Bh4 domain peptides and uses thereof

Abstract

Stapled peptides and mutant BCL-2 family peptides and compositions comprising the same are provided. Also provided are methods of using the stapled peptides and mutant BCL-2 family peptides for selectively protecting sensory neurons from age-related and / or chemotherapy induced axonal degeneration in a human subject in need thereof, treating or preventing chemotherapy induced peripheral neuropathy (CIPN) in a human subject in need thereof, averting neuropathic side effects of chemotherapy and / or other causes of axonal degeneration in a human subject in need thereof, and treating or preventing hearing loss in a human subject in need thereof. The methods involve administering to the human subject a stapled peptide and a mutant BCL-2 family peptide disclosed herein.

Description

[0001] BH4 DOMAIN PEPTIDES AND USES THEREOF

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 675,531, filed July 25, 2024, the contents of which are incorporated by reference herein in its entirety.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] This invention was made with government support under R35CA197583, R50CA211399, R01CA205255 and K00AG078230 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

[0006] SEQUENCE LISTING

[0007] This application contains a Sequence Listing that has been submitted electronically as an XML file named 00530-0424W01_SL.xml. The XML file, created on July 23, 2025, is 185,475 bytes in size. The material in the XML text file is hereby incorporated by reference in its entirety.

[0008] BACKGROUND

[0009] Axons that span long distances enable rapid communication within a neural circuit, but they are particularly vulnerable to injury and degeneration. Therefore, axonal degeneration impairs circuit functionality and is a prominent component of multiple neurologic disorders. Many chemotherapeutic drugs required to treat cancers can injure long-range axons (e.g., peripheral sensory or motor neuron axons), causing chemotherapy-induced peripheral neuropathy (CIPN) with impaired tactile sensation and persistent pain. CIPN is a painful and debilitating side effect of cancer treatment and often leads to dose reduction or cessation of potentially curative therapy. The prevalence of CIPN has been estimated to be as high as 68% within the first month of exposure to offending agents, with symptoms persisting beyond 6 months in approximately 30% of patients. Currently, the molecular mechanisms of CIPN are not understood, and there are no available treatments. There is an unmet need to provide compositions and methods for treating or preventing CIPN.

[0010] The ability to sense, respond to, and perceive music, warning cues, language, and other environmental stimuli depends on the auditory nervous system. The human sense of hearing is made possible by the conversion of sound stimuli into patterns of neural activity that can be integrated with other sensory information to guide behavior, intraspecies communication, and other critical functions. This remarkably rapid processing, which can occur in the space of 10 microseconds, relies on hair cells and their proper innervation by precisely wired spiral ganglion neurons within the cochlea of the inner ear. Successful transmission of the intensity, frequency, and timing of stimuli from hair cells to the brain is contingent on the proper assembly of these auditory circuits during development.

[0011] Through precisely organized circuits within the inner ear, sensory information represented by specific displacements of the inner and outer hair cells along the basilar membrane is converted into electrical signals that propagate along the spiral ganglia. Peripheral spiral ganglion axons transmit acoustic information from the hair cells to central projections within the eighth (VIlTh) nerve which innervates the brainstem at the anteroventral cochlear nucleus, the posteroventral cochlear nucleus, and the dorsal cochlear nucleus. Integrating centers within the superior olivary complex and inferior colliculus of the midbrain allow organisms to localize sources in space and process complex features such as the temporal and harmonic properties of speech and music. As spiral ganglion neurons form appropriate connections to provide the critical bridge from the inner ear to the central nervous system, understanding how these neurons initiate their assembly and maintain their processes over time is critical to efforts aimed at producing therapies to treat defects in the auditory nervous system.

[0012] WO 2018 / 039545 A2 and Pease-Raissi et al. (Pease-Raissi et al., 2017, Neuron, 96, 373-386) disclose specific Bcl-w-based peptides for treating or preventing chemotherapy-induced peripheral neuropathy (CIPN) and / or hearing loss. However, there remains a need for specific variants that can treat these conditions as well as alternative peptide scaffolds that can be engineered to treat CIPN and / or hearing loss. In particular, there remains a need for therapeutics with demonstrable neuroprotective effects against a diversity of neurotoxic drugs that induce degeneration of axons.

[0013] SUMMARY

[0014] The present disclosure is based, at least in part, on the finding that certain positions (e.g., L at position 3, V at position 4, D at position 6, F at position 7, V at position 8, G at position 9, Y at position 10, Q at position 14, G at position 16, or V at position 18 of SEQ ID NO:21) of a stapled bcl-w BH4 domain peptide tolerate substitution without abrogating binding of the substituted stapled BCL-w BH4 domain peptide to inositol 1,4, 5 -trisphosphate receptor, type 1 (IP3R1). The present disclosure is also based, at least in part, on the finding substitution of certain positions (e.g., R at position 1, Y at position 10, K at position 11, R at position 13, K at position 15, and Y at position 17 of SEQ ID NO:21) of a stapled bcl-w BH4 domain peptide abrogate binding of the substituted stapled bcl-w BH4 domain peptide to IP3R1 . Surprisingly, the present disclosure is also based, at least in part, on the finding that the binding profile of a BCL-2 BH4 domain peptide can be modified to gain the ability to bind IP3R1 by substituting S at position 12 of SEQ ID NO: 1 (a bcl-2 BH4 domain peptide) with a positive amino acid (e.g., His, Lys, Arg, or Orn (ornithine)).

[0015] Thus, provided herein are stapled peptides (e.g., stapled mutant BCL-2 peptides and stapled mutant BCL-w peptides) and mutant BCL-2 peptides. The stapled peptides and mutant BCL-2 peptides provided herein are useful in selectively protecting sensory neurons from age-related and / or chemotherapy induced axonal degeneration in a human subject in need thereof, treating or preventing CIPN in a human subject in need thereof, averting neuropathic side effects of chemotherapy and / or other causes of axonal degeneration in a human subject in need thereof, and treating or preventing hearing loss in a human subject in need thereof. The methods involve administering to the human subject a stapled peptide or a mutant BCL-2 peptide described herein. Non-limiting aspects and embodiments of these methods are described herein. Any of the aspects described below can be used in any combination in the methods described herein.

[0016] Provided herein is a BCL-2 mutant peptide, the BCL-2 mutant peptide comprising the amino acid sequence EIVMKYIHYKLSQRGYEWDA (SEQ ID NO: 1) with S at position 12 substituted by a positively charged amino acid and optionally further including 1 to 4 other amino acid substitutions within SEQ ID NO: 1, optionally wherein the BCL-2 mutant peptide binds inositol 1,4,5-trisphosphate receptor, type 1 (IP3R1) or BAX. In some instances, S at position 12 is substituted with one of His, Lys, Arg, or Orn. In some instances, S at position 12 is substituted with Arg. In some instances, (i) Leu at position 11 and Gly at position 15, or (ii) Gin at position 13 and Glu at position 17, or (iii) Leu at position 11 and Trp at position 18 of SEQ ID NO: 1 are each substituted with an a,a-disubstituted non-natural amino acid. In some instances, the M at position 4 of SEQ ID NO:1 is substituted with norleucine. In some instances, one or more of positions 2, 3, 6, 7, 9, 10, 14, and 16 of SEQ ID NO: 1 are not substituted. In some instances, one or more of positions 10, 14, and 16 of SEQ ID NO:1 are not substituted. In some instances, one or more of positions 2, 3, 6, 7, 8, 10, 14, and 16 of SEQ ID NO: 1 are conservatively substituted. In some instances, one or more of positions 10, 14, and 16 of SEQ ID NO:1 are conservatively substituted. In some instances, one or more of positions 10, 14, and 16 of SEQ ID NO: 1 is substituted with an amino acid selected from K, R, Y, F, and W. In some instances, one or more of positions 10, 14, and 16 of SEQ ID NO:1 is substituted with an amino acid selected from K and R. In some instances, one or more of positions 10, 14, and 16 of SEQ ID NO: lis substituted with an amino acid selected from Y, F, and W. In some instances, the BCL-2 mutant peptide includes an Arg immediately N-terminal to Glu at position 1 of SEQ ID NO: 1. In some instances, the BCL-2 mutant peptide comprises a sequence selected from the group consisting of:

[0017] EIVMKYIHYKLRQRGYEWDA (SEQ ID NO:2),

[0018] EIVBKYII IYKLRQRGYEWDA (SEQ ID NO: 3), REIVMKYIHYKLRQRGYEWDA (SEQ ID NO: 4), and REIVBKYIHYKLRQRGYEWDA (SEQ ID NO: 5), with 2 to 4 amino acid substitutions, wherein B is norleucine; wherein none of the 2 to 4 amino acid substitutions replace amino acids at positions 10, 12, 14, or 16 of SEQ ID NO:2 or 3, or positions 11, 13, 15, or 17 of SEQ ID NO:4 or 5 or if positions 10, 12, 14, or 16 of SEQ ID NO:2 or 3, or positions 11 , 13, 15, or 17 of SEQ ID NO:4 or 5 are substituted, it is with conservative amino acid substitutions; and optionally wherein 2 of the 2 to 4 amino acid substitutions replace amino acids three or six amino acids apart within any one of SEQ ID NOs:2-5 with a,a-disubstituted non-natural amino acids. In some instances, the BCL-2 mutant peptide binds to IP3R1, BAX, or IP3R1 and BAX. In some instances, the BCL-2 mutant peptide is 20 to 50, 20 to 40, 20 to 30, 20 to 25, 21, 20, less than 50, less than 40, less than 30, or less than 25 amino acids in length.

[0019] Also provided herein are BCL-2 stapled peptides. In some instances, the BCL-2 stapled peptide comprises the amino acid sequence EIVMKYIHYKXi SQRX2YEWDA (SEQ ID NO:6), EIVMKYIHYKLSX1RGYX2WDA (SEQ ID NO:7), or EIVMKYIHYK8SQRGYEXDA (SEQ ID NO: 8) with S at position 12 of any one of SEQ ID NOs: 6-8 substituted by a positively charged amino acid and optionally further including 1 or 2 other amino acid substitutions within any one of SEQ ID NOs:6-8, wherein the 1 or 2 other substitutions are not at Xi or X2 of SEQ ID NOs: 6 and 7 and are not at 8 or X of SEQ ID NO:8, wherein Xi, X2, 8, and X are a,a-disubstituted non-natural amino acids, and wherein the side chains of Xi and X2 or 8 and X, respectively, are cross linked, and optionally wherein the stapled peptide binds IP3R1. In some instances, S at position 12 of any one of SEQ ID NOs:6-8 is substituted with one of His, Lys, Arg, and Orn. In some instances, S at position 12 of any one of SEQ ID NOs:6-8 is substituted with Arg. In some instances, Xi and X2 are each (S)-2-(4-pentenyl)alanine, and wherein 8 is (R)-a-(7'-octenyl)alanine and X is (S)-2-(4-pentenyl)alanine. In some instances, M at position 4 of any one of SEQ ID NOs:6-8 is substituted with norleucine. In some instances, one or more of positions 2, 3, 6, 7, 9, 10, 11, 15, and 16 of SEQ ID NOs:6-8 are not substituted. In some instances, one or more of positions 10, 14, and 16 of SEQ ID NOs: 6-8 are not substituted. In some instances, one or more of positions 2, 3, 6, 7, 8, 10, 14, and 16 of SEQ ID NO: 1 are conservatively substituted. In some instances, one or more of positions 10, 14, and 16 of SEQ ID NO: 1 are conservatively substituted. In some instances, one or more of positions 10, 14, and 16 of SEQ ID NO: 1 is substituted with an amino acid selected from K, R, Y, F, and W. In some instances, one or more of positions 10, 14, and 16 of SEQ ID NO: 1 is substituted with an amino acid selected from K and R. In some instances, one or more of positions 10, 14, and 16 of SEQ ID NO: lis substituted with an amino acid selected from Y, F, and W. In some instances, the stapled peptide includes an Arg immediately N-terminal to Glu at position 1 of any one of SEQ ID NOs:6-8. In some instances, the stapled peptide comprises a sequence selected from the group consisting of:

[0020] EIVMKYIHYKX1RQRX2YEWDA (SEQ ID NON), EIVBKYIHYKX1RQRX2YEWDA (SEQ ID NO: 10), EIVMKYIHYKLRX1RGYX2WDA (SEQ ID NO: 11 ), EIVBKYIHYKLRX1RGYX2WDA (SEQ ID NO: 12), EIVMKYIHYK8RQRGYEXDA (SEQ ID NO: 13), EIVBKYIHYK8RQRGYEXDA (SEQ ID NO: 14), REIVMKYIHYKX1RQRX2YEWDA (SEQ ID NO: 15), REIVBKYIHYKX1RQRX2YEWDA (SEQ ID NO: 16), REIVMKYIHYKLRX1RGYX2WDA (SEQ ID NO: 17), REIVBKYIHYKLRX1RGYX2WDA (SEQ ID NO: 18), REIVMKYIHYK8RQRGYEXDA (SEQ ID NO: 19), and REIVBKYIHYK8RQRGYEXDA (SEQ ID NO:20), with 0 to 2 amino acid substitutions relative to SEQ ID NOs: 9, 11, 13, 15, 17, and 19, or 0 or 1 amino acid substitution relative to SEQ ID NOs: 10, 12, 14, 16, 18, and 20, wherein B is norleucine. In some instances, the stapled peptide binds IP3R1, BAX, or IP3R1 and BAX. In some instances, the stapled peptide is 20 to 50, 20 to 40, 20 to 30, 20 to 25, 21, 20, less than 50, less than 40, less than 30, or less than 25 amino acids in length.

[0021] Also provided herein are BCL-w stapled peptides. In some instances, the BCL-w stapled peptide comprises the amino acid sequence RALVADFVGYKX1RQKX2YV (SEQ ID NO:21) with 1 to 3 amino acid substitutions, wherein Xi and X2 are a,a-disubstituted non-natural amino acids, wherein the side chains of Xi and X2 are cross linked, and wherein positions 12 and 16 of SEQ ID NO:21 are not substituted, optionally wherein the stapled peptide also binds IP3R1, and further optionally wherein the stapled peptide binds one or both of BAX and BAK. In some instances, one or more of L at position 3, V at position 4, D at position 6, F at position 7, V at position 8, G at position 9, Y at position 10, Q at position 14, G at position 16, or V at position 18 of SEQ ID NO:21 are substituted with an alanine, glycine, valine, or leucine. In some instances, one of L at position 3, V at position 4, D at position 6, F at position 7, V at position 8, G at position 9, Y at position 10, Q at position 14, G at position 16, or V at position 18 of SEQ ID NO:21 is substituted with an alanine, glycine, valine, or leucine. In some instances, one or more of R at position 1, Y at position 10, K at position 11, R at position 13, K at position 15, and Y at position 17 of SEQ ID NO:21 are not substituted or, if substituted, R at position 1, K at position 11, R at position 13, or K at position 15 of SEQ ID NO:21 is substituted with a positively charged amino acid, and Y at position 10 of SEQ ID NO:21 is substituted with a hydrophobic, particularly aromatic amino acid. In some instances, Xi and X2 of SEQ ID NO:21 are each (S)-a-(4'- pentenyl)alanine. In some instances, the stapled peptide binds IP3R1, BAX, or IP3R1 and BAX. In some instances, the stapled peptide is 20 to 50, 20 to 40, 20 to 30, 20 to 25, 21, 20, 19, 18, less than 50, less than 40, less than 30, or less than 25 amino acids in length.

[0022] In some instances, the BCL-w stapled peptide comprises the amino acid sequence RALVADFVGYK8RQKGYVX (SEQ ID NO:22) with 1 to 3 amino acid substitutions, wherein 8 and X are a, a-di substituted non-natural amino acids, wherein the side chains of 8 and X are cross linked, and wherein positions 12 and 19 of SEQ ID NO:22 are not substituted, optionally wherein the stapled peptide binds IP3R1, and further optionally wherein the stapled peptide binds one or both of BAX and BAK. In some instances, one or more of D at position 6 and G at position 9 of SEQ ID NO:22 are substituted with an alanine, glycine, valine, or leucine. In some instances, one or more of R at position 1, Y at position 10, K at position 11, R at position 13, K at position 15, and Y at position 17 of SEQ ID NO:22 are not substituted, or, if substituted, R at position 1, K at position 11, R at position 13, or K at position 15 of SEQ ID NO:22 is substituted with a positively charged amino acid, and Y at position 10 of SEQ ID NO:22 is substituted with a hydrophobic, particularly aromatic amino acid. In some instances, 8 and X of SEQ ID NO:22 are (R)-a-(7'-octenyl)alanine and (S)-a-(4'-pentenyl)alanine, respectively. In some instances, the stapled peptide binds IP3R1, BAX, or IP3R1 and BAX. In some instances, the stapled peptide is 20 to 50, 20 to 40, 20 to 30, 20 to 25, 21, 20, 19, less than 50, less than 40, less than 30, or less than 25 amino acids in length.

[0023] Also provided herein are stapled peptides described or depicted in the figures or working examples herein.

[0024] Also provided herein is a method of making a stapled peptide, the method comprising:

[0025] (a) providing:

[0026] (i) a BCL-2 mutant peptide described herein,

[0027] (ii) a peptide comprising the amino acid sequence EIVMKYIHYKX1SQRX2YEWDA (SEQ ID NO: 30), EIVMKYIHYKLSX1RGYX2WDA (SEQ ID NO: 31), or EIVMKYIHYK8SQRGYEXDA (SEQ ID NO:32) with S at position 12 of any one of SEQ ID NOs:30-32 substituted by a positively charged amino acid and optionally further including 1 or 2 other amino acid substitutions within any one of SEQ ID NOs:30-32, wherein the 1 or 2 other substitutions are not at Xi or X2 of SEQ ID NOs: 30 and 31 and are not at 8 or X of SEQ ID NO:32, wherein Xi , X2 , 8, and X are a, a-di substituted non-natural amino acids,

[0028] (iii) a peptide comprising the amino acid sequence RALVADFVGYKX1RQKX2YV (SEQ ID NO:33) with 1 to 3 amino acid substitutions, wherein Xi and X2 are a, a-di substituted non-natural amino acids, and wherein positions 12 and 16 of SEQ ID NO:33 are not substituted, or

[0029] (iv) a peptide comprising the amino acid sequence RALVADFVGYK8RQKGYVX (SEQ ID NO:34) with 1 to 2 amino acid substitutions, wherein 8 and X are a, a-di substituted non-natural amino acids, wherein the side chains of 8 and X are cross linked, and wherein positions 12 and 19 of SEQ ID NO:34 are not substituted; and

[0030] (b) crossdinking the peptide thereby making the stapled peptide. In some instances, the cross-linking is via a ring-closing metathesis (RCM) reaction. In some instances, the method further comprises formulating the stapled peptide as a sterile pharmaceutical composition.

[0031] Also provided herein is a pharmaceutical composition comprising any one of the aforementioned mutant BCL-2 peptides, and a pharmaceutically acceptable carrier.

[0032] Also provided herein is a pharmaceutical composition comprising any one of the aforementioned stapled peptides, and a pharmaceutically acceptable carrier.

[0033] Also provided herein is a method of selectively protecting sensory neurons from age-related and / or chemotherapy induced axonal degeneration in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of any one of the aforementioned mutant BCL-2 peptides, any one of the aforementioned stapled peptides, or any one of the aforementioned pharmaceutical compositions.

[0034] Also provided herein is a method of treating CIPN in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of any one of the aforementioned mutant BCL-2 peptides, any one of the aforementioned stapled peptides, or any one of the aforementioned pharmaceutical compositions.

[0035] Also provided herein is a method of preventing CIPN in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of any one of the aforementioned mutant BCL-2 peptides, any one of the aforementioned stapled peptides, or any one of the aforementioned pharmaceutical compositions.

[0036] Also provided herein is a method for averting neuropathic side effects of chemotherapy and / or other causes of axonal degeneration in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of any one of the aforementioned mutant BCL-2 peptides, any one of the aforementioned stapled peptides, or any one of the aforementioned pharmaceutical compositions.

[0037] Also provided herein is a method for treating or preventing hearing loss in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of any one of the aforementioned mutant BCL- 2 peptides, any one of the aforementioned stapled peptides, or any one of the aforementioned pharmaceutical compositions.

[0038] Also provided herein is a pharmaceutical composition comprising (a) means for selectively protecting sensory neurons from age-related and / or chemotherapy induced axonal degeneration in a human subject in need thereof, and (b) a pharmaceutically acceptable carrier.

[0039] Also provided herein is a pharmaceutical composition comprising (a) means for treating CIPN in a human subject in need thereof, and (b) a pharmaceutically acceptable carrier.

[0040] Also provided herein is a pharmaceutical composition comprising (a) means for preventing CIPN in a human subject in need thereof, and (b) a pharmaceutically acceptable carrier.

[0041] Also provided herein is a pharmaceutical composition comprising (a) means for averting neuropathic side effects of chemotherapy and / or other causes of axonal degeneration in a human subject in need thereof, and (b) a pharmaceutically acceptable carrier. Also provided herein is a pharmaceutical composition comprising (a) means for treating or preventing hearing loss in a human subject in need thereof, and (b) a pharmaceutically acceptable carrier.

[0042] Also provided herein is a pharmaceutical composition comprising (a) means for binding human IP3R1, and (b) a pharmaceutically acceptable carrier, optionally wherein the binding is in the D3 arm of IP3R1.

[0043] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the exemplary methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present application, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0044] Other features and advantages of the invention will be apparent from the following detailed description and from the claims.

[0045] BRIEF DESCRIPTION OF THE DRAWINGS

[0046] FIGs. 1A-1K: Binding activities of a stapled BCL-w BH4 library to endogenous and recombinant IP3R1.

[0047] FIG. 1A is a cartoon highlighting the BH4 domain of BCL-w, which is composed of the al helix and a portion of the unstructured loop between al and a2, with amino acids 11-28 of the helical portion subjected to all-hydrocarbon stapling.

[0048] FIG. IB: Structure of the IP3 receptor type 1 (IP3RI) depicting its structural and functional domains. Domain 3, which has been implicated as a cytosolic surface site of BCL-2 and BCL-XL BH4 interaction, is highlighted. FIG. 1C and ID: Compositions of / ', i+4 (FIG. 1C) and i, i+ 7 (FIG. ID) staple scanning libraries of the BCL-w BH4 domain sequence. The stapling amino acids are indicated as X for S-pentenylalanine and 8 for R-octenylalanine. The sequences in FIG. 1C are from top to bottom SEQ ID NOs:63-73, 1 18, and 74-76, respectively; these peptides are labeled as 1-15. The sequences in FIG. ID are from top to bottom SEQ ID NOs:77-87 and 119, respectively; these peptides are labeled 16-27.

[0049] FIG. IE: Helical wheel depiction of the helical portion of the BCL-w BH4 domain, highlighting regions of hydrophobicity, positive and negative charge, and hydrophilic residues.

[0050] FIGs. 1F-1G: Cerebellar lysates were incubated with biotinylated and i, i+4 (FIG. IF) or i, i+ 7 (FIG. 1G) stapled BCL-w BH4 peptides followed by streptavidin pull-down, gel electrophoresis, and anti-IP3Rl western blot. Band intensities were quantified by ImageJ analysis and normalized to the band of highest intensity for plotting. Error bars are mean ± SEM for experiments performed in triplicate with three independent preparations of protein and peptides. In FIG. IF, 1-15 correspond to peptides of SEQ ID NOs: 63-73, 118, and 74-76, respectively (see FIG. 1C). In FIG. 1G, 16-27 correspond to peptides of SEQ ID NOs: 77-87 and 119, respectively (see FIG. ID)

[0051] FIG. 1H: The influence of stapling at a particular amino acid position of the BCL-w BH4 peptide based on its helical structure, whereby an adverse “hit” is scored if a staple that begins or ends at a particular residue results in 25% or less endogenous IP3R1 pull-down activity. Residues with 2-3 “hits” (Rl l, Y20, K21, R23, K25, Y27), or 1 “hit” (Al 5, Fl 7, VI 8, G19, Q24, V28) are highlighted and shown as stick representations. Residues A12, L13, V14, D16, L22 and G26 show no “hits” so are most amenable to crosslinking in an (z, i+4 configuration.

[0052] FIG. 1I-1J: Recombinant IP3R1 D3 protein (i.e., the D3 domain of IP3R1) was incubated with biotinylated and i, i+4 (FIG. II) or i, i+7 (FIG. 1J) stapled BCL-w BH4 peptides followed by streptavidin pull-down, gel electrophoresis, and anti-GST western blot. Band intensities were quantified by ImageJ analysis and normalized to the band of highest intensity for plotting. Error bars are mean ± SEM for experiments performed in triplicate with three independent preparations of protein and peptides. In FIG. II, 1-15 correspond to peptides of SEQ ID NOs: 63-73, 118, and 74-76, respectively (see FIG. 1C). In FIG. 1 J, 16-27 correspond to peptides of SEQ ID NOs: 77-87 and 119, respectively (see FIG. ID).

[0053] FIG. IK: The influence of stapling at a particular amino acid position of the BCL-w BH4 peptide based on its helical structure, whereby an adverse “hit” is scored if a staple that begins or ends at a particular residue results in 25% or less recombinant IP3R1 D3 pull-down activity. Residues with 2-3 “hits” (Rl l, Y20, Y27), or 1 “hit” (A12, L13, A15, V18, G19, K21, R23, K25) are highlighted and shown as stick representations. Residues V14, D16, F17, L22, Q24, G26 and V28 show no “hits” so are most amenable to crosslinking in an (z, z+7) configuration.

[0054] FIGs. 2A-2H: Molecular Dynamics Simulation of the Interaction between IP3R1 D3 and the BH4 Domain of BCL-w.

[0055] FIG. 2A: Sample conformer of the BCL-w BH4 domain based on a molecular dynamics simulation of amino acid residues 11-28.

[0056] FIG. 2B: Clustering of coarse-grained simulation trajectories that capture binding of the BCL-w BH4 peptide to the ARM2 region of IP3R1 (amino acids 1200-1566).

[0057] FIG. 2C: A low energy conformer of the BCL-w BH4 helix at a surface groove of the IP3R1 ARM2 domain.

[0058] FIGs. 2D-2E: Contact probabilities of BCL-w BH4 (FIG. 2D) and IP3R1 ARM2 domain (FIG. 2E) residues. Data are aggregated over 80 ns of an all-atom, equilibrium, molecular dynamics simulation and aggregated for three different trajectories.

[0059] FIG. 2F: Hydrophobic interactions at the BCL-w BH4 / IP3R1 helix-in-groove interface.

[0060] FIG. 2G: Hydrophilic and electrostatic interactions at the BCL-w BH4 / IP3R1 helix-in-groove interface.

[0061] FIG. 2H: Relative location of the BCL-w BH4 binding site at a surface groove of the ARM2 domain, within the cytosolic portion of IP3R1. FIG. 3A-3G: Point Mutagenesis Reveals Key Binding Determinants for BCL-w BH4 / IP3 R 1 - ARM2 Interact! on .

[0062] FIGs. 3A-3B: Sequence compositions of the alanine scanning libraries of the i, i+4 stapled SAH-BCL-w BH4-12 (FIG. 3 A) and i, i+ 7 SAH-BCL-w BH4-27 (FIG. 3B) peptides. Stapling amino acids S-pentenyl alanine and R-octenyl alanine are labeled X and 8, respectively. The sequences in FIG. 3A are from top to bottom SEQ ID NOs: 118 (labeled 12) and 88-101 (labeled with the alanine substitution), respectively. The sequences in FIG. 3B are from top to bottom SEQ ID NOs: 119 (labeled 27) and 102-116 (labeled with the alanine substitution).

[0063] FIGs. 3C-3D: Recombinant GST-IP3R D3 protein was incubated with biotinylated and z, i+4 (FIG. 3C) or / , i+7 (FIG. 3D) stapled BCL-w BH4 peptides followed by streptavidin pull-down, gel electrophoresis, and anti-GST western blot. Band intensities were quantified by ImageJ analysis and normalized to the band of highest intensity for plotting. Error bars are mean ± SEM for experiments performed in triplicate with three independent preparations of protein and peptides. FIG. 3C from left to right corresponds to peptides of SEQ ID NOs: 118 and 88-101, respectively (see FIG. 3A). FIG. 3D from left to right corresponds to peptides of SEQ ID NOs: 119 and 102-116, respectively (see FIG. 3B).

[0064] FIG. 3E: The influence of alanine mutagenesis at a particular amino acid position of the BCL-w BH4 peptide is depicted based on its helical structure. Amino acids at or below a 25% binding threshold in one or both datasets (K21, R23, K25) or at or below a 50% binding threshold in one or both datasets (R11, Y27) are highlighted and shown as stick representations. The remaining amino acids, with above 50% binding activity across both datasets, are most amenable to alanine mutation.

[0065] FIG. 3F: Sequence compositions of BCL-w and BCL-2 BH4 peptides bearing the indicated staples and point mutations. The sequences in FIG. 3F from top to bottom correspond to SEQ ID NOs: 23, 1, 118, 74, 117, 121 and 120, respectively.

[0066] FIG. 3G: Cerebellar lysates were incubated with the indicated peptides followed by streptavidin pull-down, gel electrophoresis, and anti-IP3Rl western blot. Band intensities were quantified by ImageJ analysis and normalized to the band of highest intensity for plotting. Error bars are mean ± SEM for experiments performed in triplicate with three independent preparations of protein and peptides. Statistical significance was calculated by one-way ANOVA with Bonferroni’s multiple comparisons test, where p is *** < o.OOl. FIG. 3G from left to right corresponds to peptides of SEQ ID NOs: 118, 74, 117, 121 and 120, respectively (see FIG. 3F)

[0067] FIGs. 4A-4D: Sequence-specific BH4 Protection from Paclitaxel-induced Axonal Degeneration.

[0068] FIG. 4A: Quantitation of axonal degeneration in response to paclitaxel (30 nM, 24 h), with or without 2 h pretreatment with the indicated SAH-BCL-w BH4 peptides (10 nM). The axonal degeneration index reflects the ratio of the area of fragmented axons to total axon area of E15 DRG neurons grown in compartmentalized cultures. Data are mean ± SEM for five independent experiments (27-29 images each) with individual data points plotted. Statistical significance was calculated by one-way ANOVA with Bonferroni’s multiple comparisons test, where p is *** < 0.001, or ns (not significant) for p > 0.05.

[0069] FIG. 4B: Binarized images of Tuj 1 immunostained axons treated in compartmentalized cultures as indicated. Scale bar, 20 mm.

[0070] FIG. 4C: Quantitation of axonal degeneration in response to paclitaxel (30 nM, 24 h), with or without 2 h pretreatment with the indicated SAH-BCL-w or SAH-BCL-2 BH4 peptides (10 nM). Data are mean ± SEM for four independent experiments (16-18 images each) with individual data points plotted. Statistical significance was calculated by one-way ANOVA with Bonferroni’s multiple comparisons test, where p is ** < 0.01, *** < 0.001, or ns (not significant) for p > 0.05.

[0071] FIG. 4D: Binarized images of Tuj 1 immunostained axons treated in compartmentalized cultures as indicated. Scale bar, 20 mm.

[0072] DETAILED DESCRIPTION The present disclosure is based, at least in part, on the finding that certain positions (e.g., L at position 3, V at position 4, D at position 6, F at position 7, V at position 8, G at position 9, Y at position 10, Q at position 14, G at position 16, or V at position 18 of SEQ ID NO:21) of a stapled bcl-w BH4 domain peptide tolerate substitution without abrogating binding of the substituted stapled BCL-w BH4 domain peptide to inositol 1,4, 5 -trisphosphate receptor, type 1 (IP3R1). The present disclosure is also based, at least in part, on the finding substitution of certain positions (e.g., R at position 1, Y at position 10, K at position 11, R at position 13, K at position 15, and Y at position 17 of SEQ ID NO:21) of a stapled bcl-w BH4 domain peptide abrogate binding of the substituted stapled bcl-w BH4 domain peptide to IP3R1. Surprisingly, the present disclosure is also based, at least in part, on the finding that the binding profile of a BCL-2 BH4 domain peptide can be modified to gain the ability to bind IP3R1 by substituting S at position 12 of SEQ ID NO: 1 (a bcl-2 BH4 domain peptide) with a positive amino acid (e.g., His, Lys, Arg, or Om (ornithine)).

[0073] Thus, provided herein are stapled peptides (e.g., stapled mutant BCL-2 peptides and stapled mutant BCL-w peptides) and mutant BCL-2 peptides. The stapled peptides and mutant BCL-2 peptides provided herein are useful in selectively protecting sensory neurons from age-related and / or chemotherapy induced axonal degeneration in a human subject in need thereof, treating or preventing CIPN in a human subject in need thereof, averting neuropathic side effects of chemotherapy and / or other causes of axonal degeneration in a human subject in need thereof, and treating or preventing hearing loss in a human subject in need thereof. The methods involve administering to the human subject a stapled peptide or a mutant BCL-2 peptide described herein. Non-limiting aspects and embodiments of these methods are described herein. Any of the aspects described below can be used in any combination in the methods described herein. MUTANT BCL-2 PEPTIDES

[0074] All proteins belonging to the Bcl-2 family contain either a BH1, BH2, BH3, or BH4 domain. The BCL-2 family proteins are classified as multidomain anti-apoptotic, multidomain pro-apoptotic, or BH3-only pro-apoptotic proteins. All anti-apoptotic proteins contain BH1 and BH2 domains, and some of them contain an additional N- terminal BH4 domain (e.g., BCL-2, BCL-XL, BCL-W), which is not seen in pro-apoptotic proteins, except for BCL-Xs. All pro-apoptotic proteins, except for BAD, and some anti- apoptotic proteins, such as BCL-2 or BCL-XL, contain a BH3 domain that is necessary for dimerization with other proteins of BCL-2 family and crucial for their killing activity; some of them also contain BH1 and BH2 domains (e.g., BAX, BAK).

[0075] The BH4 domain of BCL-2 has the amino acid sequence: EIVMKYIHYKLSQRGYEWDA (SEQ ID NO: 1). The BH4 domain of bcl-w has the amino acid sequence: RALVADFVGYKLRQKGYV (SEQ ID NO:23). An alignment of the BH4 domain of BCL-2 and BCL-w is depicted in FIG. IF.

[0076] Provided herein are BCL-2 mutant peptides. In some instances, the BCL-2 mutant peptide comprises the amino acid sequence EIVMKYIHYKLSQRGYEWDA (SEQ ID NO: 1) with S at position 12 substituted by a positively charged amino acid (e.g., His, Lys, Arg, or Orn). In some instances, the S at position 12 of SEQ ID NO: 1 is substituted with Arg. In some instances, the BCL-2 mutant peptide binds inositol 1,4,5-trisphosphate receptor, type 1 (IP3R1). In some instances, the BCL-2 mutant peptide binds BAX. In some instances, the BCL-2 mutant peptide binds IP3R1 and BAX. Methods for determining binding of a BCL-2 mutant peptide described herein to IP3R1 or BAX are known in the art and described in the working examples herein. In some instances, the BCL-2 mutant peptide is 20 to 50, 20 to 40, 20 to 30, 20 to 25, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, less than 50, less than 40, less than 30, or less than 25 amino acids in length.

[0077] In some instances, the BCL-2 mutant peptide further includes 1 to 4 (e.g., 1, 2, 3, 4) other amino acid substitutions within SEQ ID NO:1. In some instances, the 1 to 4 other amino acid substitutions are at any one of positions 1, 4, 5, 8, and 17-20 of SEQ ID NO: 1. In some instances, the 1 to 4 other amino acid substitutions are with conservative amino acids, non-conservative amino acid substitutions, or a combination thereof. In some instances, the 1 to 4 other amino acid substitutions are not at one or more of positions 2, 3, 6, 7, 9, 10, 1 1, 13, 14, 15, and 16 of SEQ ID NO: 1 . In some instances, the 1 to 4 other amino acid substitutions are not at one or more of positions 10, 14, and 16 of SEQ ID NO: 1. In some instances, the 1 to 4 amino acid substitutions are at any of positions 2, 3, 6, 7, 9, 10, 11, 13, 14, 15, and 16 of SEQ ID NO: 1 with conservative amino acid substitutions. In some instances, the 1 to 4 amino acid substitutions are at one or more of positions 10, 14, and 16 of SEQ ID NO: 1 with an amino acid selected from K, R, Y, F, and W. In some instances, the 1 to 4 amino acid substitutions are at one or more of positions 10, 14, and 16 of SEQ ID NO: 1 with an amino acid selected from K and R. In some instances, the 1 to 4 amino acid substitutions are at one or more of positions 10, 14, and 16 of SEQ ID NO: 1 with an amino acid selected from Y, F, and W. In some instances, (i) Leu at position 11 and Gly at position 15, (ii) Gin at position 13 and Glu at position 17, or (iii) Leu at position 11 and Trp at position 18 of SEQ ID NO: 1 are each substituted with a,a-disubstituted non-natural amino acids, e.g., a, a-di substituted nonnatural amino acids comprising olefinic side chains that are capable of being cross-linked to each other, e.g., to form a hydrocarbon staple. In some instances, Gin at position 13 and Glu at position 17 of SEQ ID NO: 1 are each substituted with a,a-disubstituted nonnatural amino acids, e.g., a, a-di substituted non-natural amino acids comprising olefinic side chains that are capable of being cross-linked to each other, e.g., to form a hydrocarbon staple.

[0078] In some instances, a conservative amino acid substitution is an amino acid substitution that does not reduce (e.g., substantially reduce) binding of the peptide or stapled peptide to its target protein (e.g., IP3R1 or BAX), and can, in some circumstances, improve binding activity. Methods for detecting any reduction in binding can include comparing binding affinity following conservative amino acid substitution, wherein any amino acid substitution that reduces (e.g., substantially reduces) binding are not conservative amino acid substitutions. In some aspects, substantially reduced binding can include binding that is 10% or less, 20% or less, 30% or less, 40% or less, 50% or less, 60% or less, 70% or less, 80% or less, 90% or less, 95% or less, 98% or less, 99% or less, or 100% less than binding of the unsubstituted peptide or stapled peptide to its target protein (e.g., IP3R1 or BAX). Methods for assessing interaction between a stapled peptide or unstapled peptide and a target protein (e.g., IP3R1 or BAX) are disclosed herein.

[0079] In some instances, a conservative amino acid substitution is a substitution in which one amino acid residue is replaced with another 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 positively charged side chains (e.g., histidine, lysine, arginine, and ornithine), basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan), beta-branched side chains (e.g., threonine, valine, and isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine).

[0080] In some instances, the BCL-2 mutant peptide has at least 80%, at least 85%, at least 90%, at least 95% or 100% sequence identity to the sequence of SEQ ID NO: 1, wherein the BCL-2 mutant peptide comprises a substitution at position S12 of SEQ ID NO: 1 with a positively charged amino acid (e.g., His, Lys, Arg, or Orn). In some instances, (i) Leu at position 11 and Gly at position 15, (ii) Gin at position 13 and Glu at position 17, or (iii) Leu at position 1 1 and Trp at position 18 of SEQ ID NO: 1 are each substituted with a,a-disubstituted non-natural amino acids, e.g., a, a-di substituted nonnatural amino acids comprising olefinic side chains that are capable of being cross-linked to each other, e.g., to form a hydrocarbon staple.

[0081] In some instances, the BCL-2 mutant peptide includes an Arg immediately upstream of the N-terminal Glu at position 1 of SEQ ID NO: 1 (e.g., REIVMKYIHYKLSQRGYEWDA (SEQ ID NO:50), with S at position 12 substituted by a positively charged amino acid (e.g., His, Lys, Arg, or Om)).

[0082] In some instances, the BCL-2 mutant peptide comprises a sequence selected from the group consisting of:

[0083] EIVMKYIHYKLRQRGYEWDA (SEQ ID NO: 2), EIVBKYIHYKLRQRGYEWDA (SEQ ID NO: 3), REIVMKYIHYKLRQRGYEWDA (SEQ ID NO:4), and REIVBKYIHYKLRQRGYEWDA (SEQ ID NO: 5), with 2 to 4 amino acid substitutions, wherein B is norleucine; wherein none of the 2 to 4 amino acid substitutions replace amino acids at positions 10, 12, 14, or 16 of SEQ ID NO:2 or 3, or positions 11, 13, 15, or 17 of SEQ ID NO:4 or 5 or if positions 10, 12, 14, or 16 of SEQ ID N0:2 or 3, or positions 11, 13, 15, or 17 of SEQ ID N0:4 or 5 are substituted, it is with conservative amino acid substitutions; and optionally wherein 2 of the 2 to 4 amino acid substitutions replace amino acids three or six amino acids apart within any one of SEQ ID NOs:2-5 with a,a-disubstituted non-natural amino acids, e.g., a,a-disubstituted non-natural amino acids comprising olefinic side chains that are capable of being cross-linked to each other, e.g., to form a hydrocarbon staple. In some instances, none of the 2-4 amino acid substitutions are at positions 10, 12, 14, and 16 of SEQ ID NO:2 or 3; or positions 11, 13, 15, and 17 of SEQ ID NO: 4 or 5. In some instances, one or more of the 2-4 amino acid substitutions are at positions 10, 12, 14, and 16 of SEQ ID NO:2 or 3; or positions 11, 13, 15, and 17 of SEQ ID NO: 4 or 5 with conservative amino acid substitutions. In some instances, the 2 to 4 amino acid substitutions are at any of positions 1 , 4, 5, 8, and 17-20 of SEQ ID NO:2 or 3 or at any of positions 2, 5, 6, 9, 13, and 18-21 of SEQ ID NO:4 or 5. In some instances, the 2 to 4 amino acid substitutions are with conservative amino acids, non-conservative amino acid substitutions, or a combination thereof. In some instances, the 1 to 4 other amino acid substitutions are not at one or more of positions 2, 3, 6, 7, 9, 10, 11, 13, 14, 15, and 16 of SEQ ID NO:2 or 3 or are not at one or more of positions 3, 4, 7, 8, 10, 11, 14, 15, 16, and 17 of SEQ ID NO:4 or 5. In some instances, the 2 to 4 amino acid substitutions are not at one or more of positions 10, 14, and 16 of SEQ ID NO:2 or 3 or are not at position 11, 15, and 17 of SEQ ID NO:4 or 5. In some instances, the 2 to 4 amino acid substitutions are at any of positions 2, 3, 6, 7, 9, 10, 11, 13, 14, 15, and 16 of SEQ ID NO:2 or 3 conservative amino acid substitutions or at any of positions 3, 4, 7, 8, 10, 1 1, 14, 15, 16, and 17 of SEQ ID NO:4 or 5 conservative amino acid substitutions. In some instances, the 2 to 4 amino acid substitutions are at one or more of positions 10, 14, and 16 of SEQ ID NO:2 or 3 or one or more of positions 11, 15, and 17 of SEQ ID NO:4 or 5 with an amino acid selected from K, R, Y, F, and W. In some instances, the 2 to 4 amino acid substitutions are at one or more of positions 10, 14, and 16 of SEQ ID NO:2 or 3 or one or more of positions 11, 15, and 17 of SEQ ID NO:4 or 5 with an amino acid selected from K and R. In some instances, the 2 to 4 amino acid substitutions are at one or more of positions 10, 14, and 16 of SEQ ID NO:2 or 3 or one or more of positions 11, 15, and 17 of SEQ ID NO:4 or 5 with an amino acid selected from Y, F, and W. In some instances, the BCL-2 mutant peptide binds inositol 1,4, 5 -trisphosphate receptor, type 1 (IP3R1). In some instances, the BCL-2 mutant peptide binds BAX. In some instances, the BCL-2 mutant peptide binds IP3R1 and BAX. In some instances, the BCL-2 mutant peptide is 20 to 50, 20 to 40, 20 to 30, 20 to 25, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, less than 50, less than 40, less than 30, or less than 25 amino acids in length.

[0084] STAPLED PEPTIDES

[0085] Also provided herein are stapled versions of the mutant BCL-2 peptides described herein.

[0086] “Peptide stapling” or “hydrocarbon stapling” is a term coined from a synthetic methodology, wherein two olefin-containing side-chains (e.g., cross-linkable side chains) present in a polypeptide chain are covalently joined (e.g., “stapled together”) using a ring-closing metathesis (RCM) reaction to form a cross-linked ring (see, e.g., Blackwell et al., J. Org. Chem., 66: 5291-5302, 2001; Angew et al., Chem. Int. Ed. 37:3281, 1994). As used herein, the term “peptide stapling” or “hydrocarbon stapling” includes the joining of two (e.g., at least one pair of) double bond-containing side-chains, triple bondcontaining side-chains, or double bond-containing and triple bond-containing side chain, which can be present in a polypeptide chain, using any number of reaction conditions and / or catalysts to facilitate such a reaction, to provide a singly “stapled” polypeptide. The term “multiply stapled” polypeptides refers to those polypeptides containing more than one individual staple, and can contain two, three, or more independent staples of various spacings and compositions. The term “peptide stitching,” as used herein, refers to multiple and tandem “stapling” events in a single polypeptide chain to provide a “stitched” (e.g, tandem or multiply stapled) polypeptide, in which two staples, e.g., are linked to a common residue. Peptide stitching is disclosed in US Patent Nos. 8,592,377 and 9,079,970, which are both hereby incorporated by reference in their entirety. In some instances, staples, as used herein, can retain the unsaturated bond or can be reduced (e.g., as mentioned below in the stitching paragraph description).

[0087] While many peptide staples have all hydrocarbon cross-links, other type of crosslinks or staples can be used. For example, triazole-containing (e g, 1, 4 triazole or 1, 5 triazole) crosslinks can be used (see, e.g., Kawamoto et al., J. Med. Chem. 55: 1137-1146, 2012; WO 2010 / 060112).

[0088] Stapling of a peptide using an all-hydrocarbon cross-link has been shown to help maintain its native conformation and / or secondary structure, particularly under physiologically relevant conditions (see, e.g., Schafmiester et al., J. Am. Chem. Soc. 122:5891-5892, 2000; Walensky et al., Science 305: 1466-1470, 2004).

[0089] Stapling the peptide herein by an all-hydrocarbon crosslink predisposed to have an alpha-helical secondary structure can constrain the peptide to its native alpha-helical conformation. The constrained secondary structure can, for example, increase the peptide’s resistance to proteolytic cleavage, can increase the peptide’s thermal stability, can increase the peptide’s hydrophobicity, can allow for better penetration of the peptide into the target cell’s membrane (e.g., through an energy-dependent transport mechanism, such as pinocytosis), and / or can lead to an improvement in the peptide’s biological activity relative to the corresponding uncross-linked (e.g., “unstitched” or “unstapled”) peptide.

[0090] Stapled peptides herein include at least two modified amino acids that together form an internal (intramolecular) cross-link (or staple), wherein the at least two modified amino acids are separated by 2 (i.e., i, i+3), 3 (i.e., i, i+4), or 6 (i.e., i, i+7) amino acids. Additional exemplary relative positions of staple(s) and / or stitch(es) that can be introduced in any of the stapled peptides described herein are known in the art. See, e.g., US 2016 / 0031959 incorporated by reference in its entirety herein.

[0091] In the case of a cross-link between i and i+3 the cross-link can be a C7 alkylene or alkenylene. In the case of a cross-link between and i+4 the cross-link can be a Cs alkylene or alkenylene. In the case of a cross-link between / and z+ 7 the cross-link can be a Ci 1, C12, or C13 alkylene or alkenylene. When the cross-link is an alkenylene, there can be one or more double bonds.

[0092] In the case of a cross-link between z and i+3 the cross-link can be a Ce, C7, or Cs alkyl or alkene e.g., a Ce alkene having a single double bond). In the case of a cross-link between i and i 1 4 the cross-link can be a Cs alkyl or alkene. In the case of a cross-link between i and i+ 7 the cross-link can be a Ci 1, C12, or C13 alkyl or alkene e.g, a C11 alkene having a single double bond). When the cross-link is an alkene, there can be one or more double bonds.

[0093] For stapled peptides where an i linked to i+7 staple is used (two turns of the helix stabilized), either one Ss amino acid ((S)-a-(4'-pentenyl)alanine) and one Rs amino acid ((R)-a-(7'-octenyl)alanine) can be used, or one Ss amino acid ((S)-a-(7'-octenyl)alanine)) and one Rs amino acid ((R)-a-(4'-pentenyl)alanine) can be used. For stapled peptides where an i linked to i+4 staple is used (one turn of the helix stabilized), either two S5 amino acids ((S)-a-(4'-pentenyl)alanine) can be used, or two Rs amino acids ((R)-a-(4'- pentenyl)alanine) can be used.

[0094] Internal cross-links (e.g., staples) can be positioned on amino acids within stapled peptide to conserve the structural relationship of amino acids in the binding or the interacting face of the peptide (e.g., to preserve the binding interface of a peptide). Alternatively, staples can placed on the interacting face as long as binding affinity or activity is not altered. The “interacting face” of the stapled peptides described herein includes those amino acid residues of the alpha helix that interact (e. ., interact specifically or bind specifically) with IP3R1 or BAX. In some aspects, the staple can be placed such that they partially or completely engage the target and enhance binding activity. For example, staples can be placed to conserve the structural relationship of amino acids in an interaction face of the stapled peptide. Such internal cross-links can include: one or more staples; one or more stitches; and / or a combination of one or more staples with one or more stitches.

[0095] Selection of amino acids for modification (e.g., to support a staple) can also be facilitated by staple scanning. The term “staple scan” refers to the synthesis of a library of stapled peptides whereby the location of the i and z+3; i and z-4; and z and z+ 7 single and multiple staple, or stitches, are positioned sequentially down the length of the peptide sequence, sampling all possible positions, to identify desired or optimal properties and activities for the stapled or stitched constructs.

[0096] Suitable tethers are described herein and in U.S. Patent Application Publication No. 2005 / 0250680, U.S. Patent No. 8,592,377, U.S. Patent Application Publication No. 2011 / 0318352, WO 2009 / 108261, and WO 2010 / 148335, each of which are herein incorporated by reference in their entireties.

[0097] Amino acid side chains suitable for use in the stapled peptides disclosed herein are known in the art. For example, suitable amino acid side chains include methyl (as the alpha- amino acid side chain for alanine is methyl), 4-hydroxyphenylmethyl (as the alpha-amino acid side chain for tyrosine is 4-hydroxyphenylmethyl) and thiomethyl (as the alpha-amino acid side chain for cysteine is thiomethyl), etc. A “terminally unsaturated amino acid side chain” refers to an amino acid side chain bearing a terminal unsaturated moiety, such as a substituted or unsubstituted, double bond (e.g., olefinic) or triple bond (e.g., acetylenic), that participates in cross-linking reaction with other terminal unsaturated moieties in the polypeptide chain. In certain aspects, a “terminally unsaturated amino acid side chain” is a terminal olefinic amino acid side chain. In certain aspects, a “terminally unsaturated amino acid side chain” is a terminal acetylenic amino acid side chain. In certain aspects, the terminal moiety of a “terminally unsaturated amino acid side chain” is not further substituted.

[0098] As noted above, an internal tether or cross-link can extend across the length of one helical turn (z.e., about 3.4 amino acids (i.e., i, i+3, or z, i+4) or two helical turns (z.e., about 7 amino acids (i.e., i, i+7). Accordingly, amino acids positioned at z and z+3; z and z+ ; or z and z+ 7 of SEQ ID NO:2 or 3 are ideal candidates for chemical modification and cross-linking. Thus, e.g., where a peptide has the sequence . ..Xaai, Xaa2, Xaaa, Xaa4, Xaas, Xaae, Xaaz, Xaas, Xaav .. (wherein . .” indicates the optional presence of additional amino acids), cross-links between Xaai and Xaa4, or between Xaai and Xaas, or between Xaai and Xaas are useful, as are cross-links between Xaa2 and Xaas, or between Xaa2 and Xaae, or between Xaa2 and Xaa9, etc.

[0099] Provided herein are BCL-2 stapled peptides. In some instances, the stapled peptide comprises the amino acid sequence EIVMKYIHYKX1SQRX2YEWDA (SEQ ID NO:6), EIVMKYIHYKLSX1RGYX2WDA (SEQ ID NO:7), or EIVMKYIHYK8SQRGYEXDA (SEQ ID NO: 8) with S at position 12 of any one of SEQ ID NOs: 6-8 substituted by a positively charged amino acid (e.g., His, Lys, Arg, and Orn), wherein Xi, X2, 8, and X are a,a-disubstituted non-natural amino acids, and wherein the side chains of Xi and X2 or 8 and X are cross linked. In some instances, the S at position 12 of any one of SEQ ID NOs: 6-8 is substituted with Arg. In some instances, the a,a- disubstituted non-natural amino acids comprise olefinic side chains that are capable of being cross-linked to each other, e.g., to form a hydrocarbon staple. In some instances, Xi and X2 are each (S)-2-(4-pentenyl)alanine. In some instances, 8 is (R)-a-(7 - octenyl)alanine and X is (S)-2-(4-pentenyl)alanine. In some instances, the stapled peptide binds IP3R1. In some instances, the stapled peptide binds BAX. In some instances, the stapled peptide binds IP3R1 and BAX. In some instances, the stapled peptide is 20 to 50, 20 to 40, 20 to 30, 20 to 25, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, less than 50, less than 40, less than 30, or less than 25 amino acids in length.

[0100] In some instances, the stapled peptide further includes 1 or 2 other amino acid substitutions within any one of SEQ ID NOs:6-8, wherein the 1 or 2 other substitutions are not at Xi or X2 of SEQ ID NOs: 6 and 7 and are not at 8 or X of SEQ ID NO: 8. In some instances, M at position 4 of any one of SEQ ID NOs:6-8 is substituted with norleucine. In some instances, the 1 or 2 other amino acid substitutions are at any one of positions 1, 4, 5, 8, and 17-20 of SEQ ID NOs:6-8. In some instances, the 1 or 2 other amino acid substitutions are with conservative amino acids, non-conservative amino acid substitutions, or a combination thereof. In some instances, the 1 or 2 other amino acid substitutions are not at one or more of positions 2, 3, 6, 7, 9, 10, 11, 13, 15, and 16 of SEQ ID NOs:6-8. In some instances, the 1 or 2 other amino acid substitutions are not at positions 10, 14, and 16 of SEQ ID NOs:6-8. In some instances, the 1 or 2 amino acid substitutions are at any of positions 2, 3, 6, 7, 9, 10, 11, 13, 14, 15, and 16 of SEQ ID NOs:6-8 with conservative amino acid substitutions. In some instances, the 1 or 2 other amino acid substitutions are at one or more of positions 2, 3, 6, 7, 8, 10, 14, and 16 of SEQ ID NOs: 6-8 with conservative amino acid substitutions. In some instances, the 1 or 2 other amino acid substitutions are at one or more of positions 10, 14, and 16 of SEQ ID NOs:6-8 with conservative amino acid substitutions. In some instances, the 1 or 2 other amino acid substitutions are at one or more of positions 10, 14, and 16 of SEQ ID NOs:6- 8 with an amino acid selected from K, R, Y, F, and W. In some instances, the 1 or 2 other amino acid substitutions are at one or more of positions 10, 14, and 16 of SEQ ID NOs:6- 8 with an amino acid selected from K and R. In some instances, the 1 or 2 other amino acid substitutions are at one or more of positions 10, 14, and 16 of SEQ ID NOs:6-8 with an amino acid selected from Y, F, and W.

[0101] In some instances, the stapled peptide includes an Arg immediately upstream of the N-terminal Glu at position 1 of SEQ ID NOs:6-8 (e.g., EIVMKYIHYKX1SQRX2YEWDA (SEQ ID NO:60), EIVMKYIHYKLSXiRGYX2WDA (SEQ ID N0:61), or EIVMKYIHYK8SQRGYEXDA (SEQ ID NO:62) with S at position 12 of any one of SEQ ID NOs: 60-62 substituted by a positively charged amino acid (e.g., His, Lys, Arg, and Orn)).

[0102] In some instances, the stapled peptide comprises a sequence selected from the group consisting of:

[0103] EIVMKYIHYKX1RQRX2YEWDA (SEQ ID NO: 9), EIVBKYIHYKX1RQRX2YEWDA (SEQ ID NO: 10), EIVMKYIHYKLRX1RGYX2WDA (SEQ ID NO: 11), EIVBKYIHYKLRX1RGYX2WDA (SEQ ID NO: 12), EIVMKYIHYK8RQRGYEXDA (SEQ ID NO: 13), EIVBKYIHYK8RQRGYEXDA (SEQ ID NO: 14), REIVMKYIHYKX1RQRX2YEWDA (SEQ ID NO: 15), REIVBKYIHYKX1RQRX2YEWDA (SEQ ID NO: 16), REIVMKYIHYKLRX1RGYX2WDA (SEQ ID NO: 17), REIVBKYIHYKLRX1RGYX2WDA (SEQ ID NO: 18), REIVMKYIHYK8RQRGYEXDA (SEQ ID NO: 19), and REIVBKYIHYK8RQRGYEXDA (SEQ ID NO:20), with 0 to 2 amino acid substitutions relative to SEQ ID NOs:9, 11, 13, 15, 17, and 19, or 0 or 1 amino acid substitution relative to SEQ ID NOs:10, 12, 14, 16, 18, and 20, wherein B is norleucine, wherein Xi, X2, 8, and X are a,a-disubstituted non-natural amino acids, and wherein the side chains of Xi and X2 or 8 and X are cross linked, and wherein the amino acid substitutions are not at any of Xi, X2, 8 or X. In some instances, the amino acid substitution(s) are at any one of positions 1, 4, 5, 8, and 17-20 of SEQ ID NOs:9-14 or at any one of positions 2, 5, 6, 9, and 18-21 of SEQ ID NOs: 15-20. In some instances, the amino acid substitution(s) are with conservative amino acids, non-conservative amino acid substitutions, or a combination thereof. In some instances, the amino acid substitution(s) are not at one or more of positions 2, 3, 6, 7, 9, 10, 11, 13, 15, and 16 of SEQ ID NOs: 9- 14 or are not at one or more of positions 3, 4, 7, 8, 10, 11, 12, 14, 16, and 17 of SEQ ID NOs: 15-20. In some instances, the amino acid substitution(s) are not at positions 10, 14, or 16 of SEQ ID NOs:9-14 or are not at positions 11, 15, or 17 of SEQ ID NOs: 15-20. In some instances, the amino acid substitution(s) are at any of positions 2, 3, 6, 7, 9, 10, 11, 13, 14, 15, and 16 of SEQ ID NOs:9-14 with conservative amino acid substitutions or are at any of positions 3, 4, 7, 8, 10, 11, 12, 14, 15, 16, and 17 of SEQ ID NOs: 15-20 with conservative amino acid substitutions. In some instances, the amino acid substitution(s) are at one or more of positions 10, 14, and 16 of SEQ ID NOs:9-14 or at one or more of positions 11, 15, and 17 of SEQ ID NOs:15-20 with an amino acid selected from K, R, Y, F, and W. In some instances, the amino acid substitution(s) are at one or more of positions 10, 14, and 16 of SEQ ID NOs:9-14 or at one or more of positions 11, 15, and 17 of SEQ ID NOs: 15-20 with an amino acid selected from K and R. In some instances, the amino acid substitution(s) are at one or more of positions 10, 14, and 16 of SEQ ID NOs:9-14 or at one or more of positions 11, 15, and 17 of SEQ ID NOs: 15-20 with an amino acid selected from Y, F, and W. In some instances, the a,a- disubstituted non-natural amino acids comprise olefinic side chains that are cross-linked to each other, e.g., to form a hydrocarbon staple. In some instances, Xi and X2 are each (S)-2-(4-pentenyl)alanine. In some instances, 8 is (R)-a-(7'-octenyl)alanine and X is (S)- 2-(4-pentenyl)alanine. In some instances, Xi and X2 are each (S)-2-(4-pentenyl)alanine. In some instances, 8 is (R)-a-(7'-octenyl)alanine and X is (S)-2-(4-pentenyl)alanine. In some instances, the stapled peptide binds inositol 1,4,5-trisphosphate receptor, type 1 (IP3R1). In some instances, the stapled peptide binds BAX. In some instances, the stapled peptide binds LP3R1 and BAX. In some instances, the stapled peptide is 20 to 50, 20 to 40, 20 to 30, 20 to 25, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, less than 50, less than 40, less than 30, or less than 25 amino acids in length.

[0104] Also provided herein are stapled peptides based on the BH4 domain of BCL-w.

[0105] In some instances, the BCL-w stapled peptide comprises the amino acid sequence RALVADFVGYKX1RQKX2YV (SEQ ID NO:21) with 1 to 3 amino acid substitutions, wherein Xi and X2 are a,a-disubstituted non-natural amino acids, wherein the side chains of Xi and X2 are cross linked, and wherein positions 12 and 16 of SEQ ID NO:21 are not substituted. In some instances, the a,a-disubstituted non-natural amino acids comprise olefinic side chains that are cross-linked to each other, e.g., to form a hydrocarbon staple. In some instances, Xi and X2 are each (S)-2-(4-pentenyl)alanine. In some instances, one or more of L at position 3, V at position 4, D at position 6, F at position 7, V at position 8, G at position 9, Y at position 10, Q at position 14, G at position 16, Y at position 17, or V at position 18 of SEQ ID NO:21 are substituted with an alanine, glycine, valine, or leucine. In some instances, one of L at position 3, V at position 4, D at position 6, F at position 7, V at position 8, G at position 9, Y at position 10, Q at position 14, G at position 16, or V at position 18 of SEQ ID NO:21 is substituted with an alanine, glycine, valine, or leucine. In some instances, one or more of R at position 1, Y at position 10, K at position 11, R at position 13, K at position 15, and Y at position 17 of SEQ ID NO:21 are not substituted. In some instances, one or more of R at position 1, K at position 11, R at position 13, or K at position 15 of SEQ ID NO:21 is substituted with a positively charged amino acid (e.g., His, Lys, Arg, or Orn), and / or Y at position 10 or Y at position 17 of SEQ ID NO:21 is substituted with a hydrophobic, particularly aromatic amino acid (e.g., D, E, H, K, N, Orn, Q, R or Y). In some instances, the stapled peptide binds IP3R1. In some instances, the stapled peptide binds IP3R1 and BAX. In some instances, the stapled peptide binds BAX, BAK, or a combination thereof. In some instances, the stapled peptide binds IP3R1, BAX, BAK, or a combination thereof. In some instances, the stapled peptide is 18 to 50, 18 to 40, 18 to 30, 18 to 25, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, less than 50, less than 40, less than 30, less than 25, or less than 20 amino acids in length.

[0106] In some instances, the BCL-w stapled peptide comprises the amino acid sequence RALVADFVGYK8RQKGYVX (SEQ ID NO:22) with 1 or 2 amino acid substitutions, wherein 8 and X are a, a-di substituted non-natural amino acids, wherein the side chains of 8 and X are cross linked, and wherein positions 12 and 19 of SEQ ID NO:22 are not substituted. In some instances, the a,a-disubstituted non-natural amino acids comprise olefinic side chains that are cross-linked to each other, e.g., to form a hydrocarbon staple. In some instances, 8 and X of SEQ ID NO:22 are (R)-a-(7'-octenyl)alanine and (S)-a-(4'- pentenyl)alanine, respectively. In some instances, one or more of D at position 6 and G at position 9 of SEQ ID NO:22 are substituted with an alanine, glycine, valine, or leucine. In some instances, one or more of R at position 1, Y at position 10, K at position 11, R at position 13, K at position 15, and Y at position 17 of SEQ ID NO:22 are not substituted. In some instances, one or more of R at position 1, K at position 11, R at position 13, or K at position 15 of SEQ ID NO:22 is substituted with a positively charged amino acid (e.g., His, Lys, Arg, or Om), and / or Y at position 10 or Y at position 17 of SEQ ID NO:22 is substituted with a hydrophobic, particularly aromatic amino acid (e g., D, E, H, K, N, Orn, Q, R or Y)). In some instances, the stapled peptide binds IP3R1. In some instances, the stapled peptide binds IP3R1 and BAX. In some instances, the stapled peptide binds BAX, BAK, or a combination thereof. In some instances, the stapled peptide binds IP3R1, BAX, BAK, or a combination thereof. In some instances, the stapled peptide is 19 to 50, 19 to 40, 19 to 30, 19 to 25, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, less than 50, less than 40, less than 30, or less than 25 amino acids in length.

[0107] In some instances, the foregoing stapled peptides comprise the structure of Formula (I), Formula (I) or a pharmaceutically acceptable salt thereof, wherein; each Ri and R2 are independently H or a Ci to C10 alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;

[0108] R3 is alkylene, alkenylene, or alkynylene (e.g., a Ce, C7, Cs, C11, C12, or C13 alkylene), or [R4 -K-R4]n; each of which is substituted with 0-6 Rs; R.4 and R.4’are independently alkylene, alkenylene, or alkynylene (e. , each are independently a Ci, C2, C3, C4, C5, C6, C7, Cs, C9, or C10 alkylene, alkenylene or alkynylene);

[0109] Rs is halo, alkyl, ORe, N(Re)2, SRe, SORe, SO2R6, CO2R6, Re, a fluorescent moiety, or a radioisotope;

[0110] O

[0111] K is O, S, SO, SO2, CO, CO2, CONRe, , aziridine, episulfide, diol, or amino alcohol;

[0112] Re is H, alkyl, or a therapeutic agent; n is 2, 3, 4, or 6; x is an integer from 2-10; w and y are each independently an integer from 0-100; z is an integer from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10); and each Xaa is independently an amino acid (e.g., one of the 20 naturally occurring amino acids or any naturally occurring non-naturally occurring amino acid).

[0113] In the case of Formula I, the following aspects are among those disclosed.

[0114] In cases where x = 2 (i.e., i+3 linkage), R3 can be a C7 alkylene or alkenylene.

[0115] Where it is an alkenylene, there can one or more double bonds. In cases where x = 6 (i.e., i+4 linkage), R3 can be a C11, C12, or C13 alkylene or alkenylene. Where it is an alkenylene, there can one or more double bonds. In cases where x = 3 (i.e., i+4 linkage), R3 can be a Cs alkylene or alkenylene. Where it is an alkenylene, there can one or more double bonds.

[0116] In certain instances, the two alpha, alpha-disubstituted stereocenters (alpha carbons) are both in the R configuration or S configuration (e.g., i, i+4 cross-link), or one stereocenter is R and the other is S (e.g., i, i+ 7 cross-link). Thus, where Formula I is depicted as the C’ and C” disubstituted stereocenters can both be in the R configuration or they can both be in the S configuration, for example, when x is 3. When x is 6, the C’ disubstituted stereocenter is in the R configuration and the C” disubstituted stereocenter is in the S configuration or the C’ disubstituted stereocenter is in the S configuration and the C” disubstituted stereocenter is in the R configuration. The Rs double bond can be in the E or Z stereochemical configuration.

[0117] In some instances Rs is [R4-K-R4’]n; and R4 and R4’are independently alkylene, alkenyl ene, or alkynylene (e.g., each are independently a Ci, C2, C3, C4, Cs, Ce, C7, Cs, C9, or C10 alkylene, alkenylene or alkynylene).

[0118] The tether can include an alkyl, alkenyl, or alkynyl moiety (e.g., Ce, Cs, or C11 alkyl, a Ce, Cs, or C11 alkenyl, or C5, Cs, or C11 alkynyl).

[0119] The stapled peptides can contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers, and diastereomeric mixtures and geometric isomers (e.g., Z or cis and E or trans) of any olefins present. For example, stapled peptides disclosed herein can exist in particular geometric or stereoisomeric forms, including, for example, cis- and trans-i somers, R- and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemic mixtures thereof, and other mixtures thereof. Enantiomers can be free (e.g., substantially free) of their corresponding enantiomer, and / or can also be optically enriched. “Optically enriched,” as used herein, means that the compound is made up of a significantly greater proportion of one enantiomer. In certain aspects substantially free means that a composition contains at least about 90% by weight of a preferred enantiomer. In other aspects the compound is made up of at least about 95%, 98%, or 99% by weight of a preferred enantiomer. Preferred enantiomers can be isolated from racemic mixtures using techniques known in the art, including, but not limited to, for example, chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts or prepared by asymmetric syntheses (see, e.g., Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen, S. H. et al., Tetrahedron 33:2725 (1977); Eliel, EX, Stereochemistry of Carbon Compounds (McGraw- Hill, NY, 1962); Wilen, S.H., Tables of Resolving Agents and Optical Resolutions p. 268 (EX. Eliel, Ed., Univ, of Notre Dame Press, Notre Dame, IN, 1972). All such isomeric forms of these stapled peptides are expressly included in the present invention.

[0120] The stapled peptides can also be represented in multiple tautomeric forms, in such instances, the invention expressly includes all tautomeric forms of the compounds described herein (e.g., isomers in equilibrium (e.g., keto-enol), wherein alkylation at multiple sites can yield regioisomers), regioisomers, and oxidation products of the stapled peptides disclosed herein (the invention expressly includes all such reaction products). All such isomeric forms of such stapled peptides are included, as are all crystal forms.

[0121] The symbol “ ” when used as part of a molecular structure, refers to a single bond or a trans or cis double bond.

[0122] The term “halo” refers to any radical of fluorine, chlorine, bromine, or iodine. The term “alkyl” refers to a hydrocarbon chain that can be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, Ci-Cio indicates that the group can have from 1 to 10 (inclusive) carbon atoms in it. In the absence of any numerical designation, “alkyl” is a chain (straight or branched) having 1 to 20 (inclusive) carbon atoms in it. The term “alkylene” refers to a divalent alkyl (i.e., -R-).

[0123] The term “alkenyl” refers to a hydrocarbon chain that can be a straight chain or branched chain having one or more carbon-carbon double bonds in either Z or E geometric configurations. The alkenyl moiety contains the indicated number of carbon atoms. For example, C2-C10 indicates that the group can have from 2 to 10 (inclusive) carbon atoms in it. The term “lower alkenyl” refers to a C2-C8 alkenyl chain. In the absence of any numerical designation, “alkenyl” is a chain (straight or branched) having 2 to 20 (inclusive) carbon atoms in it. The term “alkynyl” refers to a hydrocarbon chain that can be a straight chain or branched chain having one or more carbon-carbon triple bonds. The alkynyl moiety contains the indicated number of carbon atoms. For example, C2-C10 indicates that the group can have from 2 to 10 (inclusive) carbon atoms in it. The term “lower alkynyl” refers to a C2-C8 alkynyl chain. In the absence of any numerical designation, “alkynyl” is a chain (straight or branched) having 2 to 20 (inclusive) carbon atoms in it.

[0124] The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclic aromatic ring system wherein 0, 1, 2, 3, 4, or 5 atoms of each ring can be substituted by a substituent. Examples of aryl groups include phenyl, naphthyl, and the like. The term “arylalkyl” or the term “aralkyl” refers to alkyl substituted with an aryl. The term “arylalkoxy” refers to an alkoxy substituted with aryl.

[0125] The term “cycloalkyl” as employed herein includes saturated and partially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons, preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, wherein the cycloalkyl group additionally can be optionally substituted. Preferred cycloalkyl groups include, without limitation, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptadienyl, cycloheptatrienyl, cyclooctyl, cyclooctenyl, cyclooctadienyl, cyclooctatrienyl, and cyclooctynyl.

[0126] The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3, or 4 atoms of each ring can be substituted by a substituent. Examples of heteroaryl groups include pyrrolyl, pyridyl, furyl or furanyl, imidazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, benzimidazolyl, pyridazyl, pyrimidyl, thiophenyl, quinolinyl, indolyl, thiazolyl, oxazolyl, isoxazolyl, and the like. The term “heteroarylalkyl” or the term “heteroaralkyl” refers to an alkyl substituted with a heteroaryl. The term “heteroarylalkoxy” refers to an alkoxy substituted with heteroaryl. The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g, carbon atoms and 1-3, 1 -6, or 1 -9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3 atoms of each ring can be substituted by a substituent. Examples of heterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl, aziridinyl, oxiryl, thiiryl, morpholinyl, tetrahydrofuranyl, and the like.

[0127] The term “substituents” refers to a group “substituted” on an alkyl, cycloalkyl, aryl, heterocyclyl, or heteroaryl group at any atom of that group. Suitable substituents include, without limitation, halo, hydroxy, mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, carboxy, alkanesulfonyl, alkylcarbonyl, azido, and cyano groups.

[0128] In some instances, the hydrocarbon tethers (i.e., cross-links) described herein can be further manipulated. In one instance, a double bond of a hydrocarbon alkenyl tether (e.g., as synthesized using a ruthenium-catalyzed ring closing metathesis (RCM)) can be oxidized (e.g, via epoxidation or dihydroxylation) to provide one of compounds below.

[0129] Either the epoxide moiety or one of the free hydroxyl moieties can be further functionalized. For example, the epoxide can be treated with a nucleophile, which provides additional functionality that can be used, for example, to attach a tag (e.g., a radioisotope or fluorescent tag). The tag can be used to help direct the compound to a desired location in the body or track the location of the compound in the body. Alternatively, an additional therapeutic agent can be chemically attached to the functionalized tether (e.g., a cytoprotective agent). Such derivatization can alternatively be achieved by synthetic manipulation of the amino or carboxy-terminus of the stapled peptide, or via the amino acid side chain. Other agents can be attached to the functionalized tether, e.g., an agent that facilitates entry of the stapled peptide into cells.

[0130] While hydrocarbon tethers (cross-links) have been described, other tethers (cross- linkes) are also envisioned. For example, the tether can include one or more of an ether, thioether, ester, amine, or amide moiety. In some cases, a naturally-occurring amino acid side chain can be incorporated into the tether. For example, a tether can be coupled with a functional group such as the hydroxyl in serine, the thiol in cysteine, the primary amine in lysine, the acid in aspartate or glutamate, or the amide in asparagine or glutamine. Accordingly, it is possible to create a tether using naturally-occurring amino acids rather than using a tether that is made by coupling two non-naturally occurring amino acids. It is also possible to use a single non-naturally occurring amino acid together with a naturally occurring amino acid.

[0131] It is further envisioned that the length of the tether (cross-link) can be varied. For instance, a shorter length of tether can be used where it is desirable to provide a relatively high degree of constraint on the secondary alpha-helical structure, whereas, in some instances, it is desirable to provide less constraint on the secondary alpha-helical structure, and thus a longer tether can be desired.

[0132] Additionally, while examples of tethers (cross-links) spanning from amino acids i to i+3, i to z+ ; and z to z+ 7 have been described in order to provide a tether that is primarily on a single face of the alpha helix, the tethers can be synthesized to span any combinations of numbers of amino acids (e.g., i to i+ 7).

[0133] In some instances, alpha-di substituted amino acids are used in the stapled peptide to improve the stability of the alpha-helical secondary structure. However, alphadisubstituted amino acids are not required, and instances using mono-alpha substituents (e.g., in the tethered amino acids) are also envisioned.

[0134] METHODS OF MAKING PEPTIDES AND STAPLED PEPTIDES

[0135] Methods of synthesizing the peptides and stapled peptides described herein are known in the art. Nevertheless, the following exemplary method can be used. It will be appreciated that the various steps can be performed in an alternate sequence or order to give the desired compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the peptides and stapled peptides described herein are known in the art and include, e.g., those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 3d. Ed., John Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser’s Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

[0136] Methods of performing different types of stapling are well known in the art (see, e.g., Lactam stapling'. Shepherd et al., J. Am. Chem. Soc., 127:2974-2983 (2005); Triazole stapling'. Kawamoto et al., J. Med. Chem., 55:1137-1146 (2011); UV- cycloaddition stapling: Madden et al., Bioorg. Med. Chem. Lett., 21 : 1472-1475 (2011); Disulfide stapling'. Jackson et al., Am. Chem. Soc.,113:9391-9392 (1991); Oxime stapling'. Haney et al., Chem. Commun., 47: 10915-10917 (2011); Thioether stapling'. Brunel and Dawson, Chem. Commun., 552-2554 (2005); Phoioswilchable stapling'. J. R. Kumita et al., Proc. Natl. Acad. Sci. U. S. A., 97:3803-3808 (2000); Double-click stapling. Lau et al., Chem. Sci., 5: 1804-1809 (2014); Bis-lactam stapling. J. C. Phelan et al.„ J. Am. Chem. Soc., 119:455-460 (1997); and Bis-arylation stapling'. A. M. Spokoyny et al., J. Am. Chem. Soc., 135:5946-5949 (2013)). I

[0137] The peptides and stapled peptides of this invention can be made by chemical synthesis methods, which are well known to the ordinarily skilled artisan. See, e.g., Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, W. H. Freeman & Co., New York, N.Y., 1992, p. 77. Hence, peptides and stapled peptides can be synthesized using the automated Merrifield techniques of solid phase synthesis with the a-NH protected by either t-Boc or Fmoc chemistry using side chain protected amino acids on, e.g., an Applied Biosystems Peptide Synthesizer Model 430A or 431. One manner of making of the peptides and stapled peptides described herein is using solid phase peptide synthesis (SPPS). The C-terminal amino acid is attached to a cross-linked polystyrene resin via an acid labile bond with a linker molecule. This resin is insoluble in the solvents used for synthesis, making it relatively simple and fast to wash away excess reagents and by-products. The N-terminus is protected with the Fmoc group, which is stable in acid, but removable by base. Any side chain functional groups are protected with base stable, acid labile groups.

[0138] Longer peptides can be made by conjoining individual synthetic peptides using native chemical ligation. Alternatively, the longer synthetic peptides can be synthesized by well-known recombinant DNA techniques. Such techniques are provided in well- known standard manuals with detailed protocols. To construct a gene encoding a peptide of this invention, the amino acid sequence is reverse translated to obtain a nucleic acid sequence encoding the amino acid sequence, preferably with codons that are optimum for the organism in which the gene is to be expressed. Next, a synthetic gene is made, typically by synthesizing oligonucleotides which encode the peptide and any regulatory elements, if necessary. The synthetic gene is inserted in a suitable cloning vector and transfected into a host cell. The peptide is then expressed under suitable conditions appropriate for the selected expression system and host. The peptide is purified and characterized by standard methods. The peptides can be made in a high-throughput, combinatorial fashion, e.g., using a high-throughput multiple channel combinatorial synthesizer available from Advanced Chemtech.

[0139] One or more peptide bonds can be replaced, e.g., to increase physiological stability of the stapled peptide, by: a retro-inverso bonds (C(O)-NH); a reduced amide bond (NH-CH2); a thiomethylene bond (S-CH2 or CH2-S); an oxomethylene bond (O- CH2 or CH2-O); an ethylene bond (CH2-CH2); a thioamide bond (C(S)-NH); a transolefin bond (CH=CH); a fluoro- substituted trans-olefin bond (CF=CH); a ketomethylene bond (C(O)-CHR) or CHR-C(O), wherein R is H or CH3; and a fluoro-ketomethylene bond (C(O)-CFR or CFR-C(O), wherein R is H, F, or CH3. The peptides and stapled peptides can be further modified by one or more of: acetylation, amidation, biotinylation, cinnamoylation, famesylation, fluoresceination, formylation, myristoylation, palmitoylation, phosphorylation (Ser, Tyr, or Thr), stearoylation, succinylation, and sul furyl ati on. As indicated above, peptides and stapled peptides can be conjugated to, for example, polyethylene glycol (PEG); alkyl groups (e. , C1-C20 straight or branched alkyl groups); fatty acid radicals; and combinations thereof.

[0140] Methods of producing stapled peptides using a, a-di substituted non-natural amino acids are known in the art (see, e.g., Bird et al., Methods EnzymoL, 446:369, 2008; Bird et al, Current Protocols in Chemical Biology, 2011; US Publication No. 2020-0352899, each of which is incorporated by reference herein in its entirety), a, a-Di substituted nonnatural amino acids containing olefinic side chains of varying length can be synthesized by known methods (see, e.g., Williams et al., J. Am. Chem. Soc. 113:9276, 1991; Schafmeister et al., J. Am. Chem Soc. 122:5891, 2000; Bird et al., Methods EnzymoL, 446:369, 2008; Bird et al, Current Protocols in Chemical Biology?, 2011). For stapled peptides where an i linked to i 1 7 staple is used (two turns of the helix stabilized), either one S5 amino acid ((S)-a-(4'-pentenyl)alanine) and one Rs amino acid ((R)-a-(7'- octenyl)alanine) can be used, or one Ss amino acid ((S)-a-(7'-octenyl)alanine)) and one Rs amino acid ((R)-a-(4'-pentenyl)alanine) can be used. For stapled peptides where an i linked to i+4 staple is used (one turn of the helix stabilized), either two S5 amino acids ((S)-a-(4'-pentenyl)alanine) can be used, or two Rs amino acids ((R)-a-(4'- pentenyl)alanine) can be used. The starting chiral auxiliary confers the R- or S- alkyl- stereoisomer. Also, 8-iodooctene can be used in place of 5 -iodopentene. Inhibitors can be synthesized on a solid support using solid-phase peptide synthesis (SPPS) on MB HA resin (see, e.g., WO 2010 / 148335).

[0141] Fmoc-protected a-amino acids (other than the olefinic amino acids Fmoc-7?s-OH, Fmoc-As-OH, Fmoc-As-OI I, and Fmoc-As-OH), 2-(6-chl oro-1 - / / -benzotri azol e-l-yl)- 1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU), and Rink Amide MBHA are commercially available from, e.g., Novabiochem (San Diego, CA). Dimethylformamide (DMF), A-methyl-2-pyrrolidinone (NMP), A,A-diisopropylethylamine (DIEA), trifluoroacetic acid (TFA), 1,2-di chloroethane (DCE), fluorescein isothiocyanate (FITC), and piperidine are commercially available from, e.g, Sigma- Aldrich. Olefinic amino acid synthesis is reported in the art (Williams et al., Org. Synth., 80:31, 2003).

[0142] In some instances, the peptides and stapled peptides are synthesized according to methods described in the working examples herein.

[0143] In some instances, a method of making a stapled peptide described herein comprises: (a) provided a peptide comprising an amino acid sequence of the stapled peptide (prior to cross-linking), and (b) cross-linking the peptide (e.g., by a ring-closing metathesis (RCM reaction) thereby making the stapled peptide. In some instances, the method further comprises formulating the stapled peptide as a sterile pharmaceutical composition. In some instances, the peptide is a BCL-2 mutant peptide described herein. In some instances, the peptide comprises the amino acid sequence EIVMKYIHYKX1SQRX2YEWDA (SEQ ID NO: 30), EIVMKYIHYKLSXIRGYX2WDA (SEQ ID NO:31), or EIVMKYIHYK8SQRGYEXDA (SEQ ID NO:32) with S at position 12 of any one of SEQ ID NOs:30-32 substituted by a positively charged amino acid (e.g., His, Lys, Arg, or Orn) and optionally further including 1 or 2 other amino acid substitutions within any one of SEQ ID NOs:30-32, wherein the 1 or 2 other substitutions are not at Xi or X2 of SEQ ID NOs: 30 and 31 and are not at 8 or X of SEQ ID NO:32, wherein Xi , X2 , 8, and X are a,a-disubstituted non-natural amino acids. In some instances, the peptide comprises the amino acid sequence RALVADFVGYKX1RQKX2YV (SEQ ID NO:33) with 1 to 3 amino acid substitutions, wherein Xi and X2 are a, a-di substituted non-natural amino acids, and wherein positions 12 and 16 of SEQ ID NO:33 are not substituted. In some instances, the peptide comprises the amino acid sequence RALVADFVGYK8RQKGYVX (SEQ ID NO:34) with 1 to 2 amino acid substitutions, wherein 8 and X are a, a-di substituted non-natural amino acids, wherein the side chains of 8 and X are cross linked, and wherein positions 12 and 19 of SEQ ID NO:34 are not substituted. In some instances, the peptides and stapled peptides can include a detectable label. A label includes a moiety that has at least one element, isotope, or functional group incorporated into the moiety which enables detection of the peptide to which the label is attached. Labels can be directly attached (i.e., via a bond) or can be attached by a linker. Labels can be attached to peptide or stapled peptide described herein at any position that does not interfere with the biological activity (e.g., binding to IP3R1 or BAX) or characteristic of the inventive stapled peptide or peptide that is being detected. Known labels include Biotin or Fluorescein, e.g., attached by Fluorescein Isothiocyanate (FITC).

[0144] Again, methods suitable for obtaining (e.g., synthesizing), stapling, and purifying the peptides and stapled peptides disclosed herein are also known in the art (see, e.g., Bird et. al., Methods in Enzymology 446:369-386 (2008); Bird et al, Current Protocols in Chemical Biology 2011; Walensky et al., Science 305: 1466-1470 (2004); Schafmeister et al., J. Am. Chem. Soc. 122:5891-5892 (2000); U.S. Patent Application Publication No. 2010 / 0168388; and U.S. Patent No. 7,723,468, each of which are hereby incorporated by reference in their entirety).

[0145] In some embodiments, the stapled peptides are substantially free of non-stapled peptide contaminants or are isolated. In some embodiments, the peptides are isolated. Methods for purifying peptides and stapled peptides include, for example, synthesizing the peptide on a solid-phase support (and, for stapled peptides, performing the cyclization). Next, the solid-phase support may be isolated and suspended in a solution of a solvent such as DMSO, DMSO / dichloromethane mixture, or DMSO / NMP mixture. The DMSO / dichloromethane or DMSO / NMP mixture may comprise about 30%, 40%, 50%, or 60% DMSO. In a specific embodiment, a 50% / 50% DMSO / NMP solution is used. The solution may be incubated for a period of 1, 6, 12, or 24 hours, following which the resin may be washed, for example with dichloromethane or NMP In one embodiment, the resin is washed with NMP. Shaking and bubbling an inert gas into the solution may be performed. Properties of the peptides and stapled peptides of the invention can be assayed, for example, using the methods described below. For example, any of the peptides and stapled peptides described herein can be tested for their ability to bind to IP3R1, bind to BAX, prevent or decrease stress-induced cell death (e.g., using methods described herein or known in the art) or to increase or induce cell death (e.g., apoptosis) (e.g., using fluorescence-assisted cell sorting, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and / or immunofluorescence microscopy, or any other methods known in the art for detecting cellular apoptosis or cell death).

[0146] Assays to determine a-helicity, melting temperature (Tm), in vitro protease resistance, and in vivo protease resistance are known in the art (see, e.g., US Patent Application Publication No. 2020-0352899, which is incorporated herein in its entirety).

[0147] In Vitro Binding Assays'. To assess the binding and affinity of peptides and stapled peptides described herein to proteins (e.g., IP3R1, BAX), a fluorescence polarization assay (FPA) can be used, for example. The FPA technique measures the molecular orientation and mobility using polarized light and fluorescent tracer. When excited with polarized light, fluorescent tracers (e.g., FITC) attached to molecules with high apparent molecular weights (e.g., FITC-labeled peptide or stapled peptide bound to a large protein) emit higher levels of polarized fluorescence due to their slower rates of rotation as compared to fluorescent tracers attached to smaller molecules (e.g., FITC- labeled peptide or stapled peptide that are free in solution). In vitro binding assays to assess the binding of a peptide or stapled peptide described herein to IP3R1 or BAX are also provided in the working examples herein.

[0148] In Vitro Displacement Assays to Characterize Antagonists of Peptide-Protein Interactions'. To assess the binding and affinity of compounds that antagonize the interaction between a peptide or stapled peptide described herein and a protein (e.g., IP3R1, BAX), a fluorescence polarization assay (FPA) utilizing a fluoresceinated peptide or stapled peptide is used, for example. The FPA technique measures the molecular orientation and mobility using polarized light and fluorescent tracer. When excited with polarized light, fluorescent tracers (e.g., FITC) attached to molecules with high apparent molecular weights (e.g. FITC-labeled peptides or stapled peptides bound to a large protein (e.g., IP3R1 protein) emit higher levels of polarized fluorescence due to their slower rates of rotation as compared to fluorescent tracers attached to smaller molecules (e.g. FTTC-labeled peptides or stapled peptides that are free in solution). A compound that antagonizes the interaction between the fluoresceinated peptides or stapled peptides and an acceptor protein will be detected in a competitive binding FPA experiment.

[0149] Binding Assays in Intact Cells'. It is possible to measure binding of peptides or stapled peptides to proteins (e.g., IP3R1, BAX) on or in intact cells by, e.g., immunoprecipitation experiments.

[0150] Cellular Penetrability Assays'. To measure the cell penetrability of the peptides or stapled peptides described herein, intact cells are incubated with fluoresceinated stapled peptides (10 pM) for 4 hours in serum-free media or in media supplemented with human serum at 37°C, washed twice with media and incubated with trypsin (0.25%) for 10 minutes at 37°C. The cells are washed again and resuspended in PBS. Cellular fluorescence is analyzed, for example, by using either a FACSCalibur flow cytometer or Cellomics KineticScan® HCS Reader.

[0151] Clinical Trials. To determine the suitability of the peptides, stapled peptides, or pharmaceutical compositions of the invention for treatment of humans, clinical trials can be performed. For example, patients having a cancer or suspected of having a cancer requiring chemotherapy are selected and separated into treatment and one or more control groups, wherein the treatment group is administered a peptide, stapled peptide, or pharmaceutical composition of the invention, while the control groups receive a placebo or a known cytoprotective drug. The treatment safety and efficacy of the peptide, stapled peptide, or pharmaceutical compositions of the invention can thus be evaluated by performing comparisons of the patient groups with respect to factors, such as prevention of symptoms, time to resolution of symptoms, and / or time to a decrease in the number, severity, or frequency of one or more symptoms of the disease. In some embodiments, subject administered a peptide, stapled peptide, or pharmaceutical composition of the invention can have a reduced number of symptoms of the disease as compared to a subject in a control group receiving a placebo.

[0152] PHARMACEUTICAL COMPOSITIONS

[0153] One or more of the peptides or stapled peptides disclosed herein can be formulated for use as or in pharmaceutical compositions comprising the peptide or stapled peptide, and a pharmaceutically acceptable carrier. Such pharmaceutical compositions can be formulated or adapted for administration to a subject via any route, c.g, any route approved by the Food and Drug Administration (FDA). Exemplary methods are described in the FDA Data Standards Manual (available at www.fda.gov / Drugs / DevelopmentApprovalProcess / FormsSubmissionRequirements / ElectronicSubmissions / DataStandardsManualmonograph s / default.htm). For example, pharmaceutical compositions can be formulated or adapted for administration by inhalation (e.g., oral and / or nasal inhalation (e.g., via nebulizer or spray)), injection (e.g., intravenously, intra-arterial, subdermally, intraperitoneally, intramuscularly, and / or subcutaneously), and / or for oral administration, transmucosal administration, and / or topical administration (including topical creams or ointments, topical (e.g, nasal) sprays, and / or topical solutions).

[0154] In some instances, pharmaceutical compositions can include an effective amount of one or more peptides or stapled peptides described herein. The terms “effective amount” and “effective to treat,” as used herein, refer to an amount or a concentration of one or more peptides or stapled peptides or a pharmaceutical composition described herein utilized for a period of time (including acute or chronic administration and periodic or continuous administration) that is effective within the context of its administration for causing an intended effect or physiological outcome (e.g., prevention or treatment of CIPN and / or hearing loss).

[0155] Pharmaceutical compositions of this invention can include one or more peptides or stapled peptides described herein and any pharmaceutically acceptable carrier, adjuvant and / or vehicle. In some instances, pharmaceutical compositions can further include one or more additional therapeutic agents in amounts effective for achieving a modulation of disease or disease symptoms.

[0156] The term “pharmaceutically acceptable carrier” includes a carrier that can be administered to a patient, together with one or more peptides or stapled peptides of this invention, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the one or more peptides or stapled peptides.

[0157] The pharmaceutical compositions of this invention can contain any conventional non-toxic pharmaceutically-acceptable carriers, adjuvants, or vehicles. In some cases, the pH of the formulation can be adjusted with pharmaceutically acceptable acids, bases, or buffers to enhance the stability of the formulated peptides(s) and / or stapled peptide(s), or its delivery form. The term “parenteral” as used herein includes subcutaneous, intra- cutaneous, intra-venous, intra-muscular, intra-articular, intra-arterial, intra-synovial, intra- stemal, intra-thecal, intra-lesional, and intra-cranial injection or infusion techniques.

[0158] Pharmaceutical compositions can be in the form of a solution, powder for inhalation and / or nasal administration, or cream or spray for topical administration. Such compositions can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents.

[0159] Pharmaceutical compositions can include preservatives and additives. Preservatives can be included to limit or prevent microbial growth or contamination.

[0160] In some instances, one or more of the peptides or stapled peptides disclosed herein can be conjugated, for example, to a carrier protein. Such conjugated compositions can be monovalent or multivalent. For example, conjugated compositions can include one peptide or stapled peptide disclosed herein conjugated to a carrier protein. Alternatively, conjugated compositions can include two or more peptides or stapled peptides disclosed herein conjugated to a carrier. In such instances, additional cytoprotective components or chemotherapeutic agents can also be coupled to the carrier protein.

[0161] As used herein, when two entities are “conjugated” to one another, they are linked by a direct or indirect covalent or non-covalent interaction. In certain aspects, the association is covalent. In other aspects, the association is non-covalent. Non-covalent interactions include hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetic interactions, electrostatic interactions, etc. An indirect covalent interaction is when two entities are covalently connected, optionally through a linker group.

[0162] Carrier proteins can include any protein that increases or enhances the cytoprotective activity of a pharmaceutical composition in a subject. Polymeric carriers can be a natural or a synthetic material containing one or more primary and / or secondary amino groups, azido groups, or carboxyl groups. Carriers can be water-soluble.

[0163] In some aspects, the present disclosure provides methods for using these pharmaceutical compositions for selectively protecting sensory neurons from age-related and / or chemotherapy induced axonal degeneration, treating or preventing the CIPN or hearing loss, or averting neuropathic side effects of chemotherapy and / or other causes of axonal degeneration.

[0164] Although effective amounts can depend, among other things, on the species of subject treated, the body weight of the subject, and the chosen treatment regimen, effective amounts can be readily determined by those in the art.

[0165] METHODS OF USE

[0166] Also provided herein are methods of using the peptides, stapled peptides, and pharmaceutical compositions described herein.

[0167] For instance, provided herein is a method of treating CIPN in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of a mutant BCL-2 peptide, stapled peptide, or pharmaceutical composition described herein. Also provided herein is a method of preventing CIPN in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of a mutant BCL-2 peptide, stapled peptide, or pharmaceutical composition described herein. Furthermore, provided herein is a method of treating CIPN in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of a mutant BCL-w stapled peptide, or pharmaceutical composition described herein. Also provided herein is a method of preventing CIPN in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of a mutant BCL-w stapled peptide, or pharmaceutical composition described herein.

[0168] Chemotherapy is typically used in the context of cancer treatment and uses one or more anti-cancer drugs referred to as chemotherapeutic agents as part of a standardized treatment regimen. Chemotherapy may be given with a curative intent (which almost always involves combinations of drugs), or it may aim to prolong life or to reduce symptoms (palliative chemotherapy).

[0169] Non-limiting examples of chemotherapeutic agents include: alkylating agents (e.g., mechlorethamine, chlorambucil, cyclophosamide, ifosfamide, melphalan, streptozocin, carmustine, lomustine, oxaliplatin, busulfan, dacarbazine, temozolomide, thiotepa, and altretamine), antimetabolites (e.g., 5-fluorouracil, capecitabine, 6- mercaptopurine, folic acid analogs (e.g., methotrexate), gemcitabine, arabinosides (e.g., cytarabine), fludarabine, and premetrexed), anthracyclines (e.g., daunorubicin, doxorubicin, epirubicin, and idarubicin), topoisomerase inhibitors (e.g., topotecan, irinotecan, and epipodophyllotoxins (e.g., etoposide and teniposode)), microtubuletargeting agents and spindle poisons (e.g., taxanes (paclitaxel and docetaxel), vinblastine, vincristine, vindesine, vinorelbine, vinflunine, discodermolide, eleutherobin, sarcodictyin, epothilone, ixaberpilone, colchicine, combretastatin, 2-m ethoxy estradiol, noscapine, and estramustine), proteasome inhibitors, EGF-R inhibitors, Eph-R inhibitors, p38 / JAK kinase inhibitors, PI3K inhibitors, MEK inhibitors, MAPK inhibitors, Trk inhibitors, proteasome inhibitors, Raf inhibitors, corticosteroids (e.g., dexamethasone, prednisolone, and methyl prednisolone), platinum compounds, and therapeutic antibodies (e.g., bevacizumab, brentuximab vedotin, cetuximab, ibritumomab tiuxetan, ipilimumab, panitumumab, rituximab, tositumomab, and trastuzumab). Chemotherapy often produces several side-effects.

[0170] Chemotherapy-induced peripheral neuropathy (CIPN), a type of chemotoxic axon injury, is a common and dose-limiting side effect of numerous cytotoxic chemotherapies in oncology. CIPN constitutes a frequent cause of axon injury and neurological impairment. Patients with CIPN experience pain, tingling, numbness, and / or impaired motor function, due to degeneration of long peripheral sensory or motor neuron axons.

[0171] Chemotherapeutic drugs that result in CIPN include microtubule-targeting agents. e.g., CIPN can result from use of chemotherapeutic agents such as, but not limited to, taxanes (e.g., paclitaxel, docetaxel), vinca alkaloids (e.g., vinblastine, vinorelbine, vindesine, vinflunine, vincristine) alkylating agents, arabinosides, proteasome inhibitors, PI3K inhibitors, Raf inhibitors, discodermolide, eleutherobin, sarcodictyin, epothilone, colchicine, combretastatin, 2-methoxyestradiol, and noscapine. Microtubule-targeting chemotherapeutic agents (e.g., those used to treat breast, ovarian, and lung cancers) cause a primarily sensory neuropathy. The mechanism for the chemotherapy-induced degeneration is not understood, and there are currently no treatments available for this common disorder.

[0172] US Patent Application Publication No. 2020 / 0352899 (which is incorporated by reference herein in its entirety) shows that microtubule targeting chemotherapeutic agents reduce axonal expression of bclw, but do not alter expression of the closely related components Bcl2 or BCIXL; that bclw protects axons from degeneration, and microtubuletargeting drugs such as paclitaxel cause degeneration by discontinuing this protection by lowering axonal levels of bclw. US Patent Application Publication No. 2020 / 0352899 describes that the BH4 domain of bclw is sufficient to reduce or prevent axonal degeneration. Accordingly, the peptides and stapled peptides described herein provide a clinically useful therapy for CIPN.

[0173] The disclosure includes methods of using an effective amount of one or more of the peptides, stapled peptides, or pharmaceutical compositions described herein for the prevention or treatment of CIPN in a subject in need thereof. In some instances, the methods involve administering a peptide or stapled peptide described herein. The method can further comprise administering other therapeutically beneficial agents to the subject. For example, metformin and / or one or more calpain inhibitors can be administered before, during, and / or after administration of the peptides, stapled peptides, or pharmaceutical compositions described herein.

[0174] The term “subject,” as used herein, refers to any mammal. In certain aspects, the term “subject,” as used herein, refers to a human (e.g., a man, a woman, or a child).

[0175] The terms “administer,” “administering,” or “administration,” as used herein refers to implanting, absorbing, ingesting, injecting, or inhaling, one or more of the inventive peptides, stapled peptides, or pharmaceutical compositions (e.g., any of those described herein). In some instances, one or more of the peptides, stapled peptides, or pharmaceutical compositions disclosed herein can be administered to a subject topically and / or orally. For example, the methods herein include administration of an effective amount of one or more peptides, stapled peptides, or pharmaceutical compositions to achieve the desired or stated effect.

[0176] Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific peptide or stapled peptide employed, the age, body weight, general health status, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the CIPN, the patient’s disposition to the treatment, and the judgment of the treating physician.

[0177] Following administration, the subject can be evaluated to detect, assess, or determine the number of symptoms and / or the severity and / or frequency of one or more symptoms of the CIPN in the subject. In some instances, treatment can continue until a reduction in the number of symptoms and / or the severity and / or frequency of one or more symptoms of CIPN is observed. Upon improvement of a patient’s condition, a maintenance dose of a mimetic, polypeptide, or pharmaceutical composition, or a combination thereof, of the disclosure can be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, can be reduced, as a function of the symptoms, to a level at which the improved condition is retained. Patients can, however, require intermittent treatment on a long-term basis upon any recurrence of CIPN.Also provided herein is a method of selectively protecting sensory neurons from age-related and / or chemotherapy induced axonal degeneration in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of a peptide, stapled peptide, or pharmaceutical composition described herein.

[0178] Also provided herein is a method of averting neuropathic side effects of chemotherapy and / or other causes of axonal degeneration in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of a peptide, stapled peptide, or pharmaceutical composition described herein.

[0179] Also provided are methods of administering chemotherapy to a subject in need thereof. The method involves administering the chemotherapy and an effective amount of one or more of the peptides, stapled peptides, or pharmaceutical compositions described herein to the subject. The chemotherapeutic agents and the peptides, stapled peptides, or pharmaceutical compositions can be administered simultaneously or sequentially (e.g, the chemotherapeutic agents can be administered prior to or after the peptides, stapled peptides, or pharmaceutical compositions). In certain instances the chemotherapeutic agent is selected from the group consisting of a microtubule-targeting agent, an alkylating agent, an antimetabolite, a folic acid analogue, a spindle poison, a platinum compound, an epipodophyllotoxin, an antibiotic, an EGF-R inhibitor, an Eph-R inhibitor, a p38 / JAK kinase inhibitor, a PI3K inhibitor, a MEK inhibitor, a MAPK inhibitor, a Trk inhibitor, a proteasome inhibitor, and a Raf inhibitor. In some instances, the subject is administered an additional therapeutic agent. For example, metformin and / or one or more calpain inhibitors can be administered before, during, and / or after administration of the chemotherapeutic agents and / or the peptides, stapled peptides, or pharmaceutical compositions. In some cases, the subject being administered chemotherapy has a hematological tumor (e.g., acute myeloid leukemia, chronic myeloid leukemia, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma, acute lymphoblastic leukemia, or chronic lymphocytic leukemia). In certain instances, the subject being administered chemotherapy has a breast cancer, ovarian cancer, or lung cancer.

[0180] Also provided are methods of selecting or identifying a subject at risk of developing CIPN. The selected subjects can then be treated using any of the abovedescribed methods. The subjects can be identified or selected on the basis of one or more of the following criteria: having reduced levels of endogenous bclw in the axons of the sensory neurons relative to a control subject who does not develop CIPN upon administration of a chemotherapeutic agent (e.g., a taxane such as paclitaxel), reduced levels of EphA4 or Eph / 5 receptors in sensory neurons relative to a control subject who does not develop CIPN upon administration of a chemotherapeutic agent (e.g., a taxane such as paclitaxel), reduced expression of transporters relative to a control subject who does not develop CIPN upon administration of a chemotherapeutic agent (e.g., a taxane such as paclitaxel), reduced expression of tubulin relative to a control subject who does not develop CIPN upon administration of a chemotherapeutic agent (e.g., a taxane such as paclitaxel), reduced expression of NGF, BDNF, and / or NT3 relative to a control subject who does not develop CIPN upon administration of a chemotherapeutic agent (e.g., a taxane such as paclitaxel); and / or genetic polymorphisms in or adjacent to genes such as EphA4 / 5 that may confer increased risk, or in the genes encoding calcium- interacting proteins such as calmodulin and parvalbumin. Subjects meeting one or more of these criteria are determined to have an enhanced risk of developing CIPN.

[0181] This disclosure also features methods of treating or preventing hearing loss in a subject in need thereof. The examples provided in US Patent Application Publication No. 2020 / 0352899 show that bclw is necessary for the maintenance of hearing during aging. Accordingly, administering a peptide or stapled peptide described herein is useful for treating or preventing hearing loss. The hearing loss may be, e.g., age-related, noise- induced, or chemotherapy-induced.

[0182] In certain instances, the peptide or stapled peptide is administered by injection (e.g., into the oval window). Subjects for this treatment can be selected on the basis of having an advanced age (e.g., age greater than or equal to 65, 70, 75, 80, 85, 90, 95, or 100) and / or hearing loss (e.g., hearing thresholds greater than or equal to 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85 db, as evaluated by standard clinical methods known in the art, e.g., pure tone audiometry for air conduction thresholds at 500, 1000, and 2000 Hz).

[0183] In general, these methods include selecting a subject and administering to the subject an effective amount of one or more of the peptides, stapled peptides, or pharmaceutical compositions described herein, and optionally repeating administration as required for the treatment of hearing loss.

[0184] Following administration, the subject can be evaluated to assess the hearing loss in the subject. In some instances, treatment can continue until hearing of the subject improves. Upon improvement of a patient’s condition, a maintenance dose of a peptide, stapled peptide, or pharmaceutical composition, or a combination thereof, of the disclosure can be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, can be reduced, as a function of the hearing capacity of the subject, to a level at which the improved condition is retained. Patients can, however, require intermittent treatment on a long-term basis upon any recurrence of hearing loss.

[0185] The following are examples of the practice of the invention. They are not to be construed as limiting the scope of the invention in any way.

[0186] EXAMPLES

[0187] STAPLE SCANNING LIBRARIES OF BCL-W BH4 PEPTIDES EXHIBIT DIFFERENTIAL BINDING TO CEREBELLAR IP3R1

[0188] Stapled peptides corresponding to the BH4 domain of BCL-w were designed (FIG. 1A) to assess binding to IP3R1 (FIG. IB). Peptides were generated with z, i+4 and i, i+ 7 staple scanning stabilized alpha-helix (SAH) libraries ofBH4 domain amino acid sequences 11 to 28 of BCL-w (equivalent to residues 1 to 18 of SEQ ID NO:23, i.e., RALVADFVGYKLRQKGYV), each capped with a biotin moiety at the N-terminus (FIG. 1C-D) A BCL-w BH4 helical wheel depiction demonstrates how reinforcement of the native helical fold positions a series of circumferential positively-charged and hydrophilic residues at the C-terminal half (e.g., Y20, K21, R23, Q24, K25, Y27), with a defined hydrophobic surface that also extends into the C-terminal region (e.g., L22, G26) but is more circumferential in the N-terminal half of the helix (e.g., A12, L13, V14, A15, F17, V18, G19) (FIG. IE). The amidated and biotinylated constructs were incubated with lysate from mouse cerebellum, which is rich in IP3R1, and then performed antibiotin pull-downs and IP3R1 western analyses to assess relative binding activities of the libraries.

[0189] Comparing the compounds within each library, the initial observation was that staples positioned in the C-terminal half of the sequence were generally more disruptive than those installed in the N-terminal half. For example, only 2 of 8 N-terminally z, i+4 stapled peptides exhibited activity below a 25% binding threshold, whereas 5 of 7 C- terminally i, i+4 stapled peptides exhibited blunted activity (FIGs. IF). A similar trend was evident for z, z+7 stapled peptides (FIGs. 1G). Notably, staples that replaced R11, Y20, K21, R23, K25 and Y27 were the most disruptive, whereas replacement of residues A15, G19, F17, V18, Q25, and V28 was moderately disruptive (FIGs. 1F-H). In contrast, staples involving positions A12, L13, V14, D16, L22, and G26 exhibited among the best IP3R1 binding activity (FIGs. 1F-H). These data suggest that binding interactions involving the cluster of Y, K, and R residues in the C-terminal half of the BH4 sequence are especially important for IP3R1 pull down.

[0190] The BH4 domains of BCL-2 and BCL-XL have been reported to target the IP3R at various sites, with domain 3 (D3) located in the central modulatory region (aa 923-1581) and forming a potential binding surface at the cytosolic face of IP3R. To determine whether the library of stapled BCL-w BH4 peptides bound to D3, the corresponding recombinant GST-fusion protein was generated, repeating the biotin pull-down experiment to observe differential binding activity to IP3R1-D3 (FIGs. 1I-K). Comparing the biotin pull-down datasets for cerebellar IP3R1 and D3, the most striking corroboration occurred for the disruptive effects of staples 1, 11, 13, 16, and 25, again implicating the importance of key residues such as R11, Y20, and Y27, which are shared among these i, i-4 and z, i+ 7 staple positions (FIG. IK).

[0191] To further validate the capacity of staple scanning to identify optimal compounds and binding determinants for IP3R targeting, SAH-BCL-w BH4-12 (aa 1 1-28, stapling positions 22 and 26), a lead z, i+4 stapled peptide, was compared to BCL-w BH4 SAHB.i, a previously reported analog bearing an alternative i, i-4 staple insertion position and sequence template (aa 12-31, stapling positions 24 and 28). Quantitative binding analysis of the interactions between the stapled peptides and recombinant IP3R1 D3 by biolayer interferometry revealed Kds of 43 nM and 99 nM for SAH-BCL-w BH4-12 and BCL-w BH4 SAHB.i, respectively, reflecting a more than two-fold enhancement of binding activity upon z, i+4 stapling of the aa 11-28 template sequence with the staple positioned at aa 22 and 26 (Figure SI A-B). SAH-BCL-w BH4-13, which has the z, i+4 staple positioned at aa 23 and 27, showed no IP3R1 D3 binding activity even at the highest dose of recombinant protein applied, consistent with the results of the pull-down assay (FIGs. IF and II) As a further negative control, the unstapled peptide (aa 11-28) likewise showed no binding activity, highlighting the importance of helical structure for IP3R1 D3 interaction.

[0192] Finally, in advance of investigating the BCL-w BH4 binding site on IP3R1, the functional relevance of the observed binding interaction of a lead stapled peptide, such as SAH-BCL-w BH4-12, in modulating intracellular calcium signaling was confirmed. Wild-type (WT) and BCL-w knock-out (KO) mouse embryonic fibroblast (MEF) cell lines from Bcl-W ' mice and littermate controls were generated to confirm the absence of BCL-w protein in the KO MEFs. Comparative calcium imaging in WT vs. Bcl-w MEFs using an in vitro calcium flux assay that monitors IP3 -induced calcium release (IICR) from endoplasmic reticulum stores upon bath-applied ATP was then performed. Calcium flux from the endoplasmic reticulum in response to ATP was performed using an ER- targeted GCaMP6 and, in parallel studies, the resultant calcium changes in the cytosol using the ratiometric calcium indicator Fura Red. In both contexts, the peak response and area under the change in response curve were significantly reduced in Bcl-w" compared to WT cells. To determine if replacement of the BCL-w BH4 domain could restore homeostatic calcium signaling, the assay was adapted to include a pre-treatment step whereby Bcl-W ' cells were exposed to vehicle or SAH-BCL-w BH4-12 followed by monitoring calcium flux in response to ATP. Strikingly, SAH-BCL-w BH4- 12 treatment significantly improved the responsiveness of Bcl-W1' cells, as reflected by shifting the peak response and area under the response curve to near WT levels. Taken together, it was discovered that a lead BCL-w BH4 domain mimetic, such as SAH-BCL-w BH4-12, can engage endogenous cerebellar IP3R1 and recombinant IP3R1 D3 in a compositionspecific manner and significantly rescue the deregulation of ATP-induced calcium signaling that occurs in the absence of BCL-w. This study identifies amino acid residues in which mutations can be performed without a large impact on IP3R1 binding and are most amenable to alanine mutation.

[0193] ASSESSMENT OF THE BCL-W BH4 DOMAIN INTERACTION WITH IP3R1-D3 BY MOLECULAR DYNAMICS SIMULATION

[0194] Next, experiments were performed to gain structural insight into how the BCL-w BH4 domain helix could bind IP3R1 and D3 in particular. Whereas early assessments of IP3R structure and function derived from analyses of the 5 domains or fragments generated upon trypsin proteolysis, the more recent cryo-electron microscopy and crystal structures have provided a higher resolution understanding of how IP3 engagement of its ligand binding site at the N-terminus of IP3R results in allosteric induction of calcium flux at its C-terminus. The modulatory region that lies between the ligand binding domain and carboxy terminus is a site of regulation by a series of accessory proteins, including members of the BCL-2 family. D3 (or fragment 3) (aa 923-1581) of this central modulatory region was previously shown by biochemical means to contain a BCL- 2 / BCL-XL BH4 binding site (aa 1389-1408) within the C-terminal portion of the a-helical armadillo 2 repeat (ARM2, aa 1030-1494). However, a structure of the complex between a BCL-2 family BH4 domain and IP3R has not been determined to date. As stapled BCL- w BH4 peptides were found to likewise bind to D3 (FIG. 1I-J), molecular dynamics (MD) simulation was pursued as a means of predicting the structural basis of BCL-w BH4 / IP3R1 D3 interaction.

[0195] First, the conformational dynamics of a BCL-w BH4 domain peptide was examined using explicit-solvent MD simulations (PDB ID 1MK3, aa 11-28; 500 ns sampling). The peptide sequence maintained an a-helical fold throughout, consistent with NMR analyses of BCL-w, which also demonstrate exposure of BH4 domain residues on the protein surface (FIG. 2A). Next, a suitable full-length IP3R1 structure was identified and a subset of its D3 as a simulation target was extracted, which contained ARM2 and its adjacent scaffolding (PDB ID 8EAR; aa 1200-1566). Binding simulations were performed using a coarse-grained (CG) replica-exchange Monte Carlo strategy. This approach folds the unstructured BCL-w BH4 peptide near different sites on ARM2 and uses the resulting data to identify stable complexes. Replicas were distributed across ten temperatures and initialized using random BCL-w BH4 peptide conformers. The temperature of each replica was then steadily decreased to a common value during exchange simulations and 1000 samples were extracted from each quench. The resulting ensemble contained 104structures that lie near conformational minima. The ensemble was then clustered into low variability sets and assigned an energetic score based on the prevalence of each cluster in the overall ensemble. The 100 lowest energy samples from each replica provide a representation of BCL-w BH4 binding to IP3R1-ARM2. The leading clusters exhibit a common binding site and orientation, placing the BCL-w BH4 peptide within a groove at the surface of the ARM2 domain (FIG. 2B). The stability of this interaction mode was assessed by fine-graining the CG models (i.e., reintroducing all atomic-scale features) and performing multiple all-atom MD simulations in explicit solvent (3x simulations per cluster; 100 ns of equilibrium sampling per simulation). The resulting trajectories support the stability of this orientation (FIG. 2C) and sample a dynamic pool of contacts between the BCL-w BH4 a-helix and ARM2 groove (FIGs. 2D-E)

[0196] The MD data reveal significant hydrophobic and electrostatic complementarity at the BCL-w BH4 / IP3R1-ARM2 binding interface (FIGs. 2F-G). Notable hydrophobic interactions are seen between residues L13, V14, F17, V18 of BCL-w BH4 and residues F1372, 11376, V1419, 11421, A1422, and 11482 of IP3R1. Of special interest are the (1) interaction involving Y20 of BCL-w BH4 and El 387 of IP3R, (2) network of electrostatic interactions involving R1 1 of BCL-w BH4 and E1418, R1364, and D1472 of IP3R1, and (3) electrostatic pairings between D16, K21, R23, and K25 with residues K1310, E1387, E1319, and E1481 of IP3R (FIGs. 2F-G). Taken together, these data suggest that ARM2 contains a dedicated BCL-w BH4 binding groove on the cytosolic surface of IP3R1 (Figure 2H).

[0197] ALANINE SCANNING REVEALS A SELECTIVITY DETERMINANT

[0198] FOR BCL-W BH4 BINDING TO IP3R1

[0199] To evaluate the model structure derived from MD simulations, alanine scanning mutagenesis was performed followed by repeat binding analyses of endogenous cerebellar IP3R1 and recombinant IP3R1-D3. Whereas staple scanning examines the influence of the all -hydrocarb on staple at sequential locations along the a-helical interface, in addition to effectively evaluating the impact of two point mutations at a time, alanine scanning expands the analysis to single amino acid resolution. To select the optimal staple positions for mutational analysis, amino acid positions were identified that, upon replacement with stapling amino acids, showed no disruption of binding activity across endogenous and recombinant pull-down datasets. For example, constructs bearing staples 12 and 27 showed no adverse effect on IP3R1 binding and are predicted to point away from the binding interface based on the helical wheel (FIG. IE) and calculated model structure of the complex (FIG. 2). In addition, staples 12 and 27 share the same N- terminal staple position (L22), with G26 serving as the C-terminal position of staple 12 and a stapling residue added beyond the template sequence serving as the C-terminal position of staple 27, resulting in this set of staple peptides yielding the largest number of alanine mutants for binding analysis (FIGs. 3A-B, Data SI). The two datasets consistently demonstrated that K21 A, R23A, and K25A mutagenesis had the most detrimental effect, impairing IP3R1 pull-down to at or below the 25% binding threshold, with R11 A and Y27A also disrupting binding to a level of approximately 50% or below that of the parent peptides (FIGs. 3C-E).

[0200] Based on the experiments described above, it was noticed that BCL-2 and BCL- XL share identical or homologous positively charged residues in the positions corresponding to BCL-w K21 and K25, whereas R23 is unique to BCL-w, with BCL-2 and BCL-XL both having a serine in this position instead (FIG. 3F). To explore whether R23 confers a binding advantage to BCL-w, a BCL-w BH4 peptide bearing staple 12 and an R23S mutation was generated and tested. Strikingly, R23S mutagenesis markedly impaired IP3R1 pull-down (FIG. 3G), as also observed for a negative control construct bearing staple 13 that replaces R23 with a stapling amino acid (FIGs. 1C, IF). Taken together, the staple and alanine scanning libraries revealed key roles for amino acids within the C-terminal portion of the BCL-w BH4 domain, namely lysine, arginine, and tyrosine residues, in mediating IP3R1 binding activity, with its unique R23 residue emerging as a selectivity factor. These structure-activity relationships are highly compatible with the calculated model structure of the BCL-w BH4 / IP3R1-ARM2 interaction (FIGs. 2F-G).

[0201] To further validate the interface of the complex derived from MD simulation, binding analyses using biolayer interferometry was performed. First, it was confirmed that R23S mutagenesis of SAH-BCL-w BH4-12 impaired binding to WT IP3R1 D3. Then, to explore the impact of protein mutagenesis, two reverse-polarity double mutants of IP3R1 D3 were generated, converting E1319 in combination with either E1387 or E1481 (shown to engage in electrostatic interactions with key lysine or arginine residues of the BH4 C-terminal region [FIG. 2G]) to arginine residues. It was confirmed that the double-mutants had similar purity and migration pattern as assessed by SDS-PAGE and Coomassie stain, and similar overall structural folding as assessed by circular dichroism. E1319R / E1387 and E1319 / E1481 mutagenesis reduced the binding affinity of SAH- BCL-w BH4-12 for IP3R1 D3 by 10-fold and 3-fold, respectively, compared to WT protein. Like the qualitative mutational binding data (FIGs. 1 and 3), these quantitative results are consistent with the model structure of BCL-w BH4 interaction with IP3R1 ARM2 (FIG. 2).

[0202] NEUROPROTECTION BY STAPLED BCL-W BH4 PEPTIDES CORRELATES WITH IP3R1 BINDING ACTIVITY

[0203] Next, it was determined if the structure-activity relationships defined for stapled BCL-w BH4 compositions and IP3R1 binding activity correlated with protection from paclitaxel-induced axonal degeneration. Appropriately designed stapled peptides with the requisite combination of hydrophobicity and pl have previously been shown to achieve cellular uptake by macropinocytosis. Here, in advance of cellular testing, uptake of SAH- BCL-w BH4 peptides by dorsal root ganglion (DRG) neurons was confirmed. Compartmentalized axons of DRG cultures were then pretreated with stapled BCL-w BH4 peptides followed by paclitaxel exposure and quantitation of degeneration. BCL-w BH4 peptides bearing lead staples 12 and 27 were compared with the corresponding i, i+4 and i, i+7 negative control constructs 13 and 25. Whereas SAH-BCL-w BH4-12 and -27 peptides fully preserved the paclitaxel-treated axons to an extent comparable to axons that were not exposed to paclitaxel, SAH-BCL-w BH4-13 and -25 peptides showed no protective effect (FIGs. 4A-B). Next, the impact of R23 mutagenesis was explored and it was observed that introduction of an R23S point mutation into SAH-BCL-w BH4-12 eliminated axonal rescue from paclitaxel treatment (FIGs. 4C-D). Taken together, these data reinforce the staple position and sequence-based specificity of lead BCL-w BH4 constructs in both targeting IP3R1 and protecting axons from paclitaxel-induced degeneration. This identified this site as a key residue in the SAH-BCL-w BH4-12 interaction with IP3R1.

[0204] GENERATION OF BCL-2 BH4 DOMAIN MUTANT WITH IP3R1-D3 BINDING PROPERTIES

[0205] Based on the experiments discussed above, the inventors considered introducing a single S24R mutation (equivalent to position 12 in SEQ ID NO:1) into a stapled construct of BCL-2 BH4 (aa 13-32, analogous staple 14) previously shown not to engage IP3R1 or protect axons from paclitaxel-induced degeneration. Surprisingly, this single point mutation instilled IP3R1 binding properties (FIG. 3G) and significantly enhanced axonal rescue (FIGs. 4C-D) It is further notable that S24 in BCL-2 is an established PKC site (consensus sequence of KXSXR), which upon phosphorylation would create a charge reversal relative to R23 in BCL-w. The data identify a novel BCL-2 mutant that could be exploited to protect axons from paclitaxel -induced degeneration whilst imparting novel properties compared to BCL-w-based peptides.

[0206] DISCUSSION

[0207] A major challenge in managing CIPN is the diversity of neurotoxic drugs, unpredictability and heterogeneity of symptoms and their progression, a lack of mechanistic understanding, and limited options for intervention aside from symptomatic rather than root cause treatments. The discovery that adult Bcl-w~ ' mice develop a progressive decline in thermosensation and nociceptor innervation of the epidermis coincident with selective axonal degeneration provided a key link between BCL-w and axonal neuroprotection. Follow-up studies demonstrated that BCL-w, but not its close homologues BCL-2 or BCL-XL, could protect DRG axons from paclitaxel-induced neuropathy, a phenomenon that could be replicated by the BCL-w BH4 domain alone. Therefore, BCL-2 or BCL-XL were not considered promising leads to manage CIPN. Indeed, BCL-w, rather than BCL-2 or BCL-XL, is enriched in the axons of sensory neurons, with local expression driven by kinesin-mediated trafficking of RNA granules that contain Bcl-w RNA. A target of BCL-w within sensory axons is IP3R1, whose phosphorylation and function are adversely altered by paclitaxel treatment, disrupting homeostatic calcium flux and axonal trafficking of RNA granules. Introduction of BCL- w protein or a stapled BCL-w BH4 peptide into compartmentalized axons but not cell bodies, selectively blocked paclitaxel-induced axonal degeneration, whereas corresponding treatments with BCL-2 and BCL-XL proteins and stapled BH4 peptides had no such effect. In contrast, a BCL-2-based peptide with the S24R mutation had the desired effect and might invoke different effects compared to BCL-w.

[0208] The canonical interactions among BCL-2 family proteins regulate the critical balance between cellular life and death by modulating the integrity of the mitochondrial outer membrane. Complexes between BCL-2 proteins and non-canonical targets continue to emerge and are informing additional roles for these apoptotic regulators in distinct signaling pathways ranging from glucose-stimulated insulin secretion to fatty acid oxidation, and many others. IP3Rs are targets of BCL-2 proteins at the endoplasmic reticulum and the BH4 domains of BCL-2, BCL-XL, and MCL-1 have been implicated as mediators of interaction at various IP3R sites. Given the massive size of tetrameric IP3R (-1200 kDa), discerning both its structure and the molecular mechanisms underlying its complex regulation have been formidable tasks. The cryo-electron microscopy structures of IP3R tetramers have provided a breakthrough.

[0209] Among the sites implicated in BCL-2 and BCL-XL BH4 domain interaction, IP3R D3 is the most surface exposed. Here, it was found that a stapled peptide library of BCL- w BH4 constructs engaged endogenous IP3R1 from cerebellar lysates and recombinant IP3R1 D3 with similar binding patterns, revealing common dependencies on key residues for protein interaction, principally involving positively charged residues R11, K21, R23, and K25, and tyrosine residues Y20 and Y27. Molecular dynamics simulations predict a compatible surface on the ARM2 domain, located within IP3R1-D3 and involving residues K1310, E1319, R1364, F1372, 11376, E1387, E1418, V1419, 11421, A1422, D1472, E1481, and 11482. Of particular interest, among the otherwise highly conserved BH4 domain sequences of BCL-2, BCL-XL, and BCL-w, R23 in BCL-w is unique, with BCL-2 and BCL-XL bearing a serine in this position. Further, the serine residues of BCL- 2 and BCL-XL are located in the context of a PKC consensus sequence, indicating the potential for a charge reversal at this position upon phosphorylation. Indeed, BCL-2 S24 is an established site of regulation by PKC phosphorylation. Interestingly, there is precedent for BH4 domain charge reversals conferring differential activities between BCL-2 and BCL-XL. Specifically, the BCL-2 BH4 sequence contains a lysine at position 17, which is an aspartic acid in the corresponding location in BCL-XL. Single reverse polarity K17D mutagenesis impairs BCL-2 protein and BCL-2 BH4 domain binding to and regulation of IP3R. However, such behavior can be unpredictable and has not been confirmed for BCL-XL. Thus, defining the sequence specificities of distinct BH4-binding and functional activities is essential both to discerning mechanisms of target regulation and approaches to molecular mimicry for therapeutic development. Here, a series of sequence determinants for BCL-w BH4 interaction with IP3R1 was defined - including a selectivity determinant - using chemical staples to reinforce the bioactive structure that enables functional interaction. Intriguingly, paclitaxel exposure and genetic deletion of Bcl-w independently render axons susceptible to degeneration, with paclitaxel-treated DRGs and Bcl-w' ' MEFs both demonstrating reduced calcium flux that was found to be reversed in MEFs upon treatment with a stapled BCL-w BH4 peptide.

[0210] Materials and Methods

[0211] Peptide Synthesis

[0212] Hydrocarbon-stapled peptides corresponding to the BH4 domains of BCL-w (aa 11-28) and BCL-2 (aa 13-32) peptides were synthesized, derivatized at the N-terminus with either Biotin-b-Ala or FITC-b-Ala, purified by LC-MS to >95% purity, and quantified by amino acid analysis according to methods known in the art. Briefly, peptides were synthesized by sequential amino acid addition to Rink Amide AM resin (EMD Biosciences) using Fmoc chemistry and N-terminal derivatization with Biotin or FITC, followed by peptide deprotection, cleavage from the resin, purification by reverse phase high performance liquid chromatography-mass spectrometry (LC-MS), and quantitation by amino acid analysis. Lyophilized peptides were reconstituted in 100% DMSO and diluted into aqueous buffers for experimentation.

[0213] Recombinant Protein Expression and Purification

[0214] IP3RI domain 3 (D3) sequence (aa 923-1581) and its E1319R / E1387R and E1319 / E1481 mutants were cloned into pGEX-69-3 vector bearing an N-terminal GST tag and transformed into One Shot BL21 Star (DE3) competent E. coll cells (Thermo Fisher). Protein expression was induced by the addition of 0.5 mM IPTG (Gold Biotechnology) at 16 °C overnight. Bacterial pellets were resuspended in lysis buffer (1% Triton X-l 00 in PBS) containing protease inhibitor tablets (Roche). Bacteria were lysed using a microfluidizer (M-l 10L, Microfluidics) and centrifuged at 20,000 x RPM for 45 minutes at 4 °C to remove insoluble debris. After bacterial lysis and centrifugation, the clarified lysate was purified by GST affinity chromatography using glutathione sepharose resin (GE Healthcare) equilibrated with lysis buffer on a gravity flow column. The GST- fusion protein was eluted with 10 mM of reduced glutathione dissolved in 50 mM Tris- HCL (pH 8.0). Protein purity and identity was respectively confirmed by Coomassie staining and western blot analysis using a rabbit polyclonal anti-GST HRP-conjugated antibody (Cat# Ab3416, Thermo Fisher; RRID: AB 303783).

[0215] Biotin Pull-downs

[0216] For endogenous IP3R1 pull-down analyses, mouse cerebellum was homogenized in lysis buffer containing 1% Triton X-100, 137 mM NaCl, 20 mM Tris pH 7.4, 10% glycerol, and EDTA-free complete mini protease inhibitor cocktail (Sigma-Aldrich). The lysate was centrifuged at 14,000 x rpm for 10 min at 4 °C to remove cell debris. Protein concentration was determined using the Bradford protein assay (Sigma- Aldrich). Lysates (300 mg) were incubated with 10 mM biotinylated peptides or vehicle (1% DMSO) in a final reaction volume of 200 mL for 2 h at 4 °C. High-capacity streptavidin agarose beads (Thermo Fisher) were pre-blocked with 3% BSA PBS-Tween for 2 h at 4 °C. The preblocked beads were then washed with lysis buffer and incubated with the lysate-peptide mixture overnight at 4 °C. The next day, lysate was removed, beads were washed with cold PBS, and protein was eluted by boiling for 5 min in LDS sample buffer with 100 mM DTT. Eluates were separated by 4-12% Bis-Tris SDS-PAGE. Inputs (5%) from the original lysate were run alongside the pull-down eluates. Proteins were transferred to nitrocellulose membrane using the iBlot2 gel transfer system (Life Technologies). The membrane was blocked in 5% milk overnight on a rocker at 4 °C. After blocking, blots were washed three times with PBS-Tween at room temperature and incubated with anti- IP3R1 antibody (Cat# PAI-901, Thermo Fisher; RRID:AB_2129984) in 3% BSA in PBS. Blots were washed 3 times with PBS-Tween at room temperature and incubated with SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher) for visualization of protein. Band intensities were quantified using ImageJ software and normalized to the band of highest intensity. Cerebella were obtained from C57BL / 6J mice in accordance with the guidelines and regulations set forth by the Institutional Animal Care and Use Committee of the Dana-Farber Cancer Institute and in compliance with approved study protocol #06-004.

[0217] For IP3R D3 pull-down analyses, recombinant IP3RI domain 3 protein (0.5 mM) was incubated with 0.5 mM biotinylated peptides or vehicle (0.05% DMSO) for 2 h at 4 °C. The protein-peptide mixtures were then combined with PBS-washed, pre-blocked high-capacity streptavidin agarose beads (Thermo Fisher) overnight at 4 °C. The next day, beads were washed three times each with 1% BSA PBS-Tween, PBS-Tween, and then PBS. Bound protein was eluted from the beads by 5 min of boiling in 3x LDS supplemented with 100 mM DTT, separated by 4-12% Bis-Tris SDS-PAGE, and probed with anti-GST HRP-conjugated antibody (Cat# Ab3416, Abeam; RRID: AB_303783). Band intensities were quantified using ImageJ software and normalized to the band of highest intensity.

[0218] Biolayer Interferometry

[0219] Quantitative binding analyses of BH4 peptides and IP3R1 D3 proteins were performed on an Octet RED384 system (Fortebio, Menlo Park, CA) at 30°C. Octet Streptavidin (SA) Biosensors tips were prewetted in PBS, pH 7.4, 0.1% BSA and then coated with BH4 peptides (0.5 mM) via their N-terminal biotin-PAla moiety. The tips were then washed with PBS, pH 7.4, 0.1% BSA and soaked in an 8-fold serial dilution of IP3R1 D3 protein for 15 min to measure association rate. The fractional response relative to buffer alone was plotted vs. protein concentration and Kd calculated in Prism 10 (Graphpad) by nonlinear regression analysis using the specific binding model with Hill slope.

[0220] Circular Dichroism

[0221] CD spectra were recorded on an Aviv Biomedical spectrometer (Model 410) equipped with a Peltier temperature controller, 1-mm path-length cells, and a thermoelectric sample changer with 5-position rotor, scanning 190-260 nm in 0.5-nm increments with a 0.5-second averaging time (five scans were averaged). BH4 peptides were dissolved in 20% acetonitrile / 80% water to a final concentration of 100 mM. IP3R1 D3 proteins were dissolved in 5 mM potassium phosphate (pH 7.5) to a final concentration of 2.5 mM.

[0222] MEF Generation and Culture

[0223] Immortalized MEFs were generated from epidermal tissue harvested from Bcl-w1' and WT littermate E12 embryos followed by SV40 transformation. The absence of BCL- w protein from Bcl-wJ' MEFs was confirmed by western analysis using the BCL-w 31H4 rabbit monoclonal antibody (Cat# 2724, Cell Signaling, RRID: AB_10691557). Following immortalization, the MEFs were expanded and stored at -80 °C. Prior to calcium flux assays, the cells were thawed and cultured at 37°C, 95% C02, 5% 02 in MEF media (DMEM, 10% FBS, 1% Glutamax, 1% Penicillin-Streptomycin) and passaged at 80% confluency. One day prior to calcium flux experiments, cells were plated on poly-l-lysine coated glass coverslips and attached overnight prior to imaging.

[0224] Calcium Flux Assays

[0225] Bcl-1' and WT MEFs stably expressing ER-GCaMP6s-150 were generated by lentivirus using the reported sequence AddGene. Briefly, MEFs were pre-treated with polybrene, exposed to either control virus or ER-GCaMP6s-150 lentivirus at a multiplicity of infection (MOI) of 5, and incubated for 24 hours. After a media change, the cells were incubated for another 24 hours and then puromycin (2 mg / ml) was added for selection. Puromycin was replenished every two days until cells infected with control virus were eliminated (~8 days). Cell lines of each genotype were expanded and stored at -80°C until experimentation. For cytosolic calcium measurements, MEFs were loaded with the ratiometric calcium indicator Fura Red (2 mM) in dye loading media prepared on the day of experimentation. Specifically, Fura Red dye was reconstituted as a 1 mM stock in Pluronic F-127 and combined with Live Cell Imaging Solution (Invitrogen), bovine serum albumin (1 mg / mL), D-Glucose (10 mM), and probenecid (1%). Cell culture media was replaced with dye loading media and the cells incubated at room temperature in the dark for 30 min. For experiments involving pretreatment with SAH- BCL-w BH4-12 peptide, stapled peptide (5 mM stock in DMSO) or vehicle was added to reduced serum media (1%) to achieve a final concentration of 5 mM peptide (0.1% DMSO). The cell culture media was replaced with peptide loading media, allowing for a 1-hour incubation (37° C; 95% CO2, 5% O2) prior to replacement with dye loading media. Prior to the start of the calcium flux assay, cell culture media or dye loading media was replaced with calcium-free imaging solution (CFIS, 140 mM NaCl, 5 mM KC1, 3.35 mM MgCb, 10 mM glucose, 10 mM HEPES, pH 7.4), followed by equilibration for 8-15 min. For Fura Red experiments, this incubation also served as a deesterification period. Glass coverslips were mounted to a recording chamber using vacuum grease and placed on a heated microscope stage (37° C). CFIS was perfused through the chamber for at least one min prior to initiating the time lapse and for at least one min prior to switching to 10 mM ATP in CFIS. Cells were bathed with ATP continuously for 90 sec and then perfusion switched back to CFIS. For Fura Red experiments sequential images were acquired with 440 nm excitation / 580 nm emission and then 480 nm excitation / 580 nm emission, at 30 msec exposure per channel. For ER- GCaMP6s-150, images were acquired with 470 nm excitation / 525 nm emission. For all experiments, 16-bit images / image-sets were acquired in one-sec intervals with 2x2 binning. ER-GCaMP6s-150 and Fura Red experiments were conducted using 60x (NA 1.4) and 40x (NA 1.3) oil immersion lenses, respectively, on a Nikon Ti2 inverted microscope with Hamamatsu ORCA-R2 CCD camera (Nikon Imaging Center, Harvard Medical School). Automated analyses of calcium transients extracted from timelapse files were accomplished using Imaged and Python. Seed images were generated from maximum projections of timelapses and parsed with Cellpose to segment the field of view into regions of interest (ROIs). These ROIs were manually reviewed for quality control to exclude those cells that were not sufficiently within the field of view. The mean pixel intensity was then extracted for each ROI, including every frame within the timelapse. For Fura Red experiments, ratiometric changes were calculated by dividing the emission values from the 440 nm excitation by the 480 nm excitation. For both Fura Red and ER-GCaMP6s-150 experiments, percent change from baseline was calculated for ratio or fluorescence intensity measurements via baseline normalization (defined as the mean value for the ROI within the 30 sec period prior to stimulus onset). Maximum response was defined as either the maximum value (Fura Red) or absolute value of the minimum value (ER-GCaMP6s) during the stimulus period. Area under the curve was calculated by summing the absolute value of the percent change from baseline for every frame within the stimulus period.

[0226] Molecular Dynamics Simulation

[0227] The relatively complete Rattus norvegicus Ca2+ / ATP / IP3 -bound IP3R Type I structure (PDB ID: 8EAR) was selected as a robust substrate for simulation. To reduce overhead, the domain spanning residues 1200-1566 were extracted for computational analyses and then used for coarse-grained (CG) simulations to identify a binding site for the BCL-w BH4 peptide. Placement was accomplished by using the CABS methodology to fold a fully flexible peptide model (HiN -RALVADFVGYKLRQKGYV-COO ) in the presence of a restrained yet flexible subset of the IP3R1 receptor ARM2 region. A series of ten CABS trajectories were calculated and 1000 models were sampled from each trajectory. The resulting ensemble of 104candidates was filtered to remove unbound systems and clustered using the k-medoids method. This delivered ten plausible peptide- receptor complexes, with the atomic details for these CG alpha-carbon traces reconstructed using the MODELLER package. All-atom molecular dynamics simulations were used to map explicit molecular interactions between IP3R1 and the BCL-w BH4. Simulations were initiated using reconstructed medoids from the top CABS clusters and rotated so that their principal axes would lie along the orthorhombic cell vectors, thus minimizing cell volume. Protein dynamics were described using the CHARMM36 protein force field. Systems were solvated using a TIP3P water (15 A padding) and supplemented with 0.15 M K7Cf to ensure electrical neutrality. The initial simulation cells measured approximately 91 x 117 x 85 A3 in volume. Simulations were driven by NAMD 2.14 code, which uses a fully periodic BBK-type integrator with velocity rescaling. Rigid bond constraints were employed to achieve numerical stability at a timestep of 8t = 2 fs. The NVT calculations applied a Langevin thermostat to heavy atoms for temperature control (T = 300.0 K; damping y = 1.0 ps-1), while NPT simulations controlled pressure with a Langevin piston (target P = 101.325 kPa; period = 100.0 fs; decay time = 50.0 fs). Anisotropic cell fluctuations were allowed and multiple timestepping was used for nonbonded interactions, with short-range interactions evaluated every 2 fs and full electrostatics every 4 fs. Short-range interactions were cut off at 1.2 nm and smoothed with sigmoidal rescaling for atoms separated by more than 1.0 nm. Smoothed particle mesh Ewald (PME) was used for long-range electrostatics. Equilibration began with a 2000 step conjugate gradient (CG) minimization followed by 1.0 ns of NVT equilibration with all protein atoms fixed. Next, a harmonic constraint potential (kprot = 5 kcal mol-1 A-2) was applied to hold protein atoms near their initial positions, and 2000 steps of CG minimization and 2.0 ns of NPT equilibration was used to relax the protein. Finally, all constraints were removed, and production calculations were run for 100 ns in the NPT ensemble. Structural samples were captured every 5 ps, and the first 20 ns of the trajectories were discounted as an initial equilibration period, giving 80 ns of production simulation. This timescale was long enough to relax the BCL- w BH4-IP3R1 complexes yet short enough to prevent unwanted displacements from excluded segments of the IP3R1 complex. Three independent simulations were run for the cluster. Confocal Microscopy

[0228] DRG neurons were incubated with FITC-tagged stapled BCL-w BH4 peptides (1 mM) or vehicle (0.0025% DMSO) for 3 hours in neural basal media supplemented with 2% B27, 1% Glutamax, 1% penicillin and streptomycin, 0.08% glucose andlO ng / ml nerve growth factor and brain-derived neurotrophic factor. DRG neurons were then washed with PBS and fixed with 4% paraformaldehyde for 20 min. Cultures were permeabilized with 0.1% Triton X-100 for 10 min, blocked in 10% normal donkey serum containing 0.1% Triton X-100 for 1 hour at room temperature, and then incubated with mouse anti-Tuj 1 (1 : 1000; Cat# 801201, Biolegend, RRID: AB_2313773) and goat anti- FITC (1 :500; Cat# Abl9224, Abeam, RRID: AB_732395) overnight at 4 °C. Cultures were then incubated with donkey anti-mouse AlexaFluor-647 (1: 1000; Cat# A31571, Thermo Fisher, RRID: AB_162542) and donkey anti-goat AlexaFluor-568 (l : 1000;Cat# Al 1057, Thermo Fisher, RRID: AB 2534104) for 1 hour at room temperature. Images of DRG neurons were acquired using NIS-elements (Nikon) on a Nikon Ni-E C2 confocal microscope.

[0229] Axonal Degeneration Assay

[0230] Dorsal root ganglia (DRG) were dissected from El 5 rat embryos, dissociated and plated in Matrigel-coated (Thermo Fisher) p35 Campenot chambers. DRG cultures were maintained in NeuroBasal medium supplemented with 2% B27, 1% Glutamax, 1% penicillin and streptomycin, 0.08% glucose, 1-100 ng / mL NGF / BDNF (PeproTech), and 0.5 mM cytarabine. Cultures were maintained in incubators at 37 °C with 7.5% CO2. Campenot devices were prepared as described. BDNF and NGF were added to the cell body compartment at a concentration of 10 ng / mL and to the axon compartment at a concentration of 100 ng / mL for 2 days. On day 5, neurotrophins were removed from the cell body compartment and reduced to 1 ng / mL in the axon compartments for 2-3 days. On day 8, the peptide (10 nM) was added to each axon compartment 2 h before the addition of paclitaxel (30 nM) or vehicle (0.0025% DMSO), followed by 24 h incubation and then fixation. Neurons in Campenot cultures were fixed at room temperature with 4% PFA diluted 1 :2 in media for 10 min and then with undiluted 4% PFA for an additional 20 min. Cultures were permeabilized and blocked in 3% BSA and 0.1% Triton X-100 for 1 h at room temperature, and then incubated with mouse anti-Tuj 1 (Cat# 801201, BioLegend; RRID: AB_2313773) overnight at 4 °C. Cultures were then washed twice with PBS and incubated with goat anti-mouse AlexaFluor-488 (Cat# A-11001, Thermo Fischer; RRID: AB 2534069) and DAPI for 1 h at room temperature. Campenot dividers were then removed and samples mounted with Fluoromount-G (Southern Biotech 0100- 01). Images of distal axon tips were obtained using a Nikon Eclipse E800 microscope (40X air objective), Nikon Digital Sight DS U2 camera at room temperature, and NIS elements imaging software (Nikon). The images were analyzed using ImageJ, binarized, and axonal degeneration quantified as a degeneration index ratio of fragmented axons (particle size: 1-1000) divided by total axon area in each image. DRGs were obtained from El 5 Sprague-Dawley rat embryos in accordance with the guidelines and regulations set forth by the Institutional Animal Care and Use Committee of the Dana-Farber Cancer Institute and in compliance with approved study protocol #01-113.

[0231] QUANTIFICATION AND STATISTICAL ANALYSIS

[0232] The number and type of replicates for each experiment are indicated in the corresponding figure legends. Mean ± SEM values were calculated using Prism software (Graphpad). Statistical significance was determined by unpaired two-tailed Student’s t- test or one-way ANOVA test using GraphPad Prism software.

[0233] OTHER ASPECTS

[0234] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. All cited sources, for example, references, publications, databases, database entries, and art cited herein, are incorporated into this application by reference, even if not expressly stated in the citation. In case of conflicting statements of a cited source and the instant application, the statement in the instant application shall control.

Claims

WHAT IS CLAIMED IS:

1. A BCL-2 mutant peptide, the BCL-2 mutant peptide comprising the amino acid sequence EIVMKYIHYKLSQRGYEWDA (SEQ ID NO: 1) with S at position 12 substituted by a positively charged amino acid and optionally further including 1 to 4 other amino acid substitutions within SEQ ID NO: 1, optionally wherein the BCL-2 mutant peptide binds inositol 1,4,5-trisphosphate receptor, type 1 (IP3R1) or BAX.

2. The BCL-2 mutant peptide of claim 1, wherein S at position 12 is substituted with one of His, Lys, Arg, or Orn.

3. The BCL-2 mutant peptide of claim 1, wherein S at position 12 is substituted with Arg.

4. The BCL-2 mutant peptide of any one of claims 1 to 3, wherein (i) Leu at position 11 and Gly at position 15, or (ii) Gin at position 13 and Glu at position 17, or (iii) Leu at position 11 and Trp at position 18 of SEQ ID NO: 1 are each substituted with an a,a-disubstituted non-natural amino acid.

5. The BCL-2 mutant peptide of any one of claims 1 to 4, wherein the M at position 4 of SEQ ID NO: 1 is substituted with norleucine.

6. The BCL-2 mutant peptide of any one of claims 1 to 5, wherein one or more of positions 10, 14, and 16 of SEQ ID NO: 1 are not substituted, or if substituted are substituted with a conservative amino acid substitution.

7. The BCL-2 mutant peptide of any one of claims 1 to 6, wherein the BCL-2 mutant peptide includes an Arg immediately N-terminal to Glu at position 1 of SEQ ID8. The BCL-2 mutant peptide of claim 1 comprising a sequence selected from the group consisting of:EIVMKYIHYKLRQRGYEWDA (SEQ ID NO: 2), EIVBKYIHYKLRQRGYEWDA (SEQ ID NO: 3), REIVMKYIHYKLRQRGYEWDA (SEQ ID NO: 4), and REIVBKYIHYKLRQRGYEWDA (SEQ ID NO: 5), with 2 to 4 amino acid substitutions, wherein B is norleucine; wherein none of the 2 to 4 amino acid substitutions replace amino acids at positions 10, 12, 14, or 16 of SEQ ID NOY or 3, or positions 11, 13, 15, or 17 of SEQ ID NO:4 or 5, or if positions 10, 12, 14, or 16 of SEQ ID NO:2 or 3, or positions 11, 13, 15, or 17 of SEQ ID NO:4 or 5 are substituted, it is with conservative amino acid substitutions; and optionally wherein 2 of the 2 to 4 amino acid substitutions replace amino acids three or six amino acids apart within any one of SEQ ID NOs:2-5 with a,a-disubstituted non-natural amino acids.

9. The BCL-2 mutant peptide of any one of claims 1 to 8, wherein the BCL-2 mutant peptide binds to IP3R1, BAX, or IP3R1 and BAX.

10. A stapled peptide, the stapled peptide comprising the amino acid sequence EIVMKYIHYKX1SQRX2YEWDA (SEQ ID NO:6), EIVMKYIHYKLSXIRGYX2WDA (SEQ ID NOY), or EIVMKYIHYK8SQRGYEXDA (SEQ ID NO:8) with S at position 12 of any one of SEQ ID NOs: 6-8 substituted by a positively charged amino acid and optionally further including 1 or 2 other amino acid substitutions within any one of SEQ ID NOs:6- 8, wherein the 1 or 2 other substitutions are not at Xi or X2 of SEQ ID NOs: 6 and 7 and are not at 8 or X of SEQ ID NO: 8, wherein Xi , X2 , 8, and X are a, a-di substituted non-natural amino acids, and wherein the side chainsof Xi and X2 or 8 and X, respectively, are cross linked, and optionally wherein the stapled peptide binds IP3R1.11 . The stapled peptide of claim 10, wherein S at position 12 of any one of SEQ ID NOs:6-8 is substituted with one of His, Lys, Arg, and Orn.

12. The stapled peptide of claim 10, wherein S at position 12 of any one of SEQ ID NOs:6-8 is substituted with Arg.

13. The stapled peptide of any one of claims 10 to 12, wherein Xi and X2 are each (S)-2-(4-pentenyl)alanine, and wherein 8 is (R)-a-(7'-octenyl)alanine and X is (S)-2-(4-pentenyl)alanine.

14. The stapled peptide of any one of claims 10 to 13, wherein M at position 4 of any one of SEQ ID NOs:6-8 is substituted with norleucine.

15. The stapled peptide of any one of claims 10 to 14, wherein one or more of positions 10, 14, and 16 of SEQ ID NOs:6-8 are not substituted, or if substituted are substituted with a conservative amino acid substitution.

16. The stapled peptide of any one of claims 10 to 15, wherein the stapled peptide includes an Arg immediately N-terminal to Glu at position 1 of any one of SEQ ID NOs:6-8.

17. The stapled peptide of claim 10, comprising a sequence selected from the group consisting of:EIVMKYIHYKX1RQRX2YEWDA (SEQ ID NO: 9), EIVBKYIHYKX1RQRX2YEWDA (SEQ ID NOTO), EIVMKYIHYKLRX1RGYX2WDA (SEQ ID NO: 11),EIVBKYIHYKLRX1RGYX2WDA (SEQ ID NO: 12), EIVMKYIHYK8RQRGYEXDA (SEQ ID NO: 13), EIVBKYIHYK8RQRGYEXDA (SEQ ID NO: 14), REIVMKYIHYKX1RQRX2YEWDA (SEQ ID NO: 15), REIVBKYIHYKX1RQRX2YEWDA (SEQ ID NO: 16), REIVMKYIHYKLRX1RGYX2WDA (SEQ ID NO: 17), REIVBKYIHYKLRX1RGYX2WDA (SEQ ID NO: 18), REIVMKYIHYK8RQRGYEXDA (SEQ ID NO: 19), and REIVBKYIHYK8RQRGYEXDA (SEQ ID NO:20), with 0 to 2 amino acid substitutions relative to SEQ ID NOs: 9, 11, 13, 15, 17, and 19, or 0 or 1 amino acid substitution relative to SEQ ID NOs: 10, 12, 14, 16, 18, and 20, wherein B is norleucine.

18. The stapled peptide of any one of claims 10 to 17, wherein the stapled peptide binds IP3R1, BAX, or IP3R1 and BAX.

19. A stapled peptide, the stapled peptide comprising the amino acid sequence RALVADFVGYKX1RQKX2YV (SEQ ID NO:21) with 1 to 3 amino acid substitutions, wherein Xi and X2 are a, a-di substituted non-natural amino acids, wherein the side chains of Xi and X2 are cross linked, and wherein positions 12 and 16 of SEQ ID NO:21 are not substituted, optionally wherein the stapled peptide also binds IP3R1, and further optionally wherein the stapled peptide binds one or both of BAX and BAK.

20. The stapled peptide of claim 19, wherein one or more of L at position 3, V at position 4, D at position 6, F at position 7, V at position 8, G at position 9, Y at position 10, Q at position 14, G at position 16, or V at position 18 of SEQ ID NO:21 are substituted with an alanine, glycine, valine, or leucine.

21. The stapled peptide of claim 19, wherein one of L at position 3, V at position 4, D at position 6, F at position 7, V at position 8, G at position 9, Y at position 10, Q at position 14, G at position 16, or V at position 18 of SEQ ID NO:21 is substituted with an alanine, glycine, valine, or leucine.

22. The stapled peptide of any one of claims 19 to 21, wherein one or more of R at position 1, Y at position 10, K at position 11, R at position 13, K at position 15, and Y at position 17 of SEQ ID NO:21 are not substituted or, if substituted, R at position 1, K at position 11, R at position 13, or K at position 15 of SEQ ID NO:21 is substituted with a positively charged amino acid, and Y at position 10 of SEQ ID NO:21 is substituted with a hydrophobic, particularly aromatic amino acid.

23. The stapled peptide of any one of claims 19 to 22, wherein Xi and X2 of SEQ ID NO:21 are each (S)-a-(4'-pentenyl)alanine.

24. A stapled peptide, the stapled peptide comprising the amino acid sequence RALVADFVGYK8RQKGYVX (SEQ ID NO:22) with 1 to 3 amino acid substitutions, wherein 8 and X are a, a-di substituted non-natural amino acids, wherein the side chains of 8 and X are cross linked, and wherein positions 12 and 19 of SEQ ID NO:22 are not substituted, optionally wherein the stapled peptide binds IP3R1, and further optionally wherein the stapled peptide binds one or both of BAX and BAK.

25. The stapled peptide of claim 24, wherein one or more of D at position 6 and G at position 9 of SEQ ID NO:22 are substituted with an alanine, glycine, valine, or leucine.

26. The stapled peptide of claim 24 or 25, wherein one or more of R at position 1, Y at position 10, K at position 11, R at position 13, K at position 15, and Y at position 17 of SEQ ID NO:22 are not substituted, or, if substituted, R at position 1 , K at position 11 , R at position 13, or K at position 15 of SEQ ID NO:22 is substituted with a positively charged amino acid, and Y at position 10 of SEQ ID NO:22 is substituted with a hydrophobic, particularly aromatic amino acid.

27. The stapled peptide of any one of claims 24 to 26, wherein 8 and X of SEQ ID NO:22 are (R)-a-(7'-octenyl)alanine and (S)-a-(4'-pentenyl)alanine, respectively.

28. The BCL-2 mutant peptide of any one of claims 1 to 9 or the stapled peptide of any one of claims 10 to 27, wherein the BCL-2 mutant peptide or the stapled peptide is 20 to 50, 20 to 40, 20 to 30, 20 to 25, 21, 20, 19, less than 50, less than 40, less than 30, or less than 25 amino acids in length.

29. A method of making a stapled peptide, the method comprising:(a) providing:(i) the BCL-2 mutant peptide of any one of claims 1 to 9,(ii) a peptide comprising the amino acid sequence EIVMKYIHYKX1SQRX2YEWDA (SEQ ID NO:30), EIVMKYIHYKLSXIRGYX2WDA (SEQ ID NO:31), or EIVMKYIHYK8SQRGYEXDA (SEQ ID NO:32) with S at position 12 of any one of SEQ ID NOs:30-32 substituted by a positively charged amino acid and optionally further including 1 or 2 other amino acid substitutions within any one of SEQ ID NOs:30-32, wherein the 1 or 2 other substitutions are not at Xi or X2of SEQ ID NOs: 30 and 31 and are not at 8 or X of SEQ ID NO:32, wherein Xi , X2, 8, and X are a,a-disubstituted non-natural amino acids,(iii) a peptide comprising the amino acid sequence RALVADFVGYKXIRQKX2YV (SEQ ID NO:33) with 1 to 3 amino acidsubstitutions, wherein Xi and X2 are a, a-di substituted non-natural amino acids, and wherein positions 12 and 16 of SEQ ID NO:33 are not substituted, or(iv) a peptide comprising the amino acid sequence RALVADFVGYK8RQKGYVX (SEQ ID NO: 34) with 1 to 2 amino acid substitutions, wherein 8 and X are a, a-di substituted non-natural amino acids, wherein the side chains of 8 and X are cross linked, and wherein positions 12 and 19 of SEQ ID NO:34 are not substituted; and(b) cross-linking the peptide thereby making the stapled peptide, optionally wherein the cross-linking is via a ring-closing metathesis (RCM) reaction, and further optionally formulating the stapled peptide as a sterile pharmaceutical composition.

30. A pharmaceutical composition comprising the mutant BCL-2 peptide of any one of claims 1 to 9 or the stapled peptide of any one of claims 10 to 28, and a pharmaceutically acceptable carrier.

31. A method of selectively protecting sensory neurons from age-related and / or chemotherapy induced axonal degeneration in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of the mutant BCL-2 peptide of any one of claims 1 to 9 or the stapled peptide of any one of claims 10 to 28, or the pharmaceutical composition of claim 30.

32. A method of treating chemotherapy induced peripheral neuropathy (CIPN) in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of the mutant BCL-2 peptide of any one of claims 1 to 9 or the stapled peptide of any one of claims 10 to 28, or the pharmaceutical composition of claim 30.

33. A method of preventing chemotherapy induced peripheral neuropathy (CIPN) in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of the mutant BCL-2 peptide of any one of claims 1 to 9 or the stapled peptide of any one of claims 10 to 28, or the pharmaceutical composition of claim 30.

34. A method for averting neuropathic side effects of chemotherapy and / or other causes of axonal degeneration in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of the mutant BCL-2 peptide of any one of claims 1 to 9 or the stapled peptide of any one of claims 10 to 28, or the pharmaceutical composition of claim 30.

35. A method for treating or preventing hearing loss in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of the mutant BCL-2 peptide of any one of claims 1 to 9 or the stapled peptide of any one of claims 10 to 28, or the pharmaceutical composition of claim 30.

36. A pharmaceutical composition comprising (a) means for selectively protecting sensory neurons from age-related and / or chemotherapy induced axonal degeneration in a human subject in need thereof, and (b) a pharmaceutically acceptable carrier.

37. A pharmaceutical composition comprising (a) means for treating chemotherapy induced peripheral neuropathy (CIPN) in a human subject in need thereof, and (b) a pharmaceutically acceptable carrier.

38. A pharmaceutical composition comprising (a) means for preventing chemotherapy induced peripheral neuropathy (CIPN) in a human subject in need thereof, and (b) a pharmaceutically acceptable carrier.

39. A pharmaceutical composition comprising (a) means for averting neuropathic side effects of chemotherapy and / or other causes of axonal degeneration in a human subject in need thereof, and (b) a pharmaceutically acceptable carrier.

40. A pharmaceutical composition comprising (a) means for treating or preventing hearing loss in a human subject in need thereof, and (b) a pharmaceutically acceptable carrier.

41. A pharmaceutical composition comprising (a) means for binding human IP3R1, and (b) a pharmaceutically acceptable carrier, optionally wherein the binding is in the D3 arm of IP3R1.

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