, half-life, or targeting such as (a transferrin polypeptide fragment, an insulin fragment, an LDL binding protein fragment, a rabies virus glycoprotein fragment); R2 is a functional group covalently bonded to the alpha-carbon, having an L orientation, and having an amino acid side chain of leucine, phenylalanine, tyrosine, valine, isoleucine, methionine, alanine, or a modified amino acid side chain; R3 is —CH3, —CH2CH3, —(CH2)2CH3, —CH(CH3)2—CH2CH(CH3)2, —CH(CH3)CH2CH3, —C6H5, —C6H4(4-OH), C6H4(3-OH), C6H4(2-OH), C6H4(2-CH3), C6H4(3-CH3), C6H4(4-CH3), C6H4(2-OCH3), C6H4(3-OCH3), C6H4(4-OCH3), C6H4(2-NH2), C6H4(3-NH2), C6H4(4-NH2), C6H4(2-NHCH3), C6H4(3-NHCH3), C6H4(4-NHCH3), C6H4(2-N(CH3)2), C6H4(3-N(CH3)2), or C6H4(4-N(CH3)2); R4 is —H, or —OCH3, ═NH, —NH2, —SH, ═O, ═S, —OCH2CH3, —O(CH2)2CH3, —OCH(CH3)2, —SCH3, SCH2CH3, —S(CH2)2CH3, —SCH(CH3)2, —OH, —CH3, —CH2CH3, —F, —Cl, —Br, or —I; R5 is —H, —OCH3, —OCH2CH3, —O(CH2)2CH3, —OCH(CH3)2, —SCH3, SCH2CH3, —S(CH2)2CH3, —SCH(CH3)2, —OH, —CH3, —CH2CH3, —CN, —CHNH, —NH2, —NHCH3, —N(CH3)2, —F, —Cl, —Br, —I; and R6 is —H, —OCH3, —OCH2CH3, —O(CH2)2CH3, —OCH(CH3)2, —SCH3, SCH2CH3, —S(CH2)2CH3, —SCH(CH3)2, —OH, —CH3, —CH2CH3, —CN, —CHNH, —NH2, —NHCH3, —N(CH3)2, —F, —Cl, —Br, or —I.
In certain embodiments, a selective inhibitor of calpain-2 according to the invention is a molecule based on the following structure of Formula I:
wherein the chiral center 1 indicated by the circle is Levorotary (L), and wherein the chiral center 2 is D- or L-, or a racemic mixture. Two embodiments of the molecule of Formula 1 are purified forms having an L- at chiral center 1 and an L-form at chiral center 2, or separately an L-form at chiral center 1 and a D-form at chiral center 2.
In certain embodiments, a selective inhibitor of calpain-2 according to the invention is a molecule based on the following structure of Formula II:
wherein the chiral center 1 indicated by the circle is Levorotary (L), and wherein the chiral center 2 is D- or L-, or a racemic mixture, and wherein chiral center 3 is D- or L-, or a racemic mixture. Four preferred embodiments of the molecule of are purified forms having an L- at chiral center 1, an L-form at chiral center 2, and an L-form at chiral center 3; or separately an L-form at chiral center 1 and a D-form at chiral center 2, and an L-form at chiral center 3; or separately an L-form at chiral center 1 and a D-form at chiral center 2, and an D-form at chiral center 3; or separately an L-form at chiral center 1 and a L-form at chiral center 2, and an D-form at chiral center 3.
As stated above, the invention accomodates compounds having the ability to selectively inhibit calpain-2 with a Ki of at least 10-fold lower for calpain-2 than for calpain-1. For example, in various embodiments, a compound capable of inhibiting calpain-2 with a Ki of at least 10-fold lower for calpain-2 than for calpain-1 has a structure having the following structure of Formula III:
wherein the chiral center 1 indicated by the circle is Levorotary (L), and wherein the chiral center 2 is D- or L-, or a racemic mixture, and wherein chiral center 3 is D- or L-, or a racemic mixture. Four preferred embodiments of the molecule above are purified forms having an L- at chiral center 1, an L-form at chiral center 2, and an L-form at chiral center 3; or separately an L-form at chiral center 1 and a D-form at chiral center 2, and an L-form at chiral center 3; or separately an L-form at chiral center 1 and a D-form at chiral center 2, and an D-form at chiral center 3; or separately an L-form at chiral center 1 and a L-form at chiral center 2, and an D-form at chiral center 3.
In another embodiment of a compound capable of inhibiting calpain-2 with a Ki of at least 10-fold lower for calpain-2 than for calpain-1 has a structure having the following structure of Formula IV:
wherein the chiral center 1 indicated by the circle is Levorotary (L), and wherein the chiral center 2 is D- or L-, or a racemic mixture. Two preferred embodiments of the molecule of above are purified forms having an L- at chiral center 1 and an L-form at chiral center 2, or separately an L-form at chiral center 1 and D-form at chiral center 2.
In another embodiment of a compound capable of selectively inhibiting calpain-2 with a Ki of at least 10-fold lower for calpain-2 than for calpain-1 has a structure having the following structure of Formula V:
wherein R1 is X1—PhCH2—, or X1—Ph(CH2)2—; where X1 is —H, or a molecule that improves absorption, bioavailability, half-life, or targeting such as (a transferrin polypeptide fragment, an insulin fragment, an LDL binding protein fragment, a rabies virus glycoprotein fragment); and wherein R2 is a functional group covalently bonded to the alpha-carbon having an L orientation, and having an amino acid side chain of leucine, or phenylalanine, or tyrosine, or valine, or isoleucine, methionine, or alanine, or a modified amino acid side chain; and R3 is —CH3, or —CH2CH3, or —(CH2)2CH3, or —CH(CH3)2 or —CH2CH(CH3)2, or —CH(CH3)CH2CH3, or —C6H5, —C6H4(4-OH), C6H4(3-OH), or C6H4(2-OH), or C6H4(2-CH3), or C6H4(3-CH3), C6H4(4-CH3), or C6H4(2-OCH3), or C6H4(3-OCH3), C6H4(4-OCH3), or C6H4(2-NH2), or C6H4(3-NH2), or C6H4(4-NH2), or C6H4(2-NHCH3), or C6H4(3-NHCH3), or C6H4(4-NHCH3), or C6H4(2-N(CH3)2), or C6H4(3-N(CH3)2), or C6H4(4- N(CH3)2); and R4 is —H, or —OCH3, ═NH, or —NH2, or —SH, or ═O, or ═S, or —OCH2CH3, or —O(CH2)2CH3, or —OCH(CH3)2, —SCH3, or SCH2CH3, or —S(CH2)2CH3, or —SCH(CH3)2, or —OH, or —CH3, or —CH2CH3, or —F, or —Cl, or —Br, or —I; and R5 is —H, or —OCH3, or —OCH2CH3, or —O(CH2)2CH3, or —OCH(CH3)2, —SCH3, or SCH2CH3, or —S(CH2)2CH3, or —SCH(CH3)2, or —OH, or —CH3, or —CH2CH3, or —CN, or —CHNH, or —NH2, or —NHCH3, or —N(CH3)2, or —F, or —Cl, or —Br, or —I; and R6 is —H, or —OCH3, or —OCH2CH3, or —O(CH2)2CH3, or —OCH(CH3)2, —SCH3, or SCH2CH3, or —S(CH2)2CH3, or —SCH(CH3)2, or —OH, or —CH3, or —CH2CH3, or —CN, or —CHNH, or —NH2, or —NHCH3, or —N(CH3)2, or —F, or —Cl, or —Br, or —I.
In another embodiment of a compound capable of selectively inhibiting calpain-2 with a Ki of at least 10-fold lower for calpain-2 than for calpain-1 has a structure having the following structure of Formula VI:
wherein R1 is PhCH2—, or Ph(CH2)2—; and wherein R2 is a functional group covalently bonded to the alpha-carbon having an L orientation, and having an amino acid side chain of leucine, or phenylalanine, or tyrosine, or valine, or isoleucine, methionine, or alanine, or a modified amino acid side chain; and R3 is —CH3, or —CH2CH3, or —(CH2)2CH3, or —CH(CH3)2 or —CH2CH(CH3)2, or —CH(CH3)CH2CH3, or —C6H5, —C6H4(4-OH), C6H4(3-OH), or C6H4(2-OH), or C6H4(2-CH3), or C6H4(3-CH3), C6H4(4-CH3), or C6H4(2-OCH3), or C6H4(3-OCH3), C6H4(4-OCH3), or C6H4(2-NH2), or C6H4(3-NH2), or C6H4(4-NH2), or C6H4(2-NHCH3), or C6H4(3-NHCH3), or C6H4(4-NHCH3), or C6H4(2-N(CH3)2), or C6H4(3-N(CH3)2), or C6H4(4-N(CH3)2); and R4 is —H, or —OCH3, ═NH, or —NH2, or —SH, or ═O, or ═S, or —OCH2CH3, or —O(CH2)2CH3, or —OCH(CH3)2, —SCH3, or SCH2CH3, or —S(CH2)2CH3, or —SCH(CH3)2, or —OH, or —CH3, or —CH2CH3, or —F, or —Cl, or —Br, or —I; and R5 is —H, or —OCH3, ═NH, or —NH2, or —SH, or ═O, or ═S, or —OCH2CH3, or —O(CH2)2CH3, or —OCH(CH3)2, —SCH3, or SCH2CH3, or —S(CH2)2CH3, or —SCH(CH3)2, or —OH, or —CH3, or —CH2CH3, or —F, or —Cl, or —Br, or —I; and R6 is —H, or —OCH3, or —OCH2CH3, or —O(CH2)2CH3, or —OCH(CH3)2, —SCH3, or SCH2CH3, or —S(CH2)2CH3, or —SCH(CH3)2, or —OH, or —CH3, or —CH2CH3, or —CN, or —CHNH, or —NH2, or —NHCH3, or —N(CH3)2, or —F, or —Cl, or —Br, or —I; R7 is —H, or —OCH3, or —OCH2CH3, or —O(CH2)2CH3, or —OCH(CH3)2, —SCH3, or SCH2CH3, or —S(CH2)2CH3, or —SCH(CH3)2, or —OH, or —CH3, or —CH2CH3, or —CN, or —CHNH, or —NH2, or —NHCH3, or —N(CH3)2, or —F, or —Cl, or —Br, or —I, or a purine.
In another embodiment of a compound capable of selectively inhibiting calpain-2 with a Ki of at least 10-fold lower for calpain-2 than for calpain-1 has a structure having the following structure of Formula VII:
wherein R1 is PhCH2—, or Ph(CH2)2—; and wherein R2 is a functional group covalently bonded to the alpha-carbon having an L orientation, and having an amino acid side chain of leucine, or phenylalanine, or tyrosine, or valine, or isoleucine, methionine, or alanine, or a modified amino acid side chain; and R3 is —CH3, or —CH2CH3, or —(CH2)2CH3, or —CH(CH3)2 or —CH2CH(CH3)2, or —CH(CH3)CH2CH3, or —C6H5, —C6H4(4-OH), C6H4(3-OH), or C6H4(2-OH), or C6H4(2-CH3), or C6H4(3-CH3), C6H4(4-CH3), or C6H4(2-OCH3), or C6H4(3-OCH3), C6H4(4-OCH3), or C6H4(2-NH2), or C6H4(3-NH2), or C6H4(4-NH2), or C6H4(2-NHCH3), or C6H4(3-NHCH3), or C6H4(4-NHCH3), or C6H4(2-N(CH3)2), or C6H4(3-N(CH3)2), or C6H4(4-N(CH3)2); and R4 is —H, or —OCH3, ═NH, or —NH2, or —SH, or ═O, or ═S, or —OCH2CH3, or —O(CH2)2CH3, or —OCH(CH3)2, —SCH3, or SCH2CH3, or —S(CH2)2CH3, or —SCH(CH3)2, or —OH, or —CH3, or —CH2CH3, or —F, or —Cl, or —Br, or —I; and R5 is —H, or —OCH3, ═NH, or —NH2, or —SH, or ═O, or ═S, or —OCH2CH3, or —O(CH2)2CH3, or —OCH(CH3)2, —SCH3, or SCH2CH3, or —S(CH2)2CH3, or —SCH(CH3)2, or —OH, or —CH3, or —CH2CH3, or —F, or —Cl, or —Br, or —I; and R6 is —H, or —OCH3, ═NH, or —NH2, or —SH, or ═O, or ═S, or —OCH2CH3, or —O(CH2)2CH3, or —OCH(CH3)2, —SCH3, or SCH2CH3, or —S(CH2)2CH3, or —SCH(CH3)2, or —OH, or —CH3, or —CH2CH3, or —F, or —Cl, or —Br, or —I; and R7 is —H, or —OCH3, or —OCH2CH3, or —O(CH2)2CH3, or —OCH(CH3)2, —SCH3, or SCH2CH3, or —S(CH2)2CH3, or —SCH(CH3)2, or —OH, or —CH3, or —CH2CH3, or —CN, or —CHNH, or —NH2, or —NHCH3, or —N(CH3)2, or —F, or —Cl, or —Br, or —I; and R8 is —H, or —OCH3, or —OCH2CH3, or —O(CH2)2CH3, or —OCH(CH3)2, —SCH3, or SCH2CH3, or —S(CH2)2CH3, or —SCH(CH3)2, or —OH, or —CH3, or —CH2CH3, or —CN, or —CHNH, or —NH2, or —NHCH3, or —N(CH3)2, or —F, or —Cl, or —Br, or —I, or a purine.
In another embodiment of a compound capable of selectively inhibiting calpain-2 with a Ki of at least 10-fold lower for calpain-2 than for calpain-1 has a structure having the following structure of Formula VIII:
wherein M1 is Y1—PhCH2—, or Y1—Ph(CH2)2—, or PhCH2—Y1—, or Ph(CH2)2—Y1—, wherein Y1 is a covalently bound polypeptide, or modified polypeptide that improves absorption, bioavailability, half-life, or targeting such as (a transferrin polypeptide fragment, an insulin fragment, an LDL binding protein fragment, a rabies virus glycoprotein fragment); or where Y1 is —H, or a substitution for linking small molecule, polypeptide, or modified polypeptide moieties for improving half-life, bioavailability or targeting, such as a transferrin polypeptide fragment, an insulin fragment, an LDL binding protein fragment, a rabies virus glycoprotein fragment; or Y1 is an —O, —N, —S, or —C substitution to link polypeptides that improve membrane permeability, or blood brain barrier passage, such as those of SEQ ID NO: 195-200, or a molecule that improves absorption, bioavailability, half-life, or targeting such as (a transferrin polypeptide fragment, an insulin fragment, an LDL binding protein fragment, a rabies virus glycoprotein fragment); and wherein R2 is a functional group covalently bonded to the alpha-carbon having an L orientation, and having an amino acid side chain of leucine, or phenylalanine, or tyrosine, or valine, or isoleucine, methionine, or alanine, or a modified amino acid side chain; and R3 is —CH3, or —CH2CH3, or —(CH2)2CH3, or —CH(CH3)2 or —CH2CH(CH3)2, or —CH(CH3)CH2CH3, or —C6H5, —C6H4(4-OH), C6H4(3-OH), or C6H4(2-OH), or C6H4(2-CH3), or C6H4(3-CH3), C6H4(4-CH3), or C6H4(2-OCH3), or C6H4(3-OCH3), C6H4(4-OCH3), or C6H4(2-NH2), or C6H4(3-NH2), or C6H4(4-NH2), or C6H4(2-NHCH3), or C6H4(3-NHCH3), or C6H4(4-NHCH3), or C6H4(2-N(CH3)2), or C6H4(3-N(CH3)2), or C6H4(4-N(CH3)2); and R4 is —H, or —OCH3, ═NH, or —NH2, or —SH, or ═O, or ═S, or —OCH2CH3, or —O(CH2)2CH3, or —OCH(CH3)2, —SCH3, or SCH2CH3, or —S(CH2)2CH3, or —SCH(CH3)2, or —OH, or —CH3, or —CH2CH3, or —F, or —Cl, or —Br, or —I; R5 is —H, or —OCH3, ═NH, or —NH2, or —SH, or ═O, or ═S, or —OCH2CH3, or —O(CH2)2CH3, or —OCH(CH3)2, —SCH3, or SCH2CH3, or —S(CH2)2CH3, or —SCH(CH3)2, or —OH, or —CH3, or —CH2CH3, or —F, or —Cl, or —Br, or —I; wherein X1 is —C6H3(3,5-R6,R7), or -2-pyridyl, or -2-pyridyl(3,5, R6,R7), or -3-pyridyl(3,5, R6, R7), or -4-pyridyl(3,5, R6, R7); wherein R6 is —H, or —OCH3, or —OCH2CH3, or —O(CH2)2CH3, or —OCH(CH3)2, —SCH3, or SCH2CH3, or —S(CH2)2CH3, or —SCH(CH3)2, or —OH, or —CH3, or —CH2CH3, or —CN, or —CHNH, or —NH2, or —NHCH3, or —N(CH3)2, or —F, or —Cl, or —Br, or —I; and R7 is —H, or —OCH3, or —OCH2CH3, or —O(CH2)2CH3, or —OCH(CH3)2, —SCH3, or SCH2CH3, or —S(CH2)2CH3, or —SCH(CH3)2, or —OH, or —CH3, or —CH2CH3, or —CN, or —CHNH, or —NH2, or —NHCH3, or —N(CH3)2, or —F, or —Cl, or —Br, or —I.
In another embodiment of a compound capable of selectively inhibiting calpain-2 with a Ki of at least 10-fold lower for calpain-2 than for calpain-1 has a structure having the following structure of Formula IX:
Wherein M1 is —O, —N, —S, or —C substituted to link blocking groups such as Y1—PhCH2—, or Y1—Ph(CH2)2—, or PhCH2—Y1—, or Ph(CH2)2—Y1—, wherein Y1 is a covalently linked polypeptide that improves absorption, bioavailability, half-life, or targeting such as (a transferrin polypeptide fragment, an insulin fragment, an LDL binding protein fragment, a rabies virus glycoprotein fragment); or where Y1 is —H, or a substitution for linking a molecule that improves absorption, bioavailability, half-life, or targeting such as (a transferrin polypeptide fragment, an insulin fragment, an LDL binding protein fragment, a rabies virus glycoprotein fragment); or Y1 is an —O, —N, —S, or —C substitution to link polypeptides that improve membrane permeability, or blood brain barrier passage, such as those of SEQ ID NO: 195-200, or a molecule that improves absorption, bioavailability, half-life, or targeting such as (a transferrin polypeptide fragment, an insulin fragment, an LDL binding protein fragment, a rabies virus glycoprotein fragment), or M1 is —O, —N, —S, or —C substituted to covalently link other small molecules polypeptides or modified polypeptides that improve membrane permeability, or blood brain barrier passage, such as those of SEQ ID NO: 195-200, or a transferrin polypeptide fragment, or an insulin fragment, or an LDL binding protein fragment, or a rabies virus glycoprotein fragment, or a molecule that improves absorption, bioavailability, half-life, or targeting such as (a transferrin polypeptide fragment, an insulin fragment, an LDL binding protein fragment, a rabies virus glycoprotein fragment); and wherein R1 is a functional group covalently bonded to the alpha-carbon having an L orientation, and having an amino acid side chain of leucine, or phenylalanine, or tyrosine, or valine, or isoleucine, methionine, or alanine, or a modified amino acid side chain; and R2 is —CH3, or —CH2CH3, or —(CH2)2CH3, or —CH(CH3)2 or —CH2CH(CH3)2, or —CH(CH3)CH2CH3, or —C6H4(4-OH), C6H4(3-OH), or C6H4(2-OH), or C6H4(2-CH3), or C6H4(3-CH3), C6H4(4-CH3), or C6H4(2-OCH3), or C6H4(3-OCH3), C6H4(4-OCH3), or C6H4(2-NH2), or C6H4(3-NH2), or C6H4(4-NH2), or C6H4(2-NHCH3), or C6H4(3-NHCH3), or C6H4(4-NHCH3), or C6H4(2-N(CH3)2), or C6H4(3-N(CH3)2), or C6H4(4-N(CH3)2); and R3 is —H, or —OCH3, ═NH, or —NH2, or —SH, or ═O, or ═S, or —OCH2CH3, or —O(CH2)2CH3, or —OCH(CH3)2, —SCH3, or SCH2CH3, or —S(CH2)2CH3, or —SCH(CH3)2, or —OH, or —CH3, or —CH2CH3, or —F, or —Cl, or —Br, or —I; R4 is —H, or —OCH3, ═NH, or —NH2, or —SH, or ═O, or ═S, or —OCH2CH3, or —O(CH2)2CH3, or —OCH(CH3)2, —SCH3, or SCH2CH3, or —S(CH2)2CH3, or —SCH(CH3)2, or —OH, or —CH3, or —CH2CH3, or —F, or —Cl, or —Br, or —I; wherein X1 is —C6H3(3,5-R5,R6), or -2-pyridyl, or -2-pyridyl(3,5, R5,R6), or -3-pyridyl(3,5, R5, R6), or -4-pyridyl(3,5, R5, R6); wherein R5 is —H, or —OCH3, or —OCH2CH3, or —O(CH2)2CH3, or —OCH(CH3)2, —SCH3, or SCH2CH3, or —S(CH2)2CH3, or —SCH(CH3)2, or —OH, or —CH3, or —CH2CH3, or —CN, or —CHNH, or —NH2, or —NHCH3, or —N(CH3)2, or —F, or —Cl, or —Br, or —I; and R6 is —H, or —OCH3, or —OCH2CH3, or —O(CH2)2CH3, or —OCH(CH3)2, —SCH3, or SCH2CH3, or —S(CH2)2CH3, or —SCH(CH3)2, or —OH, or —CH3, or —CH2CH3, or —CN, or —CHNH, or —NH2, or —NHCH3, or —N(CH3)2, or —F, or —Cl, or —Br, or —I.
In another embodiment of a compound capable of selectively inhibiting calpain-2 with a Ki of at least 10-fold lower for calpain-2 than for calpain-1 has a structure having the following structure of Formula X:
wherein M1 is —O, —N, —S, or —C substituted to link blocking groups such as Y1—PhCH2—, or Y1—Ph(CH2)2—, or PhCH2—Y1—, or Ph(CH2)2—Y1—, wherein Y1 is a polypeptide, covalently linked to a molecule that improves absorption, bioavailability, half-life, or targeting such as (a transferrin polypeptide fragment, an insulin fragment, an LDL binding protein fragment, a rabies virus glycoprotein fragment); or where Y1 is —H, or a substitution for linking a molecule that improves absorption, bioavailability, half-life, or targeting such as (a transferrin polypeptide fragment, an insulin fragment, an LDL binding protein fragment, a rabies virus glycoprotein fragment); or Y1 is an —O, —N, —S, or —C substitution to link polypeptides that improve membrane permeability, or blood brain barrier passage, such as those of SEQ ID NO: 195-200, or a molecule that improves absorption, bioavailability, half-life, or targeting such as (a transferrin polypeptide fragment, an insulin fragment, an LDL binding protein fragment, a rabies virus glycoprotein fragment), or M1 is —O, —N, —S, or —C substituted to covalently link other small molecules polypeptides or modified polypeptides that improve membrane permeability, or blood brain barrier passage, such as those of SEQ ID NO: 195-200, or a molecule that improves absorption, bioavailability, half-life, or targeting such as (a transferrin polypeptide fragment, an insulin fragment, an LDL binding protein fragment, a rabies virus glycoprotein fragment); and wherein R1 is a functional group covalently bonded to the alpha-carbon having an L orientation, and having an amino acid side chain of leucine, or phenylalanine, or tyrosine, or valine, or isoleucine, methionine, or alanine, or a modified amino acid side chain; and R2 is —CH3, or —CH2CH3, or —(CH2)2CH3, or —CH(CH3)2 or —CH2CH(CH3)2, or —CH(CH3)CH2CH3, or —C6H5, —C6H4(4-OH), C6H4(3-OH), or C6H4(2-OH), or C6H4(2-CH3), or C6H4(3-CH3), C6H4(4-CH3), or C6H4(2-OCH3), or C6H4(3-OCH3), C6H4(4-OCH3), or C6H4(2-NH2), or C6H4(3-NH2), or C6H4(4-NH2), or C6H4(2-NHCH3), or C6H4(3-NHCH3), or C6H4(4-NHCH3), or C6H4(2-N(CH3)2), or C6H4(3-N(CH3)2), or C6H4(4-N(CH3)2); and R3 is —H, or —OCH3, ═NH, or —NH2, or —SH, or ═O, or ═S, or —OCH2CH3, or —O(CH2)2CH3, or —OCH(CH3)2, —SCH3, or SCH2CH3, or —S(CH2)2CH3, or —SCH(CH3)2, or —OH, or —CH3, or —CH2CH3, or —F, or —Cl, or —Br, or —I; wherein X1 is —C6H3(3,5-R4,R5), or —CHR6—C6H3-(3,5-R4,R5), or -2-pyridyl, or -2-pyridyl(3,5, R4,R5), or —CHR6-2-pyridyl(3,5, R4,R5), or -3-pyridyl(3,5, R4, R5), —CHR6-3-pyridyl(3,5,R4,R5), or -4-pyridyl(3,5, R4, R5), or —CHR6-4-pyridyl(3,5,R4,R5); wherein R4 is —H, or —OCH3, or —OCH2CH3, or —O(CH2)2CH3, or —OCH(CH3)2, —SCH3, or SCH2CH3, or —S(CH2)2CH3, or —SCH(CH3)2, or —OH, or —CH3, or —CH2CH3, or —CN, or —CHNH, or —NH2, or —NHCH3, or —N(CH3)2, or —F, or —Cl, or —Br, or —I; and R5 is —H, or —OCH3, or —OCH2CH3, or —O(CH2)2CH3, or —OCH(CH3)2, —SCH3, or SCH2CH3, or —S(CH2)2CH3, or —SCH(CH3)2, or —OH, or —CH3, or —CH2CH3, or —CN, or —CHNH, or —NH2, or —NHCH3, or —N(CH3)2, or —F, or —Cl, or —Br, or —I; wherein R6 is —H, or —OCH3, or —OCH2CH3, or —O(CH2)2CH3, or —OCH(CH3)2, —SCH3, or SCH2CH3, or —S(CH2)2CH3, or —SCH(CH3)2, or —OH, or —CH3, or —CH2CH3, or —CN, or —CHNH, or —NH2, or —NHCH3, or —N(CH3)2, or —F, or —Cl, or —Br, or —I.
In another embodiment of a compound capable of selectively inhibiting calpain-2 with a Ki of at least 10-fold lower for calpain-2 than for calpain-1 has a structure having the following structure of Formula XI:
wherein M1 is —O, —N, —S, or —C substituted to link blocking groups such as Y1—PhCH2—, or Y1—Ph(CH2)2—, or PhCH2—Y1—, or Ph(CH2)2—Y1—, wherein Y1 is a polypeptide, or modified polypeptide covalently linked for improving half-life, bioavailability or targeting; or where Y1 is —H, or a substitution for linking small molecule, polypeptide, or modified polypeptide moieties for improving half-life, bioavailability or targeting, such as (a transferrin polypeptide fragment, an insulin fragment, an LDL binding protein fragment, a rabies virus glycoprotein fragment); or Y1 is an —O, —N, —S, or —C substitution to link polypeptides that improve membrane permeability, or blood brain barrier passage, such as those of SEQ ID NO: 195-200, or a molecule that improves absorption, bioavailability, half-life, or targeting such as (a transferrin polypeptide fragment, an insulin fragment, an LDL binding protein fragment, a rabies virus glycoprotein fragment), or M1 is —O, —N, —S, or —C substituted to covalently link other small molecules polypeptides or modified polypeptides that improve membrane permeability, or blood brain barrier passage, such as those of SEQ ID. NO: 195-200, or a molecule that improves absorption, bioavailability, half-life, or targeting such as (a transferrin polypeptide fragment, an insulin fragment, an LDL binding protein fragment, a rabies virus glycoprotein fragment); and wherein R1 is a functional group covalently bonded to the alpha-carbon having an L orientation, and having an amino acid side chain of leucine, or phenylalanine, or tyrosine, or valine, or isoleucine, methionine, or alanine, or a modified amino acid side chain; and R2 is —CH3, or —CH2CH3, or —(CH2)2CH3, or —CH(CH3)2 or —CH2CH(CH3)2, or —CH(CH3)CH2CH3, or —C6H5, —C6H4(4-OH), C6H4(3-OH), or C6H4(2-OH), or C6H4(2-CH3), or C6H4(3-CH3), C6H4(4-CH3), or C6H4(2-OCH3), or C6H4(3-OCH3), C6H4(4-OCH3), or C6H4(2-NH2), or C6H4(3-NH2), or C6H4(4-NH2), or C6H4(2-NHCH3), or C6H4(3-NHCH3), or C6H4(4-NHCH3), or C6H4(2-N(CH3)2), or C6H4(3-N(CH3)2), or C6H4(4-N(CH3)2); wherein X1 is —C6H3(3,5-R3,R4), or -2-pyridyl, or -2-pyridyl(3,5, R3,R4), or -3-pyridyl(3,5, R3, R4), or -4-pyridyl(3,5, R3, R4); wherein R3 is —H, or —OCH3, or —OCH2CH3, or —O(CH2)2CH3, or —OCH(CH3)2, —SCH3, or SCH2CH3, or —S(CH2)2CH3, or —SCH(CH3)2, or —OH, or —CH3, or —CH2CH3, or —CN, or —CHNH, or —NH2, or —NHCH3, or —N(CH3)2, or —F, or —Cl, or —Br, or —I; and R4 is —H, or —OCH3, or —OCH2CH3, or —O(CH2)2CH3, or —OCH(CH3)2, —SCH3, or SCH2CH3, or —S(CH2)2CH3, or —SCH(CH3)2, or —OH, or —CH3, or —CH2CH3, or —CN, or —CHNH, or —NH2, or —NHCH3, or —N(CH3)2, or —F, or —Cl, or —Br, or —I.
In another embodiment of a compound capable of inhibiting calpain-2 with a Ki of at least 10-fold lower for calpain-2 than for calpain-1 has a structure having the following structure of Formula XII:
The compound in this example was characterized as having an calpain-1 Ki of 15 μM and Ki for calpain-2 of 0.05 μM in Table I, column 14 of U.S. Pat. No. 6,235,929, and the same compound is disclosed in Li et al., 1996, which features the same inventors, as having a μ-calpain Ki of 0.35 μM and m-calpain Ki of 0.05 μM (compound 53 on page 4092 of Li et al, 1996).
In another embodiment of a compound capable of inhibiting calpain-2 with a Ki of at least 10-fold lower for calpain-2 than for calpain-1 has a structure having the following structure of Formula XIII:
wherein R7 is
wherein Z is a carbon or a nitrogen.
In another embodiment of a compound capable of selectively inhibiting calpain-2 with a Ki of at least 10-fold lower for calpain-2 than for calpain-1 has a structure having the following structure of Formula XVI:
wherein R8 is
Methods of identifying m-calpain specific substrates are also disclosed herein. The methods comprise contacting the substrate PTEN (SEQ ID NO: 1) or fragment or modified fragment thereof with active μ-calpain or another protein, or another prospective protein that may be cleaved by calpain-1 or calpain-2, or both, and determining if the substrate is specific for calpain-1 or calpain-2. If calpain-1 but not calpain-2 cleaves the substrate, then the substrate is a calpain-1 specific substrate. If calpain-2 but not calpain-1 cleaves the substrate, then the substrate is an calpain-2 specific substrate. The rate of substrate cleavage may also indicate the substrate is specific, or highly specific for calpain-1 or calpain-2. For example, the Kcat, or Km, or Ki can be determined in the presence of a labeled substrate prospective substrate, or in the presence of the potentially specific substrate and a second labeled non-specific substrate. Kcat, or Km, or the inhibitory constant (Ki), or another measure known in the art to define the rate of substrate cleavage can be used to indicate the rate of substrate cleavage, or the inhibitory properties of a substrate in the presence of another substrate not selective for calpain-1 or calpain-2.
In one embodiment, if the Km for a substrate is at least about 7-fold lower for calpain-1 than for calpain-2, then it is determined to be a calpain-1 specific substrate. Such substrates have the potential to serve as specific inhibitors of calpain-1. In another embodiment, if the rate of catalysis Km is at least about 20-fold lower for calpain-2 than for calpain-1, then it is determined to be a highly specific calpain-2 substrate. Such substrates have the potential to serve as highly specific inhibitors of calpain-2. In another embodiment, if the Km for the substrate is at least about 10-fold lower for calpain-1 than for calpain-2, then it is determined to be a calpain-1 specific substrate. Such substrates have the potential to serve as specific inhibitors of calpain-1. In another embodiment, if the Km is at least about 20-fold lower for calpain-1 than for calpain-2, then it is determined to be a highly specific calpain-1 substrate. Such substrates have the potential to serve as highly selective inhibitors of calpain-1.
In various aspects of the invention, a compound inhibitor highly selective to calpain-2, but not calpain-1, can be identified or designed based on PTEN cleavage site(s). Thus, methods of identifying a calpain-2 selective inhibitor are also disclosed herein. These methods comprise contacting a substrate, for example PTEN (SEQ ID NO: 1) or fragment or modified fragment thereof with active calpain-1 (SEQ ID NO: 2-6), or calpain-2 (SEQ ID NO: 69-73), or fragments thereof, and determining the rate of cleavage, or Kcat. Purified proteins, polypeptides, or modified polypeptides) can be contacted with a composition comprising calpain-1 or calpain-2, or purified or purified recombinant calpain-1 or calpain-2. After proteolysis, the fragments are analyzed by gel electrophoresis, collected, and subjected to Edmund degradation, or alternatively analyzed by 2-D gel and Edmund degradation, or alternatively by mass spectrometry to determine the precise cutting site of the polypeptides of the invention. Polypeptide fragments, small molecules mimicking the polypeptide fragments, or modifications of polypeptide fragments containing structure mimicking the cutting sites or peptides or polypeptides flanking the cutting sites can be used as inhibitors.
Various methods of identifying calpain cleavage sites are known in the art. For instance, site-directed mutagenesis can be used to determine the essential elements of a calpain cleavage site (Stabach et al, 1997, incorporated herein). Isolation of cleaved fragments and subsequent Edmund degradation (Xu et al, 2007) or mass spectroscopy can be used (Chou et al, 2011). If a fragment is identified that is cleaved by calpain-2 more rapidly than by calpain-1, such fragment can be used to inhibit the cleavage of specific substrates (Xu et al, 2007). In another embodiment, such fragments can be used as an inhibitor of specific calpain isoforms.
Methods of Identifying Proteins with Calpain-1 or Calpain-2 Specificity
Recombinant PTEN can be expressed with a GST-tag in E. coli BL-21 cells in the presence of IPTG (PET15 vector) and purified with glutathione-conjugated beads or columns. In various embodiments, recombinant PTEN can be purified, isolated, and exposed to calpain-1 and calpain-2 separately and the rate of cleavage of each can be determined by measuring the appearance of cleavage products, or alternatively the cleavage rate can be measured in the presence of succinyl-Leu-Tyr-AMC or another fluorescent or difluorescent polypeptide sequence that is not selective for calpain-2 or calpain-1. The Ki of a protein suspected to exhibit calpain-1 or calpain-2 specificity can be tested by comparing the changes in the non-selective substrates exposed to calpain-2 or calpain-1.
The literature indicates that calpains play a direct role in the mediation of cell death-related signaling through extrasynaptic NMDA receptors (Li & Ju, 2012, incorporated by reference), while preliminary data indicates that synaptic activation of NMDA receptors, which induces LTP, also results in activation of the ERK pathway, and in neuroprotection. Thus, calpains are involved in competing and opposite pathways. An explanation for these dual, disparate roles is that, like other signal transduction pathways, calpainn activity is made specific through discrete scaffolding of calpains and their various substrates. Thus, for instance, even common signal transduction proteins can be differentially scaffolded to create discrete signal transducing pathways. A seminal example of this was described in the yeast high-osmolarity and mating MAPK pathways, which contain a common MAPKKK protein. Differential scaffolding of MAPKKK into each pathway mediated each independent response, with no cross-talk (Park et al, 2003, incorporated by reference). Moreover, PDZ proteins have been found to be central to separating signal transduction pathways and eliminating cross-talk (Good et al, 2011, incorporated by reference).
Scaffolding of signal transduction pathways in various cell types has been shown to be the means by which specific signals or signaling cascades are made discrete from each other. Scaffolding, or the bundling by physical association of signal transducing elements to create discrete signaling cascades that do not cross-talk has been shown to be mediated through PDZ domain-containing proteins (Good et al, 2011). It is not recognized that calpain-1 and calpain-2 have PDZ-binding domains and are scaffolded to create separate signaling cascades for calpain-1 and calpain-2. The invention also identifies calpain-1 and calpain-2 PDZ-binding domain specific peptides that displace calpain from their respective protein scaffolds: for calpain-2, TIQLDLISWLSFSVL, or fragment or modification thereof; for calpain-1 PDZ-binding domain specific peptides: VTFDLFKWLQLTMFA, or fragment or modification thereof.
As discussed above, both calpain-2 and calpain-1 have PDZ-binding domains. The PDZ-binding domains of calpain-2 versus calpain-1 are significantly different from each other, with calpain-2 being a class I PDZ-binding domain and calpain-1 domain complying with the requirements of a class II PDZ-binding domain, and thus they likely do not share PDZ domain binding partners. Since their discovery in the 1990s (Kornau et al, 1995; Woods & Bryant, 1991, all incorporated by reference), PDZ proteins have become nearly ubiquitous in eukaryotic organisms, but are much more prevalent in vertebrates. An examination of calpain-2 and calpain-1 amino acid sequences indicates that, in calpain-2 for instance, a type-I PDZ binding domain is preserved across a wide range of vertebrates. Note, for instance, that rainbow trout (Oncorhynchus mykiss), and Zebra fish (Danio rerio) present a different C-terminal sequence than the mammalian and avian vertebrates shown, but they still preserve the requirements for a type-I PDZ binding domain (S/T-X-ψ; (Kang et al, 2003, incorporated by reference)). Note also the strong conservation of the type-II PDZ binding domain of calpain-1 across species (see Table 1). Thus, these sequences are strongly conserved in vertebrates, which indicates a critical functional role.
binding
Homo sapiens
Rattus
norvegicus
Ovis aries
Bos taurus
Sus scrofa
Gallus gallus
Oncorhynchus
mykiss
indicates data missing or illegible when filed
Peptides that interfere with calpain-PDZ protein association can be easily designed and are embodiments of the invention herein. Examples of peptide inhibitors of calpain-land calpain-2 scaffolding are included herein as SEQ ID Nos: 7-68 and 74-145. and are useful in the methods of administering disclosed herein.
In various embodiments of the invention, a PDZ-binding domain of calpain-2 is used in a method of un-scaffolding calpain-2, or a phospho-mimic (replacement of serines/threonines with aspartates or glutamates) of a PDZ binding domain of calpain-2. In another embodiment, a polypeptide comprised of a calpain-2 PDZ-binding domain (SEQ ID NOs: 74-145), or peptidomimetic thereof, or a phospho-mimic of a PDZ binding domain of calpain-2 is a product that can be used in the methods of treating described herein. Peptidomimics are understood in the art as molecules that are not conventional polypeptides, but bind specifically to the same proteins of a particular polypeptide with high specificity. Scaffolding of both calpain-2 and calpain-1 help define the postsynaptic compartment space that is potentiated. A method of un-tethering calpain-2 and by administration of a calpain-2 PDZ domain is described herein. In other embodiments, a calpain-2 PDZ-binding domain is useful in a method of treating diseases of LTP impairment as described herein. A method of treating PTSD by administering a polypeptide comprised of the PDZ-binding domain of calpain-2 or peptidomimetic is an preferred embodiment of this invention. Products for the treatment of the diseases and disorders taught herein are peptides, polypeptides, modifications thereof, or peptidomimetics of the PDZ-binding domain of calpain-2. PDZ-binding domains are combined with polypeptides and modified polypeptides, or small molecule combinations or liposomes or encapsulators to enhance organ targeting, subcellular targeting, bioavailability, half-life, or potency. PDZ-binding domains can also be linked to selective inhibitors or highly selective inhibitors or formulated with selective inhibitors or highly-selective inhibitors.
In various embodiments, the PDZ-binding domain of calpain-1 is used in a method of un-scaffolding calpain-1. In another embodiment, a polypeptide comprised of a calpain-1 PDZ-binding domain (SEQ ID NOs: 7-68) or peptidomimic thereof, or a phospho-mimic of a PDZ binding domain of calpain-1. Peptidomimics are understood in the art as molecules that are not conventional polypeptides, but bind specifically to the same proteins of a particular polypeptide with high specificity. Scaffolding of both calpain-2 and calpain-1 defines the postsynaptic compartment that is potentiated. In effect, scaffolding is participating in defining the LTP space created by activated calpain-1 versus activated calpain-2. Un-scaffolding calpain-1 with a calpain-1 PDZ-peptide results in greater LTP in rat hippocampal slices, neuroprotection by activation of the ERK/AKT pathway, and protection from serum starvation and hydrogen peroxide in culture (
Other polypeptides comprising fusion proteins of the invention improve delivery across the blood-brain barrier, bioavailability and stability of the small molecules polypeptides, nucleic acids, modified polypeptides, or modified nucleic acids of the invention. Further embodiments of the calpain isoform-selective inhibitors are small molecule modifications and polypeptide sequences linked through a peptide linkage, or other modification. Small molecules, polypeptides, or modified polypeptides that improve passage into cells are optionally added to the inventions to improve the bioavailability of the calpain-1 selective or calpain-2 selective inhibitors of the invention. Small molecules, polypeptides, or modified polypeptides that improve delivery across the blood-brain barrier are optionally added as well. Polypeptides that can be optionally added either through a peptide bond or other modification include but are not limited to the polypeptides of SEQ ID NOs: 195-200. Approaches to maximizing delivery to the brain are optionally part of isoform-selective calpain inhibitors of the invention as well (Bertrand et al, 2010; Dufes et al, 2013; Gabathuler, 2009; Gabathuler, 2010a; Gabathuler, 2010b) and are incorporated herein by reference. Such polypeptides include polypeptide fragments from insulin, IGF-1, IGF-2, and transferrin, LDL-binding peptides, rabies virus glycoprotein.
Liposomal, nanocontainer, and encapsulating formulations with or without conjugates that improve cellular or organ targeting comprising the small molecules, polypeptides, or modified polypeptides of the invention are also embodiments of the invention. Liposomes, and conjugates for targeting across the blood brain barrier are described in the art, for example, in U.S. Pat. Nos. 6,759,058; 6,761,901; 6,849,269; 7,387,791; USPN 2011/0305751 A1; and (Schnyder & Huwyler, 2005, all of which are incorporated by reference), the contents of which are incorporated by reference herein. In another embodiment, small molecules, polypeptides and modified polypeptides of the invention are combined with carrier molecules such as liposomes or other containers or encapsulators described. In yet another embodiment, small molecules, polypeptides, nucleic acids, modified nucleic acids, or modified polypeptides of the invention are combined with pharmaceutically acceptable nanocontainers comprising a ligand for a glutathione transporter for delivery across the blood brain barrier, an insulin fragment, an insulin-like growth factor (IGF) fragment, a transferrin protein fragment, a humanized antibody to transferrin receptor, a humanized anti-E Selectin antibody a low-density lipoprotein (LDL) Receptor binding protein fragment, or a rabies glycoprotein polypeptide fragment.
Nucleic acids that hybridize with the coding or untranslated 5′ or 3′ regions of calpain-2 mRNA are also embodiments of the invention. Nucleic acids are shRNA, microRNA, antisense DNA oligonucleotides or modifications thereof. Modifications that impart delivery of nucleic acids to the brain and to the locus of operational space of calpain-2 mRNA are preferred modifications, and include but are not limited to the polypeptide and liposomal conjugates disclosed herein. Methods of treating with said nucleic acids are also embodiments of the invention, and include methods of enhancing LTP, enhancing consolidation of LTP, enhancing consolidation of stimuli that normally don't induce LTP, improving memory, treating memory impairment, treating said psychiatric and neurological disease disclosed herein
Calpain-2 selective inhibitors are neuroprotective in cultured neurons and enhance Long-term potentiation (LTP), a cellular model of learning and memory, in acute hippocampal slices. Calpain-,2 inhibitors are useful as methods of enhancing LTP, and methods of enhancing LTP consolidation. Calpain-2 inhibitors according to the invention are useful for improving learning and reducing neurodegeneration. Therefore, they are expected to be used effectively for treatments of diseases related to synaptic dysfunction, synaptic degeneration, or neurodegeneration, including idiotypic and familial forms of Alzheimer's disease and Parkinson's disease, and dementia, Huntington's disease, Amyotropic Lateral Sclerosis (ALS), seizure, encephalitis, stroke, vasospasm, hypovolemic shock, traumatic shock, traumatic brain injury, reperfusion injury, multiple sclerosis, AIDS related dementia, neurotoxicity, head trauma, and spinal cord injury, glaucoma, open-angle glaucoma, angle-closure glaucoma, normal tension glaucoma, congenital glaucoma, pigmentary glaucoma, pseudoexfoliative glaucoma, traumatic glaucoma, neovascular glaucoma, irido corneal endothelial syndrome, ischemia in the eye, ischemia in the retina.
In various embodiments, calpain-2 inhibitors according to the invention are useful for effectively treating hearing loss, including hearing loss as a consequence of ototoxicity due to damage of the auditory nerve, for example, as a side effect of a drug or toxin. In various other embodiments, calpain-2 inhibitors according to the invention are useful for effectively treating hearing loss as a consequence of neurodgeneration.
In various embodiments, calpain-2 inhibitors according to the invention are useful for effectively treating Wolfram syndrome 1, while in other embodiments, calpain-2 inhibitors according to the invention are useful for effectively treating Wolfram syndrome 2. In certain embodiments calpain-2 inhibitors according to the invention treats neurodegeneration associated with Wolfram Syndrome 1 or 2 by inhibiting calpain-2 activity, including calpain-2 activity that is increased as a consequence of disregulation of either WSF1 or WSF2 gene expression.
In various embodiments, an effective amount of calpain-2 inhibitor according to the invention is administered to a patient in need thereof to inhibit neuronal cell death. In various other embodiments, an effective amount of calpain-2 inhibitor according to the invention is administered to a patient in need thereof to enhance memory. In yet other embodiments, an effective amount of calpain-2 inhibitor according to the invention is administered to a patient in need thereof to treat a neurological disorder. In yet another embodiment, an effective amount of calpain-2 inhibitor according to the invention is administered to a patient in need thereof to treat glaucoma.
In various other embodiments of the invention, effective amounts of a calpain-2 selective inhibitor, according to the invention, are used to effectively to treat diseases of synaptic and behavioral dysfunction, which include but are not limited to Schizophrenia, Autism Spectral Disorders, Bipolar Illness, drug-induced psychosis, Post-Traumatic Stress Disorder (PTSD), depression and suicidal thoughts, phobias, obsessive-compulsive disorder, trisomy 21, ADHD, and ADD. Autism Spectral Disorder includes Autistic disorder, (classic autism), Angelman Syndrome, Asperger's disorder (Asperger syndrome), Pervasive developmental disorder not otherwise specified (PDD-NOS), Rett's disorder (Rett syndrome), Childhood disintegrative disorder (CDD).
The pharmaceutical compositions of the invention may be prepared by methods known in the pharmaceutical formulation art, for example, see Remington's Pharmaceutical Sciences, 22nd Ed., (Pharmaceutical Press, 2012), which is incorporated herein by reference. In a solid dosage form, a compound of the invention may be admixed with at least one pharmaceutically acceptable excipient such as, for example, sodium citrate or dicalcium phosphate or (a) (a) fillers or extenders, such as, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b) binders, such as, for example, cellulose derivatives, starch, aliginates, gelatin, polyvinylpyrrolidone, sucrose, and gum acacia, (c) humectants, such as, for example, glycerol, (d) disintegrating agents, such as, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, croscarmellose sodium, complex silicates, and sodium carbonate, (e) solution retarders, such as, for example, paraffin, (f) absorption accelerators, such as, for example, quaternary ammonium compounds, (g) wetting agents, such as, for example, cetyl alcohol, and glycerol monostearate, magnesium stearate and the like (h) adsorbents, such as, for example, kaolin and bentonite, and (i) lubricants, such as, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets, and pills, the dosage forms may also comprise buffering agents.
Pharmaceutically acceptable adjuvants known in the pharmaceutical formulation art may also be used in the pharmaceutical compositions of the invention. These include, but are not limited to, preserving, wetting, suspending, sweetening, flavoring, perfuming, emulsifying, and dispensing agents. Prevention of the action of microorganisms may be ensured by inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. If desired, a pharmaceutical composition of the invention may also contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, antioxidants, and the like, such as, for example, citric acid, sorbitan monolaurate, triethanolamine oleate, butylated hydroxytoluene, etc.
Solid dosage forms as described above may be prepared with coatings and shells, such as enteric coatings and others, as is known in the pharmaceutical art. They may contain pacifying agents, and can also be of such composition that they release the active compound or compounds in a certain part of the intestinal tract in a delayed manner. Non-limiting examples of embedded compositions that may be used are polymeric substances and waxes. The active compounds may also be in microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.
Suspensions, in addition to the active compounds, may contain suspending agents, such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like. Liquid dosage forms may be aqueous, may contain a pharmaceutically acceptable solvent as well as traditional liquid dosage form excipients known in the art which include, but are not limited to, buffering agents, flavorants, sweetening agents, preservatives, and stabilizing agents.
In addition to the topical method of administration described above, there are various methods of administering the compounds of the invention topically to the lung. One such means could involve a dry powder inhaler formulation of respirable particles comprised of the compounds of the invention, which the patient being treated inhales. It is common for a dry powder formulation to include carrier particles, to which the compound particles can adhere to. The carrier particles may be of any acceptable pharmacologically inert material or combination of materials. For example, the carrier particles may be composed of one or more materials selected from sugar alcohols; polyols, for example sorbitol, mannitol or xylitol, and crystalline sugars, including monosaccharides and disaccharides; inorganic salts such as sodium chloride and calcium carbonate; organic salts such as sodium lactate; and other organic compounds such as urea, polysaccharides, for example cyclodextrins and dextrins. The carrier particles may be a crystalline sugar, for example, a monosaccharide such as glucose or arabinose, or a disaccharide such as maltose, saccharose, dextrose or lactose, The compound of the invention would be dispersed into the respiratory tract, and subsequently contact the lower lung in a pharmaceutically effective amount.
In addition to the topical method of administration described above, there are various methods of administering the compounds of the invention systemically by such methods. One such means would involve an aerosol suspension of respirable particles comprised of the compounds of the invention, which the patient being treated inhales. The compound would be absorbed into the bloodstream via the lungs in a pharmaceutically effective amount. The respirable particles can be liquid or solid, with a particle size sufficiently small to pass through the mouth and larynx upon inhalation.
Dosage forms for oral administration, which includes capsules, tablets, pills, powders, granules, and suspensions may be used. Dosage forms for pulmonary administration, which includes metered dose inhaler, dry powder inhaler or aerosol formulations may be used. In such solid dosage forms, the active compound may be mixed with at least one inert, pharmaceutically acceptable excipient (also known as a pharmaceutically acceptable carrier).
A compound according to the invention may also be used to formulate liquid or injectable pharmaceutical compositions. Administration of a compound of the invention in pure form or in an appropriate pharmaceutical composition may be carried out via any of the accepted modes of administration or agents for serving similar utilities. Thus, administration may be, for example, orally, buccally, nasally, pulmonary, parenterally (intravenous, intramuscular, intraperitoneal, or subcutaneous), topically, transdermally, intravaginally, intravesically, intrasystemically, ophthalmically or rectally, in the form of solid, semi-solid, lyophilized powder, or liquid dosage forms, such as, for example, tablets, suppositories, pills, soft elastic and hard gelatin capsules, powders, solutions, suspensions, or aerosols, or the like, such as, for example, in unit dosage forms suitable for simple administration of precise dosages. One route of administration may be oral administration, using a convenient daily dosage regimen that can be adjusted according to the degree of severity of the condition to be treated.
A Small Molecule/Modified Polypeptide Highly Selective for Calpain-2
Formula 1, where chiral center 1 is the L-form and chiral center 2 is a racemic mixture of D- and L- in this example was introduced at various concentrations (from 1 nM to 10 μM) into an in vitro mix comprising succinic-Leu-Tyr-AMC and calpain-1 or calpain-2 (Sasaki et al, 1984), and the kinetics of the loss of fluorescence were determined for each of the calpains (Powers et al, 2000). Ki values obtained for the compound in the literature are 2.3 μM for calpain-1 and 0.022 μM for calpain-2 (Li et al, 1996). However, the Ki of calpain-1 was re-determined herein to be 1.29 μM±0.7 μM, and the Ki for calpain-2 was determined to be 0.025 μM±0.02 μM. Therefore, the assessment of selectivity described herein was different than the prior teaching. This compound is an inhibitor highly selective for calpain-2 because its Ki is more than 50-fold lower for calpain-2 than for calpain-1.
Generic calpain inhibitors block LTP when administered before LTP induction. Field recording of excitatory postsynaptic potentials (EPSPs;
A calpain 2-selective inhibitor enhances LTP. Acute hippocampal slices were prepared and bathed in ACSF. 200 nM calpain-2-selective inhibitor of Formula 1, which inhibits calpain-2 50-100 fold more than calpain-1, was administered prior to Theta-burst stimulation, which has the ability to elicit LTP (see line #1 of
A calpain 2-specific inhibitor rescues LTP impairment in hippocampal slices from a mouse model of Angelman Syndrome. Field recording of excitatory postsynaptic potentials (EPSPs) was performed in stratum radiatum of field CA1 in acute hippocampal slices prepared from male UBEA mutant mice or their wild-type littermates. After 20 min of baseline recording, theta burst stimulation (TBS, arrow of
A selective calpain-2 inhibitor blocks neuronal death mediated by extrasynaptic NMDA receptor activation. Cortical neuronal cultures (14 DIV) were treated to induce selective activation of extrasynaptic NMDA receptors, which results in neuronal cell death. Application of the highly selective calpain-2 inhibitor of Formula 1 reduced the neuronal cell death associated with extrasynaptic NMDA receptor activation in a dose-dependent fashion from 200 nM to 5 μM (
Highly-selective calpain 2 inhibitors do not interfere with synaptic activity resulting in neuroprotection. Calpain inhibitor-III (which is not a selective calpain inhibitor), but not mCalp-I (200 nM) blocked Bic- and 4-AP-induced neuroprotection against starvation in cultured cortical neurons. Neuronal death was observed and quantified by Hoechst staining. 300-500 neurons were counted for each group in three to 6 independent experiments. *p<0.05; ns, no significant difference; one-way ANOVA followed by Bonferroni test. n=3-6. Error Bar indicates SEM (
Formula 1 Enhances Memory. Formula 1 was found to have a biphasic effect on learning and memory in the fear conditioning protocol. In this protocol, mice were trained to learn the association between a context or a tone with a painful stimulus. Various doses of the compound of Formula 1 (mCalp-I) were injected i.p. 30 min before training. Animals were tested 24 h later in the context (
Intraperitoneal injection of calpain-2 selective inhibitor is protective against NMDA-induced retinal damage. Either 2 μI PBS or 2 μI NMDA (2.5 mM) was injected intravitreally into the retinas of wild-type mice that had been intraperitoneally injected with vehicle (20% DMSO), a calpain-2 selective inhibitor (C2l, Z-Leu-Abu-CONH—CH2-C6H3 (3, 5-(OMe)2)13,14-0.3 mg/kg) or the pan-calpain inhibitor calpeptin (10 mg/kg) at 30 min before and 6 h after NMDA injection. H&E staining was done at 7 days after NMDA injection. See
Calpain-1 and calpain-2 play opposite roles in retinal damage induced by intravitreal NMDA injection. Calpain activity is involved in retinal cell death induced by NMDA Receptor (R) activation. To test the specific roles of calpain-1 and calpain-2 in this process, wild-type (WT) mice were injected systemically with a calpain-2 selective inhibitor (C2l), Z-Leu-Abu-CONH—CH2-C6H3 (3, 5-(OMe)2)13,14, 30 min before NMDA intravitreal injection, as described in Example 8. Levels of spectrin breakdown products (SBDP), the cleaved products of spectrin by both calpain-1 and -2, and of PH domain and Leucine-rich repeat Protein Phosphatase 1 (PHLPP1), a protein degraded by calpain-1 following NMDAR activation, were determined in retinal extracts 6 h after NMDA injection (
Six days after intravitreal injection of NMDA or PBS to WT mice, frozen retinal sections were prepared and H&E staining was performed to evaluate cell densities in the ganglion cell layer (GCL) and the thickness of the Inner Plexiform Layer (IPL), which contains RGC dendrites. NMDA injection (NMDA plus Vehicle) significantly reduced cell density in the GCL and IPL thickness, while PBS injection (PBS plus Vehicle) had no effect on these parameters (New
In calpain-1 KO mice, GCL cell density and IPL thickness were not affected by vehicle injection. However, the effects of NMDA injection on GCL cell density and IPL thickness were larger than in WT mice (compare New
Sequential activation of calpain-1 and calpain-2 in retina after acute IOP elevation. The following IOP elevation studies were performed using a model of acute angle closure glaucoma consisting of increasing intraocular pressure (IOP) to 110 mm Hg for 60 min by inserting a needle connected to an elevated reservoir of saline into the anterior chamber. This model reproduced several features of acute angle closure, including, ischemia of retina and iris as noted by absence of red reflex and pupillary response to light. Anterior chamber synechae, resulting in a narrow angle and adhesions between the iris and the cornea, increased cells and flare in the anterior chamber and increased corneal thickness due to corneal edema. Some of these changes persisted over 3 days of observation. Eyes were collected at 0, 2, 4 and 6 h after IOP elevation and retinal frozen sections were prepared and processed for immunohistochemistry with SBDP antibody. In WT mice, SBDP was clearly present in IPL at 2, 4 and 6 h after IOP elevation. However in calpain-1 KO mice, SBDP was only evident in the IPL at 4 and 6 h but not at 2 h (
To verify the time course of calpain-2 activation in retina after increased IOP, retinal sections were immunostained with an antibody against full-length PTEN, a substrate of calpain-2 but not calpain-117. In both WT and calpain-1 KO mice, PTEN-immunoreactivity in IPL was unchanged at 2 h, but was significantly reduced at 4 and 6 h after IOP elevation (
Calpain-1 and calpain-2 play opposite roles in RGC death induced by acute IOP elevation. To evaluate elevated IOP-induced retinal damage, IOP of the right eye was elevated to 110 mm Hg for 60 min, while a sham procedure was performed in the left eye. Retinal sections were collected for H&E staining 3 days after surgery (
In calpain-1 KO mice, cell count in GCL of IOP-elevated eye was significantly lower than that of sham eye (37.7±10.4 vs. 130.3±7.0 cells/mm, n=4). Importantly, the cell survival rate in calpain-1 KO mice was significantly lower than that in WT mice (
To specifically examine the effect of C2l on RGCs, which constitute approximately 40% of the cells in mouse GCL18, retinal sections from WT mice injected with vehicle or C2l 2 h after acute IOP elevation were immunostained with an antibody against brn-3a, a selective RGC marker19 (
To explore the nature of the signaling pathways downstream of calpain-1 and calpain-2, retinas in WT, calpain-1 KO and C2l-injected WT mice were collected 3 h after IOP elevation or sham surgery, homogenized and aliquots processed for Western blots (
Intravitreal injection of C2l reduces cell death in GCL and prevents loss of vision caused by acute IOP elevation. We used intravitreal C2l injection in order to locally deliver C2l to retina. First, we tested the delivery efficiency by injecting different doses of C2l intravitreally 2 h after IOP elevation in calpain-1 KO mice and analyzing SBDP levels in IPL at 4 h (
To examine vision of mice after glaucoma, we tested the optokinetic reflex (OKR) in mice (
Diastereoisomers of Formula 1 have profoundly different Inhibitory activities.
Formula 1A, where chiral center 1 is the S-form and chiral center 2 is the S-form was separated from the S-R-form (Formula 1B) using methods that are well-known methods for separating diastereoisomers. Formula 1A, which is also referred to herein as compound 18A, in this example was introduced at various concentrations into an in vitro mix comprising succinic-Leu-Tyr-AMC and μ-calpain or m-calpain (Sasaki et al, 1984), and the kinetics of the loss of fluorescence were determined for each of the calpains. The Ki of Formula 1A for μ-calpain was determined to be 181±73 nM for calpain-1, and the Ki for calpain-2 was determined to be 7.8±2.5 nM (see Table I). In contrast, the Ki of Formula 18 (also referred to herein as compound 188) for calpain-1 was determined herein to be 514±151 μM and the Ki for calpain-2 was determined to be 15.6±9.2 μM. This represents an unexpected 2000-fold difference in the activity between the two diasteriomers with respect to the inhibition of calpain-2.
This application claims priority to U.S. Application Ser. No. 62/078,221, filed 11 Nov. 2014, and includes its disclosure herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US15/60157 | 11/11/2015 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62078221 | Nov 2014 | US |
