Stapled bad BH3 helices targeting BCL-2 mutants that cause venetoclax resistance

Abstract

Stapled BAD BH3 peptides and compositions comprising the same that are useful in overcoming venetoclax resistance are provided. Also provided are methods of using the stapled BAD BH3 peptides for treating a BCL-2 expressing and / or dependent cancer (e.g., a hematologic cancer or a solid tumor) in a human subject in need thereof. In some cases, the cancer is a venetoclax-resistant cancer due to acquired mutations in the BCL-2 protein. The methods involve administering to the human subject a stapled BAD BH3 peptide or pharmaceutical composition or delivery vehicle comprising the stapled BAD BH3 peptide disclosed herein.

Description

[0001] Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0002] STAPLED BAD BH3 HELICES THAT TARGET BCL-2 MUTANTS THAT CAUSE CANCER RESISTANCE TO VENETOCLAX

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Application No. 63 / 714,610, filed on October 31, 2024. The entire contents of the foregoing are incorporated herein by reference.

[0005] SEQUENCE LISTING

[0006] This application contains a Sequence Listing that has been submitted electronically as an XML file named 00530-0425W01_SL.xml. The XML file, created on October 29, 2025, is 131,072 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0007] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0008] This invention was made with government support under R35CA197583 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

[0009] BACKGROUND

[0010] The B-cell lymphoma-2 (BCL-2) family of proteins regulates cell death by direct binding interactions that regulate mitochondrial outer membrane permeabilization (MOMP), which represents the irreversible release of intermembrane space proteins and subsequent caspase activation leading to apoptosis. The affinities and relative abundance of the BCL-2 family proteins dictate the interactions between different members of the BCL-2 family proteins that regulate MOMP.

[0011] The BCL-2 family is divided into three groups based on their primary' function. The first group are the anti-apoptotic proteins and include the BCL-2, BCL-XL, BCL-W, Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0012] MCL-1, and BFL-1 / A1 proteins. The second group are the pro-apoptotic pore-former proteins and include the BAX, BAK, and BOK proteins. Finally, the third group are the pro-apoptotic BH3-only proteins, which can be further categorized into “activators” or “sensitizers” based on their primary interaction schemes with pro- or anti-apoptotic groups, respectively. BH3-only activators include the BID, BIM, NOXA, and PUMA proteins, while BH3-only sensitizers include the BAD, BIK, BMF, BNIP3, HRK, and NOXA proteins. Some BH3-only proteins, such as NOXA, are often classified as context-dependent.

[0013] All BCL-2 family proteins contain a BCL-2 homology 3 (BH3) domain which is one of four BH domains involved in interactions between these proteins. The anti- apoptotic and pore-forming proteins contain all four BH domains (and thus are referred to as multi-BH domain proteins) and adopt a highly conserved tertiary structure forming a hydrophobic BH3 domain-binding groove that acts as a receptor for BH3 domains of other family members. The BH3 domain is required for the primary apoptotic function of BCL-2 family members and the interactions between them in the cytosol and at intracellular membranes, such as the mitochondria. The BH3 domain of activator BH3- only proteins target an N-terminal surface groove on BAX to trigger its pro-apoptotic activity. This leads to the activation of BAX and results in a series of conformational changes that lead to homo-oligomerization and pore formation within the mitochondrial outer membrane (MOM). The BH3 domain-binding surface groove of the anti-apoptotic proteins binds and traps the BH3 domains of pro-apoptotic members, inhibiting their function and thus apoptosis.

[0014] Venetoclax is a highly selective and effective BCL-2 inhibitor, which is able to reinstate the apoptotic potential of cancer cells. This BH3 mimetic binds to the BCL-2 surface groove with high affinity, disrupting the capacity of BCL-2 to suppress pro- apoptotic members and thus lowering the threshold for apoptosis induction. This drug has positively impacted the therapeutic landscape in hematological and other malignancies. Despite its promising efficacy, patients can relapse due to multiple mechanisms contributing to drug escape. One of the primary resistance mechanisms is Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO the development of mutations in the BCL-2 gene conferring reduced venetoclax binding while preserving BH3-binding capacity.

[0015] Thus, there exists a need in the art to develop compositions and methods to counter the mutants of BCL-2 that confer venetoclax resistance.

[0016] SUMMARY

[0017] The present disclosure is based, at least in part, on the identification of stapled peptides that selectively inhibit BCL-2 and its venetoclax-resistance mutants. These stapled peptides have clinical applications in treating human cancers. Anti-apoptotic BCL-2 proteins contain a surface groove that traps the BH3 “killer domains” of pro- apoptotic family members, preventing this alpha-helical structure from activating cell death (Kale et al, Cell Death & Diff, 2018, 25(l):65-80). Venetoclax, a small -molecule BCL-2 inhibitor with therapeutic activity in acute and chronic leukemia (Souers et al, Nat Med, 2013, 19(2):202-8; Roberts et al, N Engl J Med, 2016, 374:311-322), blocks prosurvival proteins from trapping the BH3 domains of pro-death BCL-2 family members. However, clinical trials have revealed the emergence of mutations in BCL-2 that prevent venetoclax from blocking BCL-2 interactions, resulting in drug resistance and relapsed disease. Efforts to overcome venetoclax-resistance mutations with next-generation small molecule inhibitors of BCL-2 still exhibit decreased affinity for mutant proteins (Guo et al, J Med Chem, 2024, 67, 10, 7836-7858; Hafezi and Rahmani, Cancers, 2021, 13(6): 1292). Disclosed herein are peptides that mimic the natural BAD BH3 motif and selectively bind to BCL-2, including venetoclax resistance mutants of BCL-2 (see, e.g., FIGS. 3, 4, and 22). Since certain cancers depend on BCL-2, and relapsed cancers can depend on mutant BCL-2, overcoming such mutations is clinically important. The compounds exhibit preference for BCL-2 over homologous anti-apoptotic proteins, which has been shown to maximize the therapeutic window (Roberts et al, J Clin Oncol, 2012, 35(4):394-401). Thus, this disclosure encompasses the compositions of selective stapled peptide inhibitors of BCL-2 (exemplary stapled peptides encompassed by this disclosure are provided in FIGS. 3A, 4A, and 22 as well as mutants thereof that bind to venetoclax- Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO resistant BCL-2), demonstrates their biochemical and functional activity, presents crystal structures of selective BCL-2 inhibitor prototypes in complex with WT and mutant BCL- 2, and reveals the conformational consequences of compound interactions with WT and venetoclax-resistance mutations of BCL-2.

[0018] Thus, provided herein are polypeptides comprising a stapled peptide, wherein the stapled peptide comprises the structure of formula (III):

[0019] Formula (III) or a pharmaceutically acceptable salt thereof (e g., hydrochloride, mesylate, hydrobromide, acetate, fumarate, sodium, calcium, and potassium) wherein: each Ri and R2 is independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, any of which is substituted or unsubstituted. In some cases, each Ri and R2 is a methyl; and each Ra is independently alkylene, alkenylene, or alkynylene, any of which is substituted or unsubstituted. In certain cases, R3 is Cs alkylene, Cs alkenylene, or Cs alkynylene. In some cases, R3 is (CH2)3-CH=CH-(CH2)3. The stapled peptide, or the pharmaceutically acceptable salt thereof, comprises 2 to 6 substitutions relative to the sequence WAAQRYGRELRRBSDEFVDSFK (SEQ ID NO:3), wherein B is norleucine. In some cases, the stapled peptide or the pharmaceutically acceptable salt thereof comprises two substitutions relative to the sequence of SEQ ID NO:3 at (i) positions 2 and 6 of the sequence of SEQ ID NO:3, or (ii) positions 6 and 10 of the sequence of SEQ ID NO:3. These two positions (i.e., 2 and 6 or 6 and 10) are substituted with 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 cases, the stapled peptide or the pharmaceutically acceptable salt thereof optionally comprises 1 to 4 additional substitutions relative to the sequence of SEQ ID NOB on the BCL-2 noninteracting alpha helical face of the BAD BH3 peptide of SEQ ID NOB. These Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO substitutions are not at positions 2 and 6, or 6 and 10 of SEQ ID NO:3 if those positions have been substituted with a, a-di substituted non-natural amino acids comprising olefinic side chains. In some cases, the 1 to 4 additional substitutions are at one or more of Wl, Q4, R5, R8, E9, R12, D15, E16, D19, or K22 (wherein the numbering is based on SEQ ID NO:3). If position D15 is substituted it is replaced by a conservative substitution (e.g., D15E). In some cases, the stapled peptide or the pharmaceutically acceptable salt thereof optionally comprises 1 to 4 additional substitutions relative to the sequence of SEQ ID NO: 3 on the BCL-2-interacting alpha helical face of the BAD BH3 peptide of SEQ ID NO:3. These substitutions are conservative amino acid substitutions. In certain cases, the stapled peptide or the pharmaceutically acceptable salt thereof optionally comprises a total of 1 to 4 additional substitutions relative to the sequence of SEQ ID NO:3 on the BCL-2-interacting and BCL-2 non-interacting alpha helical faces of the BAD BH3 peptide of SEQ ID NO:3. In some cases, [Xaa]x is (i) positions 3-5 of the sequence of SEQ ID NO:1, or (ii) positions 7-9 of the sequence of SEQ ID NO:3.The polypeptide or the pharmaceutically acceptable salt thereof binds to both wild type BCL-2 and mutant BCL-2 proteins that are resistant to venetoclax. In some cases, the mutant BCL-2 is G101V, GIOIA, D103Y, D103E, D103V, A113G, V156D, R129L, LI 19V, F104C, F104L, F104S, or the in-frame insertion ArglO7_Argl lOdup. In some cases, the polypeptide or the pharmaceutically acceptable salt thereof exhibits reduced or no binding to BCL-w, BCL-XL, MCL-1, and / or BFL-1 / A1.

[0020] In some instances, Ri is methyl, Rs is (CH2)s-CH=CH-(CH2)3, and R2 is methyl. In some instances, (a) [Xaa]wconsists of the sequence W, [Xaa]x consists of the sequence AQR, and [Xaa]yconsists of the sequence GRELRRBSDEFVDSFK (SEQ ID NO: 41); or [Xaa]wconsists of the sequence WAAQR (SEQ ID NO: 42), [Xaa]xconsists of the sequence GRE, and [Xaa]yconsists of the sequence RRBSDEFVDSFK (SEQ ID NO: 43).

[0021] Also provided herein are polypeptides comprising a peptide, wherein the peptide comprises the sequence of SEQ ID NOs: 6 or 10, with 0, 1, 2 3, or 4 amino acid substitutions relative to the sequence of SEQ ID NOs:6 or 10, respectively, wherein the peptide binds to both wild type BCL-2 and mutant BCL-2 proteins that are resistant to Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO venetoclax. In some cases, the 1, 2 3, or 4 amino acid substitutions relative to the sequence of SEQ ID NOs:6 or 10 are on the BCL-2 non-interacting face of SEQ ID NO: 6 or 10. In some cases, the 1, 2, 3, or 4 amino acid substitutions are at one or more of Wl, Q4, R5, R8, E9, R12, D15, E16, D19, or K22 (wherein the numbering is based on SEQ ID NO: 6 or 10). If the substitution is at DI 5, then the substitution is generally a conservative substitution, e.g., D15E. In some cases, the 1, 2, 3, or 4 amino acid substitutions relative to the sequence of SEQ ID NOs:6 or 10 are on the BCL-2 interacting face of SEQ ID NO: 1 which do not inhibit the ability of the peptide to bind to BCL-2 or mutant BCL-2 proteins that are resistant to venetoclax. Such substitutions are generally conservative amino acid substitutions. In certain cases, the peptide comprises the sequence of SEQ ID NO: 6. In other cases, the peptide comprises the sequence of SEQ ID NO: 10. In some cases, the peptide exhibits reduced or no binding to BCL-w, BCL-XL, MCL-1, and / or BFL-1 / A1.

[0022] Also featured herein are polypeptides comprising a stapled peptide, wherein the stapled peptide comprises the sequence of SEQ ID NOs: 6 or 10, with 0, 1, 2 3, or 4 amino acid substitutions relative to the sequence of SEQ ID NOs:6 or 10, respectively, or a pharmaceutically acceptable salt thereof (e.g., hydrochloride, mesylate, hydrobromide, acetate, fumarate, sodium, calcium, and potassium). The stapled peptide or the pharmaceutically acceptable salt thereof binds to both wild type BCL-2 and mutant BCL- 2 proteins that are resistant to venetoclax. These substitutions are not at positions 2 and 6 of SEQ ID NO:6, or positions 6 and 10 of SEQ ID NO: 10 as those positions are substituted with a, a-di substituted non-natural amino acids comprising olefinic side chains. In some cases, the 1, 2, 3, or 4 amino acid substitutions relative to the sequence of SEQ ID NOs:6 or 10 are on the BCL-2 non-interacting face of SEQ ID NO: 1. In certain cases, optionally wherein the 1, 2 3, or 4 amino acid substitutions are at one or more of Wl, Q4, R5, R8, E9, R12, D15, E16, D19, or K22 (wherein the numbering is based on SEQ ID NO:6 or 10). If the substitution is at DI 5, then the substitution is generally a conservative substitution, e.g., D15E. In some cases, the 1, 2, 3, or 4 amino acid substitutions relative to the sequence of SEQ ID NOs:6 or 10 are on the BCL-2 interacting face of SEQ ID NO: 1. These substitutions do not inhibit the ability of the Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO peptide to bind to BCL-2 or mutant BCL-2 proteins that are resistant to venetoclax. Generally, the substitutions made to the BCL-2 interacting face of the BAD BH3 helix are conservative amino acid substitutions. In certain cases, the peptide comprises the sequence of SEQ ID NO: 6. In other cases, the peptide comprises the sequence of SEQ ID NO: 10. Also provided herein are polypeptides comprising a stapled peptide, wherein the stapled peptide comprises the sequence of SEQ ID NOs: 6 or 10, optionally wherein the stapled peptide comprises the sequence of SEQ ID NO: 6. “B” in these sequences is norleucine and the two X’s in each sequence are a,a-disubstituted non-natural amino acids comprising olefinic side chains that are capable of being cross-linked to each other (e.g., (S)-2-(4’-pentenyl)alanine), e.g., to form a hydrocarbon staple. In some cases, the stapling amino acids (X) are both (S)-2-(4’-pentenyl)alanine. In some cases, the stapling amino acids (X) are both (R)-2-(4’-pentenyl)alanine. In some cases, the stapled peptide is 22 to 50 amino acids in length.

[0023] Also provided herein are polypeptides comprising a peptide, wherein the peptide comprises the sequence of SEQ ID NOs: 6 or 10, with 1, 2 3, or 4 amino acid substitutions relative to the sequence of SEQ ID NOs:6 or 10, respectively, wherein the peptide binds to both wild type BCL-2 and mutant BCL-2 proteins that are resistant to venetoclax. In some cases, the 1, 2, 3, or 4 amino acid substitutions relative to the sequence of SEQ ID NOs:6 or 10 are on the BCL-2 non-interacting face of SEQ ID NO: 6 or 10. In some cases, the 1, 2, 3, or 4 amino acid substitutions are at one or more of Wl , Q4, R5, R8, E9, R12, D15, E16, D19, or K22 (wherein the numbering is based on SEQ ID NO: 6 or 10). If the substitution is at DI 5, then the substitution is generally a conservative substitution, e.g., D15E. In some cases, the 1, 2, 3, or 4 amino acid substitutions relative to the sequence of SEQ ID NOs:6 or 10 are on the BCL-2 interacting face of SEQ ID NO: 1 which do not inhibit the ability of the peptide to bind to BCL-2 or mutant BCL-2 proteins that are resistant to venetoclax. Such substitutions are generally conservative amino acid substitutions. In some instances, the 1, 2, 3, or 4 amino acid substitutions relative to the sequence of SEQ ID NO: 6 include a substitution at position 10 (numbered according to SEQ ID NO: 6) with phenylalanine further substituted with a chlorine moiety (e.g., 4-C1-F), e.g., as in the peptide BAD 4.2-C (SEQ Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0024] ID NO:56). In some instances, the 1, 2, 3, or 4 amino acid substitutions relative to the sequence of SEQ ID NOs: 6 or 10 include a substitution at position 17 (numbered according to SEQ ID NO:6) with tryptophan, e.g., as in the peptide BAD 4.2-D (SEQ ID NO: 57). In some instances, the 1, 2, 3, or 4 amino acid substitutions relative to the sequence of SEQ ID NOs: 6 or 10 include a substitution at position 17 (numbered according to SEQ ID NO:6) with beta-(4-biphenyl)-L-alanine, e.g., as in the peptide BAD 4.2-F (SEQ ID NO: 59). In some instances, the 1, 2, 3, or 4 amino acid substitutions relative to the sequence of SEQ ID NO: 6 include a substitution at position 10 and a substitution at position 17 (both numbered according to SEQ ID NO:6) with phenylalanine and tryptophan, respectively, e.g., as in the peptide BAD 4.2-G, SEQ ID NO: 60. In certain cases, the peptide comprises the sequence of SEQ ID NO: 56. In certain cases, the peptide comprises the sequence of SEQ ID NO: 57. In certain cases, the peptide comprises the sequence of SEQ ID NO: 59. In certain cases, the peptide comprises the sequence of SEQ ID NO: 60. In some cases, the peptide exhibits reduced or no binding to BCL-w, BCL-XL, MCL-1, and / or BEL-1 / Al. In some cases, the peptide preferentially binds to BCL-2 over BCL-XL, e.g., as determined according to an assay described in the working examples herein.

[0025] Also featured herein are polypeptides comprising a stapled peptide, wherein the stapled peptide comprises the sequence of SEQ ID NOs: 6 or 10, with 1, 2, 3, or 4 amino acid substitutions relative to the sequence of SEQ ID NOs:6 or 10, respectively, or a pharmaceutically acceptable salt thereof (e.g., hydrochloride, mesylate, hydrobromide, acetate, fumarate, sodium, calcium, and potassium). The stapled peptide or the pharmaceutically acceptable salt thereof binds to both wild type BCL-2 and mutant BCL- 2 proteins that are resistant to venetoclax. These substitutions are not at positions 2 and 6 of SEQ ID NO:6, or positions 6 and 10 of SEQ ID NO: 10 as those positions are substituted with a, a-di substituted non-natural amino acids comprising olefinic side chains. In some cases, the 1, 2, 3, or 4 amino acid substitutions relative to the sequence of SEQ ID NOs: 6 or 10 are on the BCL-2 non-interacting face of SEQ ID NO: 1. In certain cases, optionally wherein the 1, 2, 3, or 4 amino acid substitutions are at one or more of Wl, Q4, R5, R8, E9, R12, D15, E16, D19, or K22 (wherein the numbering is based on Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0026] SEQ ID NO: 6 or 10). If the substitution is at DI 5, then the substitution is generally a conservative substitution, e.g., D15E. In some cases, the 1 , 2, 3, or 4 amino acid substitutions relative to the sequence of SEQ ID NOs:6 or 10 are on the BCL-2 interacting face of SEQ ID NO: 1. These substitutions do not inhibit the ability of the peptide to bind to BCL-2 or mutant BCL-2 proteins that are resistant to venetoclax. Generally, the substitutions made to the BCL-2 interacting face of the BAD BH3 helix are conservative amino acid substitutions. In some instances, the 1, 2, 3, or 4 amino acid substitutions relative to the sequence of SEQ ID NO: 6 include a substitution at position 10 (numbered according to SEQ ID NO:6) with phenylalanine further substituted with a chlorine moiety (e.g., 4-C1-F), e.g., as in the peptide BAD 4.2-C (SEQ ID NO:56). In some instances, the 1, 2, 3, or 4 amino acid substitutions relative to the sequence of SEQ ID NOs: 6 or 10 include a substitution at position 17 (numbered according to SEQ ID NO:6) with tryptophan, e.g., as in the peptide BAD 4.2-D (SEQ ID NO: 57). In some instances, the 1, 2, 3, or 4 amino acid substitutions relative to the sequence of SEQ ID NOs: 6 or 10 include a substitution at position 17 (numbered according to SEQ ID NO:6) with beta-(4-biphenyl)-L-alanine, e.g., as in the peptide BAD 4.2-F (SEQ ID NO: 59). In some instances, the 1, 2, 3, or 4 amino acid substitutions relative to the sequence of SEQ ID NO: 6 include a substitution at position 10 and a substitution at position 17 (both numbered according to SEQ ID NO:6) with phenylalanine and tryptophan, respectively, e.g., as in the peptide BAD 4.2-G (SEQ ID NO: 60). In certain cases, the peptide comprises the sequence of SEQ ID NO: 56. In certain cases, the peptide comprises the sequence of SEQ ID NO: 57. In certain cases, the peptide comprises the sequence of SEQ ID NO: 59. In certain cases, the peptide comprises the sequence of SEQ ID NO: 60. “B” in these sequences is norleucine and the two X’s in each sequence are a,a-disubstituted non-natural amino acids comprising olefinic side chains that are capable of being crosslinked to each other (e.g., (S)-2-(4’-pentenyl)alanine), e.g., to form a hydrocarbon staple. In some cases, the stapling amino acids (X) are both (S)-2-(4’-pentenyl)alanine. In some cases, the stapling amino acids (X) are both (R)-2-(4’-pentenyl)alanine. In some cases, the stapled peptide is 22 to 50 amino acids in length. In some cases, the peptide exhibits reduced or no binding to BCL-w, BCL-XL, MCL-1, and / or BFL-1 / A1. In some cases, Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO the peptide preferentially binds to BCL-2 over BCL-XL, e.g., as determined according to an assay described in the working examples herein.

[0027] Also provided herein are delivery vehicles comprising the comprising the polypeptide or the pharmaceutically acceptable salt thereof described herein., In some cases, the delivery vehicle is a nanoparticle that encapsulates the polypeptide or the pharmaceutically acceptable salt thereof In certain cases, the nanoparticle is a lipid nanoparticle.

[0028] Also provided herein are compositions comprising a delivery vehicle (e.g., a nanoparticle such as a lipid nanoparticle) and means for binding to both wild type BCL-2 and mutant BCL-2 proteins that are resistant to venetoclax. The means for binding to both wild type BCL-2 and mutant BCL-2 proteins that are resistant to venetoclax is encapsulated within the delivery vehicle.

[0029] Also provided herein are compositions comprising a delivery vehicle (e g., a nanoparticle such as a lipid nanoparticle) and means for binding to both wild type BCL-2 and mutant BCL-2 proteins that are resistant to venetoclax and showing reduced or no binding to BCL-w, BCL-XL, MCL-1, and / or BFL-1 / A1. The means for binding to both wild type BCL-2 and mutant BCL-2 proteins that are resistant to venetoclax and showing reduced or no binding to BCL-w, BCL-XL, MCL-1, and / or BFL-1 / A1 is encapsulated within the delivery vehicle.

[0030] Also provided herein are pharmaceutical compositions comprising the polypeptides or the pharmaceutically acceptable salts thereof or the delivery vehicles described herein; and a pharmaceutically acceptable carrier.

[0031] Also provided herein are pharmaceutical compositions comprising (i) means for binding to both wild type BCL-2 and mutant BCL-2 proteins that are resistant to venetoclax; and (ii) a pharmaceutically acceptable carrier.

[0032] Also provided herein are pharmaceutical compositions comprising (i) means for binding to both wild type BCL-2 and mutant BCL-2 proteins that are resistant to venetoclax and showing reduced or no binding to BCL-w, BCL-XL, MCL-1, and / or BFL-1 / A1; and (ii) a pharmaceutically acceptable carrier. Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0033] Also provided herein are methods of making a stapled peptide, the method comprising (a) providing a peptide comprising the sequence of SEQ ID NO:6 or 10, with 0, 1, 2, or 3 amino acid substitutions. In some cases, the 1, 2, or 3 amino acid substitutions are on the BCL-2 non-interacting face of the alpha helix of the sequence set forth in SEQ ID NO:6 or 10. In certain cases, the 1, 2, or 3 amino acid substitutions are on the BCL-2-interacting face of the alpha helix of the sequence set forth in SEQ ID NO:6 or 10. The method further comprises cross-linking the peptide thereby making the stapled peptide. In some cases, both the stapling amino acids (X) are (S)-2-(4’- pentenyl)alanine. In certain cases, the cross-linking is by a ruthenium catalyzed metathesis reaction. In some cases, the method further comprises formulating the stapled peptide as a sterile pharmaceutical composition.

[0034] Also provided herein are methods of making a stapled peptide, the method comprising (a) providing a peptide comprising the sequence of SEQ ID NO:6 or 10, with 0, 1, 2, or 3 amino acid substitutions. In some cases, the 1, 2, or 3 amino acid substitutions with respect to SEQ ID NO: 6 include (i) a substitution at position 10 (numbered according to SEQ ID NO:6) with phenylalanine further substituted with a chlorine moiety (e.g., 4-C1-F), e.g., as in the peptide BAD 4.2-C (SEQ ID NO:56), (ii) a substitution at position 17 (numbered according to SEQ ID NO:6) with tryptophan, e.g., as in the peptide BAD 4.2-D (SEQ ID NO: 57), (iii) a substitution at position 17 (numbered according to SEQ ID NO:6) with beta-(4-biphenyl)-L-alanine, e.g., as in the peptide BAD 4.2-F (SEQ ID NO: 59), or (iv) a substitution at position 10 and a substitution at position 17 (both numbered according to SEQ ID NO:6) with phenylalanine and tryptophan, respectively, e.g., as in the peptide BAD 4.2-G (SEQ ID NO: 60). In some cases, the 1, 2, or 3 amino acid substitutions with respect to SEQ ID NO: 10 include (i) a substitution at position 17 (numbered according to SEQ ID NO:6) with tryptophan, e.g., as in the peptide BAD 4.2-D (SEQ ID NO: 57), or (ii) a substitution at position 17 (numbered according to SEQ ID NO: 6) with beta-(4-biphenyl)-L-alanine, e.g., as in the peptide BAD 4.2-F (SEQ ID NO: 59). The method further comprises cross-linking the peptide thereby making the stapled peptide. In some cases, both the stapling amino acids (X) are (S)-2-(4’-pentenyl)alanine. In certain cases, the cross-linking Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO is by a ruthenium catalyzed metathesis reaction. In some cases, the method further comprises formulating the stapled peptide as a sterile pharmaceutical composition.

[0035] Also provided herein are methods of treating a cancer that expresses BCL-2 or is dependent on BCL-2 in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of (i) the polypeptides or the pharmaceutically acceptable salt thereof, (ii) the delivery vehicles or compositions; or (iii) the pharmaceutical compositions described herein. In some cases, the cancer is a hematological cancer. In other cases, the cancer is a solid tumor. In some cases, the cancer comprises a mutant BCL-2 that has one or more of the following mutations: G101V, GIOIA, D103Y, D103E, D103V, A113G, V156D, R129L, LI 19V, F104C, F104L, F104S, or the in-frame insertion ArglO7_Ar l lOdup.

[0036] Also provided herein are methods of treating a venetoclax-resistant cancer in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of (i) the polypeptides or the pharmaceutically acceptable salt thereof, (ii) the delivery vehicles or compositions; or (iii) the pharmaceutical compositions described herein. In some instances, the cancer is a hematological malignancy. In some cases, the cancer is chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), small lymphocytic lymphoma (SLL), or multiple myeloma (MAI) such as relapsed or refractory' MM. In some instances, the method is for the treatment of patients with chronic lymphocytic leukemia (CLL) with 17p deletion, as detected by an FDA-approved test, who have received at least one prior therapy. In some instances, the method is for the treatment of newly diagnosed acute myeloi d leukemia (AML) in adults 75 years or older, or who have comorbidities that preclude use of intensive induction chemotherapy, in combination with azacitidine, or decitabine, or low- dose cytarabine. In some cases, the venetoclax-resistant cancer comprises a mutant BCL- 2 that has one or more of the following mutations: G101V, GIOIA, D103Y, D103E, D103V, A113G, V156D, R129L, LI 19V, F104C, F104L, F104S, or the in-frame insertion ArglO7_Argl lOdup.

[0037] Also provided herein are methods of inhibiting the interaction of BCL2 or a mutant BCL2 with a proapoptotic BCL2 family member protein in a human subject in Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO need thereof, the method comprising administering to the human subject a therapeutically effective amount of (i) the polypeptides or the pharmaceutically acceptable salt thereof, (ii) the delivery vehicles or compositions; or (iii) the pharmaceutical compositions described herein. In some cases, the mutant BCL-2 is G101V, GIOIA, D103Y, D103E, D103V, A113G, V156D, R129L, LI 19V, F104C, F104L, F104S, or the in-frame insertion ArglO7_Argl lOdup.

[0038] Also featured herein are methods of treating a BCL-2 expressing solid tumor in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of (i) the polypeptides or the pharmaceutically acceptable salt thereof, (ii) the delivery vehicles or compositions; or (iii) the pharmaceutical compositions described herein. In some cases, the solid tumor comprises a mutant BCL-2 that is resistant to venetoclax. In some cases, the mutant BCL-2 is G101V, GIOIA, D103Y, D103E, D103V, Al 13G, V156D, R129L, LI 19V, F104C, F104L, F104S, or the in-frame insertion ArglO7_Argl lOdup. In some cases, the solid tumor is a breast cancer (e.g., ER+metastatic breast cancer), a non-small cell lung cancer (NSCLC), an ovarian cancer (e.g., platinum-resistant ovarian cancer), or a pancreatic cancer.

[0039] In certain instances, in the above methods of treatment, the subject can be administered a combination therapy with other classes of agents. In certain cases, the combination therapy comprises one or more of: chemotherapy, targeted therapy, hormonal therapy, radiation therapy, immunotherapy, antibody drug conjugates, epigenetic modulators, and / or anti-angiogenesis agents.

[0040] 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 Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO control. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0041] Other features and advantages of the invention will be apparent from the following detailed description and from the claims.

[0042] BRIEF DESCRIPTION OF THE DRAWINGS

[0043] FIG. 1 presents the crystal structure of venetoclax bound to BCL-2 G101V (PDB: 6O0L), highlighting the location of the GIO IV mutation that arose upon venetoclax exposure and weakened the interaction by 177-fold.

[0044] FIG. IB presents the crystal structure of another small-molecule BH3 mimetic, S55746, bound to BCL-2 G101V (PDB: 6000), highlighting the proximity of its binding mode to the G101V mutation, resulting in a 100-fold decrement in binding activity relative to wild-type BCL-2.

[0045] FIG. 1C provides data reflecting the decreased ability of small molecule BH3 mimetics to inhibit BCL-2 G101V, as demonstrated by fold change in Ki (binding: competitive SPR against BIM-BH3), ECso (cytotoxicity: KMS-12-PE cell line ofB-cell lineage), and LCso (cytotoxicity: CLL cells harboring G101V from patients at progression relative to their CLL cells collected at study entry). (Venetoclax: Blombery et. al, Cancer Di scov, 2019, 9(3) 342-353; S55746: Birkinshaw et. al, Nat Comm, 2019, 10:2385)

[0046] FIG. 2A shows the BAD protein sequence (Uniprot: Q92934) (SEQ ID NO:2) and highlights the BH3 domain sequence (SEQ ID NO:1) selected for synthesis of a staple scanning library.

[0047] FIG. 2B demonstrates the surface groove on BCL-2 for interaction with a BAD BH3 peptide.

[0048] FIG. 2C demonstrates the comparatively smaller surface area for binding interactions on BCL-2 with venetoclax, a selective small molecule BCL-2 inhibitor (PDB: 6O0K). Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0049] FIG. 2D provides data comparing the total surface area of a BAD BH3 peptide and venetoclax, and the surface area and relative coverage of these compounds on BCL- 2, as determined by PISA analysis.

[0050] FIG. 3A lists a library of i, i-4 stapled BAD BH3 compositions (wherein X is (S)-2-(4-pentenyl)alanine) and corresponding binding assay ICso values, as determined by fluorescence polarization (FP) assay of wild-type BCL-2 using FITC-BIM BH3 as the competitor peptide (n=4).

[0051] FIG. 3B shows an immunoprecipitation experiment using individual biotinylated BAD BH3 i, i+4 peptides incubated with 293 T cell lysate, followed by western blotting for FLAG-tagged, full-length BCL-2 with an a-FLAG antibody. The data showed stark differences in binding activity between the individual stapled peptides and the BCL-2 protein.

[0052] FIG. 3C shows a helical wheel depiction of the BAD BH3 sequence, highlighting the location of four lead i, i I 4 staple positions relative to the peptides’ BCL-2 binding surface. Unexpectedly, several of the best binders positioned the staple directly toward the binding interface, which would otherwise be expected to cause steric hindrance and disrupt binding activity.

[0053] FIG. 4A lists a library of i, i~7 stapled BAD BH3 compositions (wherein 8 is (R)-2-(7-octenyl)alanine and X is (S)-a-(4-pentenyl)alanine) and corresponding binding assay ICso values, as determined by fluorescence polarization (FP) assay of wild-type BCL-2 using FITC-BIM BH3 as the competitor peptide (n=4).

[0054] FIG. 4B shows an immunoprecipitation experiment using individual biotinylated BAD BH3 i, i+ 7 peptides incubated with 293T cell lysate, followed by western blotting for FLAG-tagged, full-length BCL-2 with an a-FLAG antibody. The data showed stark differences in binding activity between the individual stapled peptides and the BCL-2 protein.

[0055] FIG. 4C shows a helical wheel depiction of the BAD BH3 sequence, highlighting the location of four lead i, i+ 7 staple positions relative to the peptides’ BCL-2 binding surface. Unexpectedly, two of the best binders positioned the staple directly toward the Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO binding interface, which would otherwise be expected to cause steric hindrance and disrupt binding activity.

[0056] FIG. 5A compares the ability of lead stapled BAD BH3 peptides to preferentially pull-down BCL-2 from an equimolar pool of anti-apoptotic protein homologues, such as BCL-XL and MCL-1, with two of the constructs showing high selectivity for BCL-2 (BAD 4.2 and BAD 4.6) in this multi-protein competitive binding assay.

[0057] FIG. 5B presents the ICso values of lead stapled BAD BH3 peptides to WT BCL- 2, as assessed by fluorescence polarization (FP) assay using FITC-BIM BH3 as the competitor peptide (n=4).

[0058] FIG. 5C presents the ICso values for the binding of a lead stapled BAD BH3 peptide, BAD 4.2, to individual anti-apoptotic BCL-2 family proteins, as assessed by fluorescence polarization (FP) assay using FITC-BIM BH3 as the competitor peptide (n=4).

[0059] FIG. 6A presents the ICso values for the competitive binding of a lead stapled BAD BH3 peptide, BAD 4.2, to a series of BCL-2 proteins, including wild-type and various clinical resistance mutants that disrupt venetoclax interaction, as assessed by FP using FITC-BIM BH3 as the competitive ligand (n=4).

[0060] FIG. 6B demonstrates the locations of venetocl ax-resistant single-point mutations on BCL-2, relative to the location of the BAD BH3 interaction site.

[0061] FIG. 7A presents the binding kinetics (15 minutes of association, 10 minutes of dissociation) of a lead stapled BAD BH3 peptide, BAD 4.2, to WT GST-BCL-2, as measured by biolayer interferometry (BLI).

[0062] FIG. 7B presents the binding kinetics (15 minutes of association, 10 minutes of dissociation) of a lead stapled BAD BH3 peptide, BAD 4.2, to GST-BCL-2 G101V, as measured by BLI.

[0063] FIG. 7C presents a second biological replicate of the binding kinetics (15 minutes of association, 10 minutes of dissociation) of a lead stapled BAD BH3 peptide, BAD 4.2, to WT GST-BCL-2, as measured by biolayer interferometry (BLI). Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0064] FIG. 7D presents a second biological replicate of the binding kinetics (15 minutes of association, 10 minutes of dissociation) of a lead stapled BAD BH3 peptide, BAD 4.2, to GST-BCL-2 GIO IV, as measured by BLI.

[0065] FIG. 7E shows equilibrium dose-response curves of best fit from the end points of binding association curves for a lead stapled BAD BH3 peptide to two biological replicates of WT and GlOlV-mutant GST-BCL-2.

[0066] FIG. 7F shows a comparison of ICso (FIG. 6A) and Kd (FIG. 7C) values for BAD 4.2 binding to BCL-2 WT and G101V proteins by competitive FP and BLI.

[0067] FIG. 8A presents the ECso values for the binding of BAD 4.2 to the earliest identified venetoclax resistant BCL-2 mutations, G101 V and F104L, as assessed by direct fluorescence polarization (FP) assay using FITC-BAD 4.2 (n=4).

[0068] FIG. 8B presents the ICso values for the binding of BAD 4.2 to the earliest identified venetoclax resistant BCL-2 mutations, G101 V and F104L, as assessed by competitive fluorescence polarization (FP) assay using FITC-BIM BH3 as the competitor peptide (n=4).

[0069] FIG. 8C shows an immunoprecipitation of WT, GIO IV, and F104L BCL-2 proteins from a 293T cell lysate by biotinylated BAD 4.2. Whereas BAD 4.2 could readily engage WT, GIO IV, and F104L mutant BCL-2 proteins, the capacity of venetoclax to compete with BAD 4.2 for binding to BCL-2 was reduced by more than 10- fold upon G101V and F104L mutagenesis.

[0070] FIG. 9A shows the comparative reactivation of tB ID-triggered, BAX-mediated liposomal release by venetoclax or BAD 4.2 blockade of the inhibitory activity of BCL-2 WT, as monitored by ANTS / DPX release over a two hour period (±SD, n=4).

[0071] FIG. 9B shows the comparative reactivation of tB ID-triggered, BAX-mediated liposomal release by venetoclax or BAD 4.2 blockade of the inhibitory activity of BCL-2 GIO IV, as monitored by ANTS / DPX release over a two hour period (±SD, n=4).

[0072] FIG. 9C shows the comparative reactivation of tB ID-triggered, BAX-mediated liposomal release by venetoclax or BAD 4.2 blockade of the inhibitory activity of BCL-2 F104L, as monitored by ANTS / DPX release over a two hour period (+SD, n=4). Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0073] FIG. 9D shows that the capacity of venetoclax to functionally reactivate tB ID- triggered, BAX-mediated liposomal release by blocking BCL-2 was impaired upon G101V or F104L mutagenesis, whereas the BAD 4.2 stapled peptide was equally capable of blocking BCL-2 or its GIO IV and F104L mutants to restore BAX-mediated poration (±SD, n=4).

[0074] FIG. 10A presents a high-resolution structure of BCL-2 WT co-crystallized with BAD 4.2 (PDB 9014, 1 .73 A resolution), demonstrating the helix-in-groove interaction, with BCL-2 surface residues shaded by hydrophobic, hydrophilic, positive charge, and negative charge features.

[0075] FIG. 10B presents a high-resolution structure of BCL-2 G101V co-crystallized with BAD 4.2 (PDB 9015, 1.99 A resolution), with BCL-2 surface residues shaded by hydrophobic, hydrophilic, positive charge, and negative charge features, highlighting the similar peptide backbone alignment within the BH3 groove as BCL-2 WT (FIG. 10A).

[0076] FIG. IOC shows the N-terminal hydrophobic residues of BAD 4.2 binding surface, including the highly conserved leucine (LI 14) and the all-hydrocarbon staple, engaging directly with a network of hydrophobic residues of the BCL-2 WT groove.

[0077] FIG. 10D shows the N-terminal hydrophobic residues of BAD 4.2 binding surface, including the highly conserved leucine (LI 14) and the all-hydrocarbon staple, engaging directly with a network of hydrophobic residues of the BCL-2 G101V groove, as also observed in the complex between BAD 4.2 and BCL-2 WT (FIG. IOC).

[0078] FIG. 10E shows a series of electrostatic and hydrophilic interactions between residues of BAD 4.2 and BCL-2 WT, including (BAD 4.2-BCL-2) DI 19-N143 / R146, E120-Y108, R115-E136 / D140, and R116-Y108 / D111.

[0079] FIG. 10F shows a series of electrostatic and hydrophilic interactions between residues of BAD 4.2 and BCL-2 G101V, including (BAD 4.2-BCL-2) DI 19-N143 / R146, E120-Y108, R115-E136 / D140, and R116-Y108 / D111, as also observed in the complex between BAD 4.2 and BCL-2 WT (FIG. 10E).

[0080] FIG. 10G shows the C-terminal phenylalanine residues of BAD 4.2 engage a network of aromatic residues, including Y108, Fl 04, Fl 98, and Y202, at the BCL-2 WT surface. Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0081] FIG. 10H shows the C-terminal phenylalanine residues of BAD 4.2 engage a network of aromatic residues, including Y108, Fl 04, Fl 98, and Y202, at the BCL-2 G101V surface, as also observed in the complex between BAD 4.2 and BCL-2 WT (FIG. 10G)

[0082] FIG. 11 presents a table of data collection and refinement statistics for X-ray crystallography structures of BCL-2 WT (FIG. 10A) and G101V (FIG. 10A) in complex with BAD 4.2.

[0083] FIG. 12A presents a difference distance matrix plot (DDMP) comparing complexes of venetoclax with BCL-2 G101V and WT (PDB 6O0L, 6O0K) proteins, highlighting in the boxed areas key changes resulting from G101V mutagenesis that persisted upon small molecule binding.

[0084] FIG. 12B presents a difference distance matrix plot (DDMP) comparing complexes of a lead stapled BAD BH3 peptide, BAD 4.2, with BCL-2 G101 V and WT (PDB 9015, 9014), illustrating that BAD 4.2 restored BCL-2 G101V to a WT-like protein conformation.

[0085] FIG. 12C presents a difference distance matrix plot (DDMP) comparing complexes of a lead stapled BAD BH3 peptide, BAD 4.2, and venetoclax with WT BCL- 2 (PDB 9014, 6O0K).

[0086] FIG. 12D presents a difference distance matrix plot (DDMP) comparing complexes of a lead stapled BAD BH3 peptide, BAD 4.2, and venetoclax with G101V BCL-2 (PDB 9015, 6O0L).

[0087] FIG. 13A shows a deuterium difference plot of the relative deuterium incorporation of BCL-2 WT bound to BAD 4.2 as compared to BCL-2 WT alone, as examined by hydrogen-deuterium exchange mass spectrometry (HDX MS). The region of protection from deuterium exchange, mapped onto a structure of BCL-2 WT (FIG. 10A), demonstrated the influence of BAD 4.2 binding on the conformational exposure of the BCL-2 WT protein.

[0088] FIG. 13B shows a deuterium difference plot of the relative deuterium incorporation of BCL-2 G101V bound to BAD 4.2 as compared to BCL-2 G101V alone, as assessed by HDX MS. The region of protection from deuterium exchange, mapped Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO onto a structure of BCL-2 G101V (FIG. 10B), demonstrated the influence of BAD 4.2 binding on the conformational exposure of the BCL-2 G101 V protein.

[0089] FIG. 13C shows a deuterium difference plot of the relative deuterium incorporation of BCL-2 G101V as compared to BCL-2 WT, mapped onto a structure of BCL-2 G101V (FIG. 10B), highlighting the impact of mutagenesis on protein conformation.

[0090] FIG. 13D shows a deuterium difference plot of the relative deuterium incorporation of BCL-2 G101V when bound to BAD 4.2 relative to that observed when BCL-2 WT was bound to BAD 4.2. Notably, there were no differences in the pattern of exchange, as mapped onto a structure of BCL-2 G101V (FIG. 10B), underscoring the capacity of BAD 4.2 to equally engage wild-type and mutant BCL-2, eliminating the differences observed when comparing the two proteins alone.

[0091] FIG. 14A presents two views of a high-resolution structure of BCL-2 R129L cocrystallized with BAD 4.2 (PDB 9016, 1.73 A resolution).

[0092] FIG. 14B illustrates the all-hydrocarbon z, i+4 staple binding directly to a region of the BCL-2 hydrophobic pocket, which was expanded by the R129L chemoresistance mutation and enabled increased hydrophobic interactions with the peptide.

[0093] FIG. 15 presents a table of data collection and refinement statistics for X-ray crystallography structures of BCL-2 R129L in complex with BAD 4.2 (FIG. 14A).

[0094] FIG. 16A presents a difference distance matrix plot (DDMP) of protein residues comparing complexes of venetoclax with BCL-2 R129L and WT (PDB 9016, 9014), highlighting regions of decreased distance arising from R129L mutagenesis.

[0095] FIG. 16B illustrates the prominent differences in intra-protein proximities as a result of R129L mutagenesis (FIG. 16A), as mapped onto a structure of a lead stapled BAD BH3 peptide, BAD 4.2, in complex with BCL-2 R129L.

[0096] FIG. 16C presents a difference distance matrix plot (DDMP) comparing the distances between BAD 4.2 residues and those of BCL-2 in the BCL-2 R129L and WT complexes (PDB 9016, 9014), highlighting the differences in peptide interaction that arose from R129L mutagenesis. Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0097] FIG. 16D illustrates the increased engagement of BAD 4.2 with the BCL-2 groove as a result of R129L mutagenesis, as measured by DDMP (FIG. 16C) and visualized by superposition of the two structures.

[0098] FIG. 17A shows a deuterium difference plot of the relative deuterium incorporation of BCL-2 WT bound to BAD 4.2 as compared to BCL-2 alone (as shown in FIG. 13A)

[0099] FIG. 17B shows a deuterium difference plot of the relative deuterium incorporation of BCL-2 R129L bound to BAD 4.2 as compared to BCL-2 R129L alone, as assessed by HDX MS. Of note, the influence of BAD 4.2 on the deuterium exchange profile of WT and mutant BCL-2 were very similar, underscoring the capacity of BAD 4.2 to effectively engage both proteins.

[0100] FIG. 17C shows a deuterium difference plot of the relative deuterium incorporation of BCL-2 R129L as compared to BCL-2 WT, highlighting the impact of mutagenesis on protein conformation, including notable allosteric consequences of R129L mutagenesis.

[0101] FIG. 17D shows a deuterium difference plot of the relative deuterium incorporation of BCL-2 R129L when bound to BAD 4.2 relative to that observed when BCL-2 WT was bound to BAD 4.2. Notably, BAD 4.2 binding mitigated the differences in the region of the canonical groove and fully eliminated the allosteric changes induced by mutagenesis.

[0102] FIG. 18A shows a deuterium difference plot of the relative deuterium incorporation of BCL-2 D103Y bound to BAD 4.2 as compared to BCL-2 D103Y alone, as assessed by HDX MS.

[0103] FIG. 18B shows a deuterium difference plot of the relative deuterium incorporation of BCL-2 Al 13G bound to BAD 4.2 as compared to BCL-2 Al 13G alone, as assessed by HDX MS.

[0104] FIG. 18C shows a deuterium difference plot of the relative deuterium incorporation of BCL-2 V156D bound to BAD 4.2 as compared to BCL-2 V156D alone, as assessed by HDX MS. Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0105] FIG. 18D illustrates the similar regions of protection outside of the 0.5-Da significance threshold across all mutants and time points (FIGS. 13B, 17B, 18A, 18B, and 18C), as mapped onto the structure of BCL-2 WT in complex with BAD 4.2, underscoring the capacity of BAD 4.2 to effectively engage all mutant proteins.

[0106] FIG. 19A shows a deuterium difference plot of the relative deuterium incorporation of BCL-2 D103Y alone as compared to BCL-2 WT alone, highlighting the impact of mutagenesis on protein conformation, including notable allosteric consequences of DI 03 Y mutagenesis.

[0107] FIG. 19B shows a deuterium difference plot of the relative deuterium incorporation of BCL-2 Al 13G alone as compared to BCL-2 WT alone, highlighting the impact of mutagenesis on protein conformation, including notable allosteric consequences of Al 13G mutagenesis.

[0108] FIG. 19C shows a deuterium difference plot of the relative deuterium incorporation of BCL-2 V156D alone as compared to BCL-2 WT alone, highlighting the impact of mutagenesis on protein conformation, including notable allosteric consequences of V156D mutagenesis.

[0109] FIG. 20A shows a deuterium difference plot of the relative deuterium incorporation of BCL-2 D103 Y when bound to BAD 4.2 relative to that observed when BCL-2 WT was bound to BAD 4.2. Notably, BAD 4.2 binding mitigated the differences in the region of the canonical groove and fully eliminated the allosteric changes induced by D103Y mutagenesis (FIG. 19A).

[0110] FIG. 20B shows a deuterium difference plot of the relative deuterium incorporation of BCL-2 Al 13G when bound to BAD 4.2 relative to that observed when BCL-2 WT was bound to BAD 4.2. Notably, BAD 4.2 binding mitigated the differences in the region of the canonical groove and fully eliminated the allosteric changes induced by Al 13G mutagenesis (FIG. 19B).

[0111] FIG. 20C shows a deuterium difference plot of the relative deuterium incorporation of BCL-2 V156D when bound to BAD 4.2 relative to that observed when BCL-2 WT was bound to BAD 4.2. Notably, BAD 4.2 binding mitigated the differences Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO in the region of the canonical groove and fully eliminated the allosteric changes induced by V156D mutagenesis (FIG. 19C).

[0112] FIG. 21A presents a heatmap of HDX MS analyses for the relative deuterium incorporation of BCL-2 G101V relative to BCL-2 WT, alone (FIG. 13C) and when bound to BAD 4.2 (FIG. 13D), highlighting how peptide binding mitigated the conformational consequences of BCL-2 G101V mutagenesis.

[0113] FIG. 21B presents a heatmap of HDX MS analyses for the relative deuterium incorporation of BCL-2 D103Y relative to BCL-2 WT, alone (FIG. 19A) and when bound to BAD 4.2 (FIG. 20A), highlighting how peptide binding mitigated the conformational consequences of BCL-2 D103Y mutagenesis.

[0114] FIG. 21C presents a heatmap of HDX MS analyses for the relative deuterium incorporation of BCL-2 Al 13G relative to BCL-2 WT, alone (FIG. 19B) and when bound to BAD 4.2 (FIG. 20B), highlighting how peptide binding mitigated the conformational consequences of BCL-2 Al 13G mutagenesis.

[0115] FIG. 21D presents a heatmap of HDX MS analyses for the relative deuterium incorporation of BCL-2 R129L relative to BCL-2 WT, alone (FIG. 17C) and when bound to BAD 4.2 (FIG. 17D), highlighting how peptide binding mitigated the conformational consequences of BCL-2 R129L mutagenesis.

[0116] FIG. 21E presents a heatmap of HDX MS analyses for the relative deuterium incorporation of BCL-2 V156D relative to BCL-2 WT, alone (FIG. 19C) and when bound to BAD 4.2 (FIG. 20C), highlighting how peptide binding mitigated the conformational consequences of BCL-2 V156D mutagenesis.

[0117] FIG. 22 lists a library of BAD 4.2 compositions with single and double point mutations (wherein X is (S)-a-(4-pentenyl)alanine, 4-CLF is 4-chloro-L-phenylalanine, DNAis D-Ala(2-naphthyl)-OH, and BIP is beta-(4-biphenyl)-L-alanine) and corresponding binding assay ICso values for WT and G101 V-mutant BCL-2, as determined by fluorescence polarization (FP) assay using FITC-BIM BH3 as the competitor peptide (n=4).

[0118] FIG. 23 lists a library of BAD 4.2 compositions with single and double point mutations (wherein X is (S)-a-(4-pentenyl)alanine, 4-CLF is 4-chloro-L-phenylalanine, Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0119] DNAis D-Ala(2-naphthyl)-OH, and BIP is beta-(4-biphenyl)-L-alanine) and corresponding binding assay ICso values for BCL-2 and BCL-XL, as determined by fluorescence polarization (FP) assay using FITC-BIM BH3 as the competitor peptide (n=4).

[0120] DETAILED DESCRIPTION

[0121] The present disclosure is based, at least in part, on the identification of stapled peptides that selectively inhibit BCL-2 as well as venetoclax-resistant BCL-2 mutants. Efforts to overcome venetocl ax-resistance mutations with next-generation small molecule inhibitors of BCL-2 still suffer from decreased affinity for mutant BCL-2 proteins. Disclosed herein are peptides that mimic the natural BAD BH3 motif and selectively bind to BCL-2, including venetoclax resistance mutants thereof. Since certain cancers depend on BCL-2, and relapsed cancers can depend on mutant BCL-2, overcoming such mutations is clinically important. The peptides disclosed herein exhibit preference for BCL-2 over homologous anti-apoptotic proteins, which has been shown to maximize the therapeutic window. Thus, this disclosure encompasses polypeptides comprising selective stapled peptide inhibitors of BCL-2 and venetoclax-resistant mutant BCL-2.

[0122] BCL-2 AND BAD

[0123] BCL-2 family proteins play an important role in the mitochondrial-mediated intrinsic apoptosis pathway. The BCL-2 family is divided into three groups based on their primary function. The first group are the anti-apoptotic proteins and include the BCL-2, BCL-XL, BCL-W, MCL-1, and BFL-1 / A1 proteins. These proteins possess four BH domains - BH1 to BH4. The second group are the pro-apoptotic pore-former proteins and include the BAX, BAK, and BOK proteins. These proteins possess at least three BH domains - BH1 to BH3. Finally, the third group are the pro-apoptotic BH3-only proteins comprising the BAD, BID, BIK, BIM, BMF, BNIP3, HRK, NOXA, and PUMA proteins, some of which are activators of BAX and BAK (such as BID, BIM, and PUMA), and others are called sensitizers or selective inhibitors of anti-apoptotic proteins (such as Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0124] BAD), whereas some BH3-only proteins can exert both activities (such as BID, BIM, PUMA, and NOXA). All BCL-2 family proteins contain a BCL-2 homology 3 (BH3) domain that is required for the primary apoptotic function of BCL-2 family members and the interactions between them in the cytosol and at intracellular membranes, such as at the mitochondria. For example, the BH3 domain of activator BH3-only proteins, such as BIM, bind to a trigger site at the N-terminal surface of pro-apoptotic BAX, which leads to the activation of BAX and results in a series of conformational changes that induce its homo-oligomerization and pore formation within the MOM. The BH3 domain-binding groove of the anti-apoptotic proteins, which includes its own BH3 domain residues that contribute to the binding surface, binds the BH3 domains of the pro-apoptotic members, including the pore-formers (such as BAX and BAK) and the BH3-only proteins (such as BAD, BIM, and BID).

[0125] Abnormal expression of BCL-2 family proteins, including overexpression of the antiapoptotic protein BCL-2, is a common finding in hematological and other malignancies. In BCL-2-dependent malignancies, BCL-2 inhibitors can exert a targeted therapeutic effect and relieve apoptosis inhibition.

[0126] Venetoclax is a highly selective and effective BCL-2 inhibitor, which is able to reinstate the apoptotic potential of cancer cells and restore apoptosis. This BH3 mimetic binds to BCL-2 with high affinity, disrupting BCL-2 signaling within the cell and inducing the apoptotic pathway. Although venetoclax has great clinical application value as a new therapeutic drug, studies have shown that some patients can experience relapse in several months to several years after remission. One of the primary resistance mechanisms is the development of mutations in the BCL-2 gene resulting in reduced venetoclax binding. Mutations in the BCL-2 gene that confer reduced venetoclax binding are known in the art, including but not limited to, e g., G101V, GIOIA, D103Y, D103E, D103V, F104C, F104L, F104S, Al 13G, LI 19V, R129L, V156D and in frame insertion ArglO7_Argl lOdup. Resistance to venetoclax can be a result of one or a combination of two or more (e.g., 2, 3, 4, 5, 6) mutations in BCL-2. For example, patients with resistance to venetoclax can have both the G101V and the D103G mutations. Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0127] BAD is a BH3-only protein that can selectively bind anti-apoptotic proteins such as BCL-2 and BCL-XL via the BH3 domain of BAD and neutralize their anti-apoptotic functions. BAD has the amino acid sequence MFQIPEFEP SEQED S S S AERGLGP SP AGDGP SGSGKHHRQ APGLLWD ASHQQEQP T S S SHHGGAGA VEIRSRHS S YP AGTEDDEGMGEEP SPFRGRSRS APPNL WAAQR YGRELRRMSDEF VD SFKKGLPRPKS AGTATQMRQ S S S WTRVFQ S WWDRNLGRG SSAPSQ (SEQ ID NO: 2). The BH3 domain of BAD has the amino acid sequence WAAQRYGRELRRMSDEFVDSFK (SEQ ID NO: 1). The sequence of BAD with the BH3 domain highlighted is depicted in FIG. 2.

[0128] Moreover, studies have identified possible consensus sequences and conserved residues in the BH3 domains of various BH3-only and BH3-only-like proteins. For example, Leu at position 10 and Asp at position 15 of SEQ ID NO: 1 have been found to be highly conserved across BH3-only proteins (See, e.g., Cell Death and Differentiation (2002) 9, 1240-1247). A consensus sequence of various BH3-only and BH3 -only-like proteins that correspond to positions 6-18 of SEQ ID NO: 1 can be iSXX cXXdAS'DZdME, wherein represents a hydrophobic residue (e.g., 2 can be a leucine), S is a small residue (G, A, S), Z is an acidic residue, and T is a hydrophilic residue (N, H, D, or Y), and X can be any amino acid. See, e.g., Oncogene, volume 27, pages S2-S19 (2008). In some cases, the conserved residues in the BH3 domain are required for BAD BH3 and BCL-2 binding.

[0129] Provided herein are BAD peptides that mimic the natural BAD BH3 motif and selectively bind to BCL-2, including venetoclax-resistant mutants thereof. In some instances, the BAD BH3 peptide comprises the amino acid sequence WAAQRYGRELRRMSDEFVDSFK (SEQ ID NO: 1). In other instances, the BAD BH3 peptide comprises the amino acid sequence WAAQRYGRELRRBSDEFVDSFK (SEQ ID NO: 3), wherein B is norleucine. In some instances, the BAD BH3 peptide binds BCL-2, including venetoclax-resistant mutants thereof. Methods for determining binding of a BAD BH3 peptide described herein to BCL-2 or mutant BCL-2 are known in the art and described in the working examples herein. In some instances, the BAD BH3 peptide is 14 to 50, 20 to 50, 20 to 40, 20 to 30, 20 to 25, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0130] 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, less than 25, less than 20, or less than 15 amino acids in length. In some cases, the BAD BH3 peptide can include 1, 2, or 3 amino acids added to the N- and / or C-terminal of SEQ ID NO: 1 or 3. In certain instances, those added amino acid sequences are the amino acid sequences from the corresponding region of the WT BAD protein.

[0131] In some instances, the BAD BH3 peptide includes 2 to 10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions within SEQ ID NO:1 or 3.

[0132] In some instances, the BAD BH3 peptide includes two amino acid substitutions within SEQ ID NO: 1 or 3, wherein the two amino acids in SEQ ID NO: 1 or 3 are 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. For example, the BAD BH3 peptide can include (i) Ala at position 2 and Tyr at position 6, (ii) Tyr at position 6 and Leu at position 10, (iii) Glu at position 9 and Met at position 13, (iv) Tyr at position 6 and Met at position 13, (v) Glu at position 9 and Glu at position 16, or (vi) Arg at position 12 and Asp at position 19 of SEQ ID NO: 1 or 3 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.

[0133] In some instances, in addition to the two amino acid substitutions with a,a- disubstituted non-natural amino acids, the BAD BH3 peptide further includes 1 to 11 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) other amino acid substitutions. In some instances, the 1 to 11 other amino acid substitutions do not interfere with the binding of the BAD BH3 peptide to BCL-2 or venetoclax -resistance mutants thereof. For example, the 1 to 11 other amino acid substitutions can be on the BCL-2 non-interacting helical face of the sequence of SEQ ID NO: 1 or 3. In some cases, the BCL-2 non-interacting helical face of SEQ ID NO: 1 and 3 includes positions 1, 4, 5, 8, 9, 11, 12, 15, 16, 19, and 22. In some instances, the 1 to 11 other amino acid substitutions are at any one or more (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) of positions 1, 4, 5, 8, 9, 11, 12, 15, 16, 19, and 22 of SEQ ID NO: 1 Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO or 3. In some instances, the 1 to 11 other amino acid substitutions are with conservative amino acids, non-conservative amino acid substitutions, or a combination thereof. Tn some instances, the substituted amino acid at positions 15 or 16 of SEQ ID NO: 1 or 3 is an acidic residue (e.g., D15E, E16D). In some instances, the 1 to 11 other amino acid substitutions are not at one or more of positions 2, 3, 6, 7, 10, 13, 14, 17, 18, 20, and 21 of SEQ ID NO: 1 or 3. In other instances, the 1 to 11 other amino acid substitutions can be at one or more of positions 2, 3, 6, 7, 10, 13, 14, 17, 18, 20, and 21 of SEQ ID NO: 1 or 3; however these substitutions are conservative amino acid substitutions. In some instances, the 1 to 11 other amino acid substitutions are not at position 15 of SEQ ID NO: 1. In some instances, the 1 to 11 other amino acid substitutions are not at position 10 of SEQ ID NO: 1. In certain cases, the substitutions to SEQ ID NO: 1 or 3 can be made by considering the alignment of the BAD BH3 domain with the BH3 domain sequences of other BCL-2 family members. In some cases, conserved residues are not substituted. In certain cases, conserved residues are substituted, e.g., with residues having similar or enhanced properties analogous to the conserved residues. For example, Leu at position 10 can be substituted with para-chlorine pheno moiety, and Asp at position 15 can be substituted with glutamic acid.

[0134] In some instances, the amino acid substitution(s) does not reduce (e.g., substantially reduce) binding of the peptide or stapled peptide to its target protein (e.g., BCL-2 or BCL-2 venetoclax resistance mutants), and can, in some circumstances, improve binding activity. Methods for detecting any reduction in binding can include comparing binding affinity following amino acid substitution, wherein any amino acid substitution that reduces (e.g., substantially reduces) binding is disfavored. 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., BCL-2 or BCL-2 mutants). Methods for assessing interaction between a stapled peptide or unstapled peptide and a target protein (e.g., BCL-2 or BCL-2 mutants) are disclosed herein. Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0135] 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).

[0136] In some instances, the BAD BH3 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 or SEQ ID NO:3. In some instances, (i) Ala at position 2 and Tyr at position 6, (ii) Tyr at position 6 and Leu at position 10 of SEQ ID NO: 1 or 3 are each substituted 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.

[0137] STAPLED PEPTIDES

[0138] Also provided herein are stapled versions of the BAD peptides described herein. Examples of such stapled peptides are provided in FIGS. 3, 4, and 22.

[0139] “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, Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO 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).

[0140] 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).

[0141] 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).

[0142] 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.

[0143] 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 Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO amino acids are separated by 2 (i.e., i, i+3), 3 (z.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.

[0144] In the case of a cross-link between z and i+3 the cross-link can be a C7 alkylene or alkenylene. In the case of a cross-link between z and i+4 the cross-link can be a Cs alkylene or alkenylene. In the case of a cross-link between z and i+ 7 the cross-link can be a C11, C12, or C13 alkylene or alkenylene. When the cross-link is an alkenylene, there can be one or more double bonds.

[0145] 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 C& alkene having a single double bond). In the case of a cross-link between z and i+4 the cross-link can be a Cs alkyl or alkene. In the case of a cross-link between z and i+ 7 the cross-link can be a C11, C12, or C13 alkyl or alkene e.g., a C 11 alkene having a single double bond). When the cross-link is an alkene, there can be one or more double bonds.

[0146] For stapled peptides where an z 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 z linked to i+4 staple is used (one turn of the helix stabilized), either two Ss amino acids ((S)-a-(4'-pentenyl)alanine) can be used, or two Rs amino acids ((R)-a-(4'- pentenyl)alanine) can be used.

[0147] 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 be placed on the interacting face as long as binding affinity or activity is not diminished. As shown in this disclosure, surprisingly and unexpectedly, staples placed on the BCL-2 interacting face of the BAD BH3 alpha helix increases the binding affinity or activity. The “interacting face” of the stapled peptides described herein includes those amino acid residues of the alpha helix that interact (e.g., interact Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO specifically or bind specifically) with BCL-2 or mutant BCL-2. In some aspects, staples 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.

[0148] 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 z and z+3; z 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.

[0149] 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, WO 2010 / 148335, and Li et al., Pharmacological Research, 203(2024) 107137, each of which are herein incorporated by reference in their entireties.

[0150] 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. Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0151] 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 (z.e., z, z+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+4; 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, Xaas, Xaa4, Xaas, Xaae, Xaa?, Xaas, Xaa9. .. (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.

[0152] Provided herein are BAD BH3 stapled peptides. In some instances, the stapled peptide comprises any of the amino acid sequences set forth in SEQ ID NOs.: 4 to 23 or SEQ ID NOs.: 24 to 40, wherein B is norleucine and X and 8 are a,a-disubstituted nonnatural amino acids, and wherein the side chains of each of the two Xs or the sidechains of 8 and X are cross linked. 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 cases, the stapled peptide comprises the sequence set forth in any one of SEQ ID NOs.: 6, 10, 13, 30, 33, or 36. In other cases, the stapled peptide comprises the sequence set forth in any one of SEQ ID NOs.: 6, 10, 13, 30, 33, or 36 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, wherein the stapled peptide binds a venetocl ax-resistant BCL-2 protein. In certain cases, the stapled peptide comprises the sequence set forth in SEQ ID NO: 6 or 10. In other cases, the stapled peptide comprises the sequence set forth in SEQ ID NO: 6 or 10 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, wherein the stapled peptide binds a venetoclax- resistant BCL-2 protein. The amino acid substitutions may be on the non-interacting helical face of the stapled peptide (i.e., the face that does not interact with BCL-2). The discussion in the section above can be used to identify amino acid(s) in the peptide to be substituted. It is of course understood that the stapling amino acids will not be substituted. In some instances, the two Xs 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 BCL-2. In some instances, the stapled peptide binds Attorney Docket No: 00530-0425W01 / DFCI 3581.W01WO mutant BCL-2. In certain cases, the mutant BCL-2 is a mutant that does not bind or has reduced binding to venetoclax (e g., relative to a protein comprising the sequence set forth in SEQ ID NO:2 or 3, or a stapled version thereof wherein the staple is either at positions 2 and 6 or at positions 6 and 10). In some instances, the venetoclax resistant BCL-2 mutant is G101V, GIOIA, D103Y, D103E, D103V, F104C, F104L, F104S, A113G, LI 19V, R129L, V156D. In some instances, the stapled peptide is 22 to 50, 22 to 40, 22 to 30, 22 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, or 22 amino acids in length. In some cases, the stapled peptide is between 8 and 22 amino acids in length, e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22 amino acids in length.

[0153] In another aspect, the disclosure features BAD stapled peptides comprising the structure of 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;

[0154] 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 R5;

[0155] R4 and R4’are independently alkylene, alkenylene, or alkynylene e.g., each are independently a Ci, C2, Cs, C4, Cs, Ce, C7, Cs, C9, or C10 alkylene, alkenylene or alkynylene);

[0156] Rs is halo, alkyl, ORe, N(Re)2, SRe, SORe, SO2R6, CO2R6, Re, a fluorescent moiety, or a radioisotope;

[0157] O

[0158] K is O, S, SO, SO2, CO, CO2, CONRe, , aziridine, episulfide, diol, or amino alcohol; Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0159] Re is H, alkyl, or a therapeutic agent; n is 2, 3, 4, or 6; x is an integer from 2-10 optionally 3 or 6; 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), optionally 1; and each Xaa is independently an amino acid (e.g., one of the 20 naturally occurring amino acids or any non-naturally occurring amino acid).

[0160] In the case of Formula I, the following aspects are among those disclosed.

[0161] In cases where x = 2 (i.e., i+3 linkage), R3 can be a C7 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 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.

[0162] In some instances, [Xaa]w is W, [Xaa]x is AQR, and [Xaa]y is GRELRRB SDEFVDSFK (SEQ ID NO: 41). In some instances, [Xaa]w is WAAQR (SEQ ID NO: 42), [Xaa]x is GRE, and [Xaa]y is RRBSDEFVDSFK (SEQ ID NO: 43). In some instances, [Xaa]w is WAAQRYGR (SEQ ID NO: 44), [Xaa]x is LRR, and [Xaa]y is SDEFVDSFK (SEQ ID NO: 45). In some instances, [Xaa]w is WAAQR (SEQ ID NO: 42), [Xaa]x is GRELRR (SEQ ID NO: 46), and [Xaa]y is SDEFVDSFK (SEQ ID NO: 45). In some instances, [Xaa]w is WAAQRYGR (SEQ ID NO: 44), [Xaa]x is LRRBSD (SEQ ID NO: 47), and [Xaa]y is FVDSFK (SEQ ID NO: 48). In some instances, [Xaa]w is WAAQRYGRELR (SEQ ID NO: 49), [Xaa]x is BSDEFV (SEQ ID NO: 50), and [Xaa]y is SFK.

[0163] In certain instances, the two alpha, al pha-di substituted 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 Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO 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 R? double bond can be in the E or Z stereochemical configuration.

[0164] In some instances R3 is [R4-K-R4’]n; and R4 and R4’are independently alkylene, alkenylene, or alkynylene (e.g., each are independently a Ci, C2, C3, C4, Cs, Ce, C7, Cs, C9, or C10 alkylene, alkenylene or alkynylene).

[0165] The tether can include an alkyl, alkenyl, or alkynyl moiety (e.g., Ce, Cs, or C11 alkyl, a Ce, Cs, or C11 alkenyl, or Cs, Cs, or C11 alkynyl).

[0166] 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 Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO 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 33'2125 (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.

[0167] 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.

[0168] The symbol “ when used as part of a molecular structure, refers to a single bond or a trans or cis double bond.

[0169] 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-).

[0170] 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. Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0171] 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.

[0172] 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.

[0173] 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.

[0174] 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. Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0175] 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.

[0176] 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.

[0177] 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.

[0178] 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 Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO 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.

[0179] While hydrocarbon tethers (cross-links) have been described, other tethers (crosslinks) 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.

[0180] 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.

[0181] Additionally, while examples of tethers (cross-links) spanning from amino acids i to i+3, i to z+ ; and i to i+ 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 z+7).

[0182] In some instances, alpha-disubstituted 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.

[0183] METHODS OF MAKING PEPTIDES AND STAPLED PEPTIDES

[0184] 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 Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO 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.

[0185] 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); Photosw itchable 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

[0186] 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-NH2 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.

[0187] 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 Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO 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.

[0188] 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.

[0189] 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-CFL); 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.

[0190] The peptides and stapled peptides can be further modified by one or more of: acetylation, amidation, biotinylation, cinnamoylation, farnesylation, fluoresceination, formylation, lipidation (e.g., myristoylation, palmitoylation, cholesterol modification), phosphorylation (Ser, Tyr, or Thr), stearoylation, succinylation, and sulfurylation. As indicated above, peptides and stapled peptides can be conjugated to, for example, Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO polyethylene glycol (PEG); alkyl groups (e.g., C1-C20 straight or branched alkyl groups); fatty acid radicals; and combinations thereof

[0191] 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 z+ 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 MBHA resin (see, e.g., WO 2010 / 148335).

[0192] Fmoc-protected a-amino acids (other than the olefinic amino acids Fmoc-7?s-OH, Fmoc-N-OH, Fmoc-As-OH, and Fmoc-M-OH), 2-(6-chl oro-1 -TZ-bcnzotri azol c- 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 / .V-di isopropyl ethyl amine (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).

[0193] In some instances, the peptides and stapled peptides are synthesized according to methods described in the working examples herein. Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0194] 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 BAD peptide described herein. In some instances, the peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs.: 4 to 40, wherein B is norleucine and each X and 8 are a, a-di substituted non-natural amino acids. In some instances, the stapled peptide comprises the amino acid sequence set forth in SEQ ID NO:6. In some instances, the stapled peptide comprises the amino acid sequence set forth in SEQ ID NO:6 with 1, 2, 3, 4, 5, 6, 7 or 8 amino acid substitution, wherein the stapled peptide binds to venetoclax-resistant BCL-2 mutants. In some instances, the peptide comprises the amino acid sequence set forth in SEQ ID NO: 10. In other instances, the peptide comprises the amino acid sequence set forth in SEQ ID NO: 10 with 1, 2, 3, 4, 5, 6, 7 or 8 amino acid substitution, wherein the stapled peptide binds to venetoclax-resistant BCL-2 mutants.

[0195] 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 BCL2 or mutant BCL2) 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).

[0196] 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. Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0197] 2010 / 0168388; and U.S. Patent No. 7,723,468, each of which are hereby incorporated by reference in their entirety).

[0198] 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.

[0199] 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 BAD BH3 peptides and stapled BAD BH3 peptides described herein can be tested for their ability to bind to BCL2, bind to mutant BCL2, prevent or decrease venetoclax resistance (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).

[0200] 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).

[0201] In Vitro Binding Assays'. To assess the binding and affinity of peptides and stapled peptides described herein to proteins (e.g., BCL-2, mutant BCL-2), 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 Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO 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 BCL-2 or mutant BCL-2 are also provided in the working examples herein.

[0202] 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., BCL-2, mutant BCL-2), 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., BCL-2 or mutant BCL-2 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 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.

[0203] Binding Assays in Intact Cells'. It is possible to measure binding of peptides or stapled peptides to proteins (e.g., BCL-2, mutant BCL-2) on or in intact cells by, e.g., immunoprecipitation experiments.

[0204] 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. Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0205] 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 an 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.

[0206] DELIVERY VEHICLES

[0207] One or more of the peptides or stapled peptides disclosed herein can be formulated in a delivery vehicle for administration to a subject. Delivery vehicles suitable for delivering therapeutic proteins are known in the art. Lipid nanoparticles is a class of delivery vehicles contemplated herein. For example, the peptides or stapled peptides disclosed herein can be loaded into liposomes, which are nanocarriers composed of phospholipid bilayers; solid lipid nanoparticles (SLN) composed of a solid lipid matrix; or nanostructured lipid carriers (NLC) made of a blend of liquid and solid liquid as matrix. Methods of producing lipid nanoparticles and formulating peptides with lipid nanoparticles for therapeutic administration are known in the art.

[0208] Based on their size and number of bilayers, liposomes can be classified into three categories: (1) unilamellar vesicles (ULVs) - vesicle has one bilayer membrane (a single phospholipid bilayer sphere enclosing the aqueous solution); (2) oligolamellar vesicles (OLVs) - vesicles with 2-5 bilayer membranes; and (3) multilam ellar vesicles (MLVs) - vesicles have five or more bilayer membranes in a structure like an onion. Usually, Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO different unilamellar vesicles encircle inside each other with successively smaller sizes, creating a multilamellar structure of concentric phospholipid spheres separated by layers of water.

[0209] The lipids compose the matrix of solid lipid nanoparticles (SLN), and are solid at room and body temperature, usually with a melting point above 40 °C, used in a concentration ranging from 5 to 40%. Distinct types of lipids are used, ranging from triglycerides, partial glycerides, and fatty acids to steroids and waxes. The choice of lipids relies on the solubility of the compound that is incorporated inside the matrix. SLN formulations include emulsifiers placed in the interface of the system that can reduce the surface tension between the aqueous and lipid phases, helping to stabilize the system. Several types of emulsifiers have been employed in SLN formulations, like as bile salts, ethoxylated alcohols, fatty acids, phospholipids, poloxamers, polyethylene glycols, polysorbates, polyvinyl alcohols, quaternary ammonium compounds, sorbitan esters, and tyloxapol. In some instances, a binary combination of emulsifiers helps to stabilize the systems more effectively and results in smaller nanoparticle sizes.

[0210] Three incorporation models for the SLN are known in the art, including (1) SLN Type I / homogeneous matrix model, (2) SLN Type II / drug enriched shell model, and (3) SLN Type 111 / drug enriched core model. In the SLN Type 1 / homogeneous matrix model, the drug is dispersed in the lipid core or as amorphous agglomerates. This type is usually produced by high pressure homogenization (HPH), either cold HPH or hot HPH with an optimized drug / lipid ratio. SLN Type I nanoparticles show good, controlled release properties. SLN Type II / drug enriched shell model includes a drug-free lipid core surrounded by an outer shell containing the drug and the lipid. This model can be used when a faster release of the encapsulated drug is desired. In the SLN Type III / drug enriched core model, the core of the nanoparticle is enriched with drug while the lipid is in the outer shell. This morphology is obtained when the drug concentration in the melted lipid mass is close to its saturation solubility and the lipid, when cooled, precipitates in the core before the lipid. This model can be suitable for drugs that require a prolonged drug release. Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0211] Nanostructured lipid carriers (NLC) are composed of an unstructured solid matrix composed of a mixture of solid and liquid lipid, and an aqueous phase containing one or more surfactants. In general, the lipids are mixed in a 70:30 up to 99.9:0.1 solid / liquid ratio and the concentration of the surfactant ranges from 1.5% to 5% (w / v). The excipients employed in the production of NLC are the ones used for SLN plus a liquid lipid, which can be, e.g., fatty alcohols, medium-chain triglycerides, paraffin oil, and squalene. Moreover, fatty acids, such as oleic, linoleic, and decanoic acid may be used since their properties as penetration enhancers.

[0212] Three types of NLCs are known in the art, including (1) the imperfect type, (2) the amorphous type, and (3) multiple oil-in-solid fat-in-water (0 / F / W) type. The imperfect type occurs when spatially different lipids are mixed, composed of fatty acids that introduce imperfections in the crystal matrix. These imperfections allow a higher drug loading capacity, which can be further increased by using different glycerides and varying the saturation and length of the carbon chain. The amorphous type uses solid special lipids as hydroxy octacosanyl hydroxy stearate or isopropyl myristate with a liquid lipid, forming a structureless amorphous matrix. The resulting amorphous state instead of an ordered state avoids -modification during storage and therefore the drug expulsion. The multiple oil-in-solid fat-in-water (O / F / W) type results in numerous nanosized liquid oil compartments disseminated in the solid matrix. In this case, the drug solubility is higher in the oil compartments, which increases the loading capacity and the prolonged release because the compartments are surrounded by solid lipids.

[0213] Other delivery vehicles suitable for peptides and stapled peptides disclosed herein include but are not limited to, a lipoplex, a polyplex, a lipidoid, a polymer, a microvesicle, an exosome, hyaluronidase, nanoparticle mimics, nanotubes, and conjugates (e.g., antibody, cell-penetrating peptide moieties).

[0214] In one instance, the disclosure encompasses a delivery vehicle (e.g., a lipid nanoparticle) comprising a polypeptide comprising a stapled peptide with the sequence of SEQ ID NO: 6 or 10 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, wherein the stapled peptide binds to BCL-2 and venetoclax-resistant BCL-2 mutants. Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0215] PHARMACEUTICAL COMPOSITIONS

[0216] 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, e.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).

[0217] 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 a BCL2-expressing cancer, a BCL-2 dependent cancer, or a venetoclax- resistant BCL-2 expressing or dependent cancer).

[0218] 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. Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0219] 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.

[0220] 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.

[0221] 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.

[0222] Pharmaceutical compositions can include preservatives and additives. Preservatives can be included to limit or prevent microbial growth or contamination.

[0223] 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.

[0224] 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 Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO interaction is when two entities are covalently connected, optionally through a linker group.

[0225] 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.

[0226] 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.

[0227] 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.

[0228] METHODS OF USE

[0229] Also provided herein are methods of using the peptides, stapled peptides, and pharmaceutical compositions described herein.

[0230] For instance, provided herein is a method of inhibiting the interaction between BCL-2 or a mutant BCL-2 (e.g., a venetoclax resistant BCL-2) and a pro-apoptotic BCL- 2 family member in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of a stapled BAD peptide or a pharmaceutical composition described herein.

[0231] Also, provided herein is a method of treating a BCL-2 expressing cancer in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of a BAD stapled peptide or a pharmaceutical composition described herein. In some cases, the cancer is a venetoclax resistant cancer.

[0232] Furthermore, provided herein is a method of treating a BCL-2 dependent cancer in a human subject in need thereof, the method comprising administering to the human Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO subject a therapeutically effective amount of a BAD stapled peptide or a pharmaceutical composition described herein. In some cases, the cancer is a venetoclax resistant cancer.

[0233] In addition, provided herein is a method of treating a venetoclax-resistant BCL-2- expressing or -dependent cancer in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of a BAD stapled peptide or a pharmaceutical composition described herein.

[0234] 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 a hematologic cancer (e.g., chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), Follicular Lymphoma (FL), acute myeloid leukemia (AML), multiple myeloma (MM) such as relapsing and / or refractory MM) in a subject in need thereof. In some cases, the hematologic cancer is resistant to venetoclax. In certain cases, the hematological cancer is BCL-2 dependent and / or expresses BCL-2. In some cases, the multiple myeloma is relapsed or refractory multiple myeloma. In some cases, the subject in need thereof has CLL with 17p deletion (e.g., as detected by an FDA-approved test). In some cases, the subject has received a prior therapy. In some cases, the methods disclosed herein are useful for the treatment of newly diagnosed acute myeloid leukemia (AM L) in adults 75 years or older, or who have comorbidities that preclude use of intensive induction chemotherapy, e.g., in combination with azacitidine, or decitabine, or low-dose cytarabine. The methods involve administering a therapeutically effective amount of a stapled peptide or a pharmaceutical composition described herein.

[0235] The disclosure also 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 a solid tumor, including, e.g., breast cancer (e.g., ER+metastatic breast cancer), non-small cell lung cancer (NSCLC), ovarian cancer (e.g., platinum-resistant ovarian cancer), and pancreatic cancer. In some cases, the peptides, stapled peptides, or pharmaceutical compositions described herein are used in combination with one or more additional anti-tumor agents, including, e.g., fulvestrant, checkpoint inhibitors, and agents targeting KRAS mutations. Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0236] In certain instances, in the above methods, the subject has one or more of the following BCL-2 mutations: G101V, GIOIA, D103Y, D103E, DI 03V, Al 13G, V156D, R129L, LI 19V, F104C, F104L, F104S, or the in frame insertion ArglO7_Ar l lOdup.

[0237] In some cases, the above methods further comprises chemotherapy. 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. In certain cases, the above methods further comprises selective anti- apoptotic inhibitors. In some cases, the above methods further comprises radiation therapy or immunotherapy. Chemotherapy, selective anti-apoptotic inhibitors, radiation therapy, or immunotherapy 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 (e.g., palliative chemotherapy). The chemotherapy, selective anti-apoptotic inhibitors, radiation therapy, or immunotherapy can be administered before, during, and / or after administration of the peptides, stapled peptides, or pharmaceutical compositions described herein.

[0238] 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-methoxyestradiol, 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 Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0239] (e.g., bevacizumab, brentuximab vedotin, cetuximab, ibritumomab tiuxetan, ipilimumab, panitumumab, rituximab, tositumomab, and trastuzumab). Chemotherapy often produces several side-effects. In certain cases, the method can further comprise administering another therapeutically beneficial agent(s) to the subject. For example, MCL-1 inhibitors, BCL-XL inhibitors, or BTK inhibitors, can be administered before, during, and / or after administration of the peptides, stapled peptides, or pharmaceutical compositions described herein.

[0240] In other cases, the method can further comprise administering combinations with other agents such as chemotherapy, targeted therapy, hormonal therapy, radiation therapy, immunotherapy, antibody drug conjugates, epigenetic modulators, antiangiogenesis agents.

[0241] In certain cases, the method involves first determining if the subject expresses BCL-2 and / or has a venetoclax-resistant BCL-2 mutation(s) before administering therapy.

[0242] 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).

[0243] 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.

[0244] 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 cancer, the patient’s disposition to the treatment, and the judgment of the treating physician. Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0245] 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 cancer 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 the cancer 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 the cancer.

[0246] In certain instances, the peptide or stapled peptide is administered by injection (e.g., into the oval window).

[0247] 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, 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).

[0248] 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 the cancer.

[0249] Following administration, the subject can be evaluated to assess the cancer in the subject. In some instances, treatment can continue until 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 condition 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 the cancer. Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0250] CERTAIN EMBODIMENTS

[0251] In certain aspects, this disclosure provides BAD BH3 peptides comprising the sequence set forth in SEQ ID NO: 6 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 substitutions, and where the two Xs in SEQ ID NO:6 are each (S)-2-(4-pentenyl)alanine, wherein B is norleucine, and wherein the stapled peptides binds to a venetocl ax-resistant BCL-2 protein (e.g., G101V, G101A, D103Y, D103E, D103V, A113G, V156D, R129L, LI 19V, F104C, F104L, F104S, or the in frame insertion Arg 107_Argl lOdup). The substitutions may be on the BCL-2 interacting and / or non-interacting face of the BAD BH3 alpha helix. In instances, where the substitution(s) are on the interacting face, they are generally conservative amino acid substitutions. In certain cases, the substitutions are at one or more ofWl, Q4, R5, R8, E9, R12, DI 5, El 6, DI 9, or K22 (wherein the numbering is based on SEQ ID NO:6). If the substitution is at position D15 of SEQ ID NO:6 it is a conservative substitution (e.g., D15E). In one instance, the peptide comprises the sequence of SEQ ID NO:6. In another instance, the peptide comprises the sequence of SEQ ID NOTO. In certain instances, the peptide is 22 to 50 amino acids in length.

[0252] In some aspects, provided herein are polypeptides comprising BAD BH3 stapled peptides comprising the sequence set forth in SEQ ID NO: 6 with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 substitutions, and where the two Xs in SEQ ID NO:6 are each (S)-2-(4- pentenyl)alanine, wherein B is norleucine, and wherein the stapled peptides binds to a venetoclax-resistant BCL-2 protein (e.g., G101V, G101A, D103Y, D103E, D103V, A113G, V156D, R129L, LI 19V, F104C, F104L, F104S, or the in frame insertion Arg 107_Argl lOdup). The substitutions do not replace the X residues in SEQ ID NO:6. In some cases, the substitutions are at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) of positions 1, 4, 5, 8, 9, 11, 12, 15, 16, 19, and 22 of SEQ ID NO:6. The substitutions can be conservative or non-conservative amino acid substitutions. In some cases, the substitutions can be at one or more positions on the BCL-2 interacting face of the BAD BH3 peptide. In those cases, the substitutions are conservative substitutions. In certain cases, the substitutions are on both the BCL-2 interacting and BCL-2 non-interacting faces of the BAD BH3 peptide. If the substitution is at position D15 of SEQ ID NO:6 it is a conservative substitution (e.g., DI 5E). The variants of the stapled BAD BH3 peptide Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO set forth in SEQ ID NO:6 bind to a venetoclax-resistant BCL-2 protein. In some cases, these variants show reduced or no binding to BCL-w, BCL-XL, MCL-1, and / or BFL-1 / - Al. In certain cases, the BAD BH3 stapled peptide is about 22 to about 50 amino acids in length.

[0253] In some aspects, provided herein is a polypeptide comprising a BAD BH3 stapled peptide comprising the structure of formula (III):

[0254] Formula (III) or a pharmaceutically acceptable salt thereof, wherein Ri is methyl, R2 is methyl, and R3 is (CH2)3-CH=CH-(CH2)3, and wherein [Xaa]wconsists of the sequence W, [Xaa]x consists of the sequence AQR with 0, 1 or 2 substitutions, and [Xaa]y consists of the sequence GRELRRBSDEFVDSFK (SEQ ID NO: 41) with 0, 1, 2, 3, or 4 substitutions. The BAD BH3 stapled peptide binds to a venetoclax-resistant BCL-2 protein. In some cases, BAD BH3 stapled peptide shows reduced or no binding to BCL-w, BCL-XL, MCL-1, and / or BFL-A1. In certain cases, the BAD BH3 stapled peptide is about 22 to about 50 amino acids in length. In some cases, the pharmaceutically acceptable salt is hydrochloride, mesylate, hydrobromide, acetate, fumarate, sodium, calcium, or potassium.

[0255] The disclosure also relates to delivery vehicles comprising a polypeptide described herein. The delivery vehicle can be a nanoparticle such as a LNP The polypeptide is encapsulated within the delivery vehicle.

[0256] Also provided are a composition comprising a nanoparticle, optionally a lipid nanoparticle, and means for binding to both wild type BCL-2 and mutant BCL-2 proteins that are resistant to venetoclax or means for binding to both wild type BCL-2 and mutant Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0257] BCL-2 proteins that are resistant to venetoclax and showing reduced or no binding to BCL-w, BCL-XL, MCL-1 and / or BFL-l / AL

[0258] Also featured herein are pharmaceutical compositions comprising the above polypeptides, stapled polypeptides, or delivery vehicles and a pharmaceutically acceptable carrier.

[0259] This disclosure also provides a pharmaceutical composition comprising means for binding to both wild type BCL-2 and mutant BCL-2 proteins that are resistant to venetoclax, and a pharmaceutically acceptable carrier.

[0260] Also provided herein are methods of making the above stapled polypeptide comprising (i) providing a peptide comprising the sequence of SEQ ID NO: 6, wherein each of the two Xs are (S)-2-(4’-pentenyl)alanine and (ii) cross-linking the peptide thereby making the stapled peptide. In certain cases, the cross-linking is by a ring closing metathesis reaction. In some cases, the method further involves formulating the stapled peptide as a sterile pharmaceutical composition.

[0261] The disclosure also encompasses methods of treating a BCL-2 dependent or BCL- 2 expressing cancer in a human subject, the method comprising administering to the subject a therapeutically effective amount of a polypeptide, stapled peptide, delivery vehicle or pharmaceutical composition described above. In some cases, the cancer is a hematological cancer (e.g., chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), small lymphocytic lymphoma (SLL) or multiple myeloma (MM) such as rel psed or refractory MM). In other cases, the cancer is a solid tumor (e.g., a breast cancer optionally ER+metastatic breast cancer, a non-small cell lung cancer (NSCLC), an ovarian cancer optionally a platinum-resistant ovarian cancer, or a pancreatic cancer). In some cases, the cancer is a venetoclax-resistant cancer (e.g., a cancer with a mutant BCL- 2, wherein the mutant BCL-2 is G101V, GIOIA, D103Y, D103E, D103V, Al 13G, V156D, R129L, L119V, F104C, F104L, F104S, or the in frame insertion ArglO7_Argl lOdup). In some cases, the methods involve combination therapy with one or more of: chemotherapy, targeted therapy, hormonal therapy, radiation therapy, immunotherapy, antibody drug conjugates, epigenetic modulators, or anti-angiogenesis Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO agents. In some instances, the combination therapy is with one or more of a MCL-1 inhibitor, a BCL-XL inhibitors, or a BTK inhibitor.

[0262] 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.

[0263] EXAMPLES

[0264] EXAMPLE 1. GENERATION OF STAPLED PEPTIDE-BASED INHIBITORS OF BCL-2

[0265] Design of stapled peptide-based inhibitors of BCL-2 to overcome venetoclax resistance mutations

[0266] Clinical BCL-2 mutants have arisen based on repeated drug exposure to the selective small molecule BCL-2 inhibitor, venetoclax, with amino acid changes localizing to the vicinity of the binding pocket and thus perturbing small molecule engagement and functional inhibition (FIGS. 1A, IB, and 1C). Whereas such small molecules can still engage BCL-2 mutants, binding activity is disrupted by over one order of magnitude, weakening pro-apoptotic function. To overcome this recent clinical challenge, stapled peptide inhibitors of BCL-2 and its mutants were designed based on the BH3 motif of the BCL-2 Associated Agonist of Cell Death (BAD) protein, which naturally blocks BCL-2 by targeting its inhibitory surface groove (FIG. 2A). An exemplary stapled BAD BH3 peptides of 22 amino acids in length occupy nearly twice the surface area of the hydrophobic groove, as compared to small molecule inhibitors (FIGS. 2B, 2C, and 2D), and mimic native BH3 binding. Without being bound by theory, whereas single point mutations can disrupt small molecule engagement, BAD BH3 peptides may be prefolded and stabilized as a native helix by insertion of the all-hydrocarbon staple and could overcome small conformational perturbations at the BH3-binding site (FIGS. 2B and 2C).

[0267] Synthesis and screening of staple scanning BAD BH3 peptide libraries Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0268] Staple scanning libraries were generated spanning one (z, i+4 staple; FIG. 3 A) or two (z, i+ 7 staple; FIG. 4A) helical turns. Compounds were initially screened by competitive fluorescence polarization (FP) binding assays to determine relative BCL-2 binding affinities based on acetylated stapled peptide competition with a FITC-BIM BH3 peptide for binding to recombinant BCL-2 (FIGS. 3A, 4A). Using an orthogonal assay, we further examined the relative ability of biotinylated and stapled BAD BH3 peptides to capture FLAG-tagged full-length BCL-2 from lysate harvested from transfected cells (FIGS. 3B, 4B). Based on this compilation of quantitative and qualitative binding data, we identified lead candidates from both the z, i+4 and z, z+ 7 libraries. Helical wheel representations demonstrated that several of the top hits were surprisingly and unexpectedly located at the BAD BH3 binding interface with BCL-2, a location that would typically disrupt binding activity due to steric hindrance (FIGS. 3C, 4C). Stapled BAD BH3 compounds 4.2, 4.6, 4.9, 7.6, 7.9, and 7.12 were selected for further analysis.

[0269] EXAMPLE 2. FEATURES OF SELECTED STAPLED BAD BH3 PEPTIDES

[0270] Relative BCL-2 selectivity of lead stapled BAD BH3 peptides

[0271] Since dual BCL-XL / BCL-2 binders such as navitoclax have been shown to induce thrombocytopenia, stapled BAD BH3 peptides that were BCL-2 selective were identified. To evaluate anti-apoptotic preference, a competitive binding assay was carried out, whereby biotinylated and stapled BAD BH3 peptides were individually incubated with an equimolar pool of recombinant BCL-2, BCL-XL, and MCL-1, followed by overnight incubation and streptavidin pull-down (FIG. 5A). Whereas BAD 4.2 and 4.6 strikingly demonstrated exclusive BCL-2 binding activity, BAD 4.9, 7.6, 7.9, and 7.12 demonstrated variable degrees of dual BCL-2 / BCL-XL binding specificity, with little to no MCL-1 interaction overall. In a quantitative, competitive fluorescence polarization (FP) assay measuring displacement of a fluorescein isothiocyanate (FITC)-labeled BIM BH3 peptide (unstapled) from recombinant BCL-2, BAD 4.2 emerged as the most potent ligand (ICso, 15.7 nM) (FIG. 5B) and was thus selected for further characterization. BAD 4.2, the top binder across the binding assays, was then subjected to FP analysis against Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO each individual anti-apoptotic target, as measured by competitive displacement of a FITC-BTM BH3 peptide, and again demonstrated a binding preference for BCL-2 over BCL-XL and BCL-w, with no interaction among MCL-1 or BFL-1 recombinant proteins (FIG. 5C)

[0272] BAD 4.2 effectively targeted the spectrum of venetoclax-resistant BCL-2 mutants and outcompeted venetoclax for binding to mutant BCL-2 in cell lysates

[0273] In contrast to the 177-fold decrease in venetoclax binding observed upon G101V mutation of BCL-2, BAD 4.2 bound with similar or better affinity across a series of clinical mutants of BCL-2, including the G101V, D103Y, F104L, Al 13G, R129L, and V156D, as measured by competitive FP (FIGS. 6A and 6B). BAD 4.2 binding was further characterized by biolayer interferometry (BLI), demonstrating time-dependent association and negligible dissociation upon exposure to WT and GlOlV-mutant GST- BCL-2 proteins (FIGS. 7A, 7B, 7C, and 7D). Equilibrium dose-response curves of best fit from the end points of binding association curves were used to determine Kd, which corroborated with ECso values (FIGS. 7E and 7F).

[0274] Direct and competitive FP analyses of BAD 4.2 binding to WT, G101V and F104L BCL-2 proteins, highlighted and corroborated the interaction potencies (FIGS. 8A and 8B). The ability of venetoclax to compete with BAD 4.2 for binding to full-length BCL-2 proteins expressed in cells was then tested. Whereas venetoclax effectively and dose-responsively competed with BAD 4.2 binding to WT BCL-2, as assessed by streptavidin pull down of biotinylated BAD 4.2 from treated lysates, the capacity of venetoclax to compete with BAD 4.2 binding to BCL-2 was reduced by more than 10- fold upon G101V or F104L mutagenesis (FIG. 8C).

[0275] BAD 4.2 neutralized the capacity of BCL-2 or its G101 V mutant to block tBID- triggered BAX poration of liposomes

[0276] BCL-2 can effectively sequester the BH3 motifs of pro-apoptotic BCL-2 proteins, such as the BH3-only protein tBID and the multidomain pro-apoptotic protein BAX to block apoptosis induction. Here, fluorophore encapsulated liposomes that mimicked the outer membrane of mitochondria were used to monitor membrane poration in response to Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO tB ID-triggered BAX activation in the presence of recombinant BCL-2 proteins and the BCL-2 inhibitors venetoclax or BAD 4.2 (FIGS. 9A, 9B, and 9C). Whereas the BAD 4.2 stapled peptide was equally capable of blocking BCL-2 or its G101V or F104L mutants to restore BAX-mediated poration, G101V or F104L mutagenesis impaired venetoclax activity (FIG. 9D). Taken together, these data demonstrate that BAD 4.2 retained both binding and functional activity irrespective of BCL-2 G101V or F104L mutagenesis, whereas, in contrast, venetoclax activity was substantially impaired.

[0277] Structural basis for BAD 4.2 binding to BCL-2 and its G101 V mutant

[0278] The crystal structures of the complexes between BAD 4.2 and both BCL-2 WT (PDB 9014, 1.73 A) and its G101V mutant (PDB 9015, 1.99 A) were solved (FIGS. 10A, 10B, and 11). In both complexes, BAD 4.2 maintained similar peptide backbone alignments within the groove, indicating that, in contrast to venetoclax, G101V mutagenesis did not affect the BAD SAHB 4.2 binding mode. We find that the allhydrocarbon i, i+4 staple was directed toward the peptide / protein binding interface and engaged a discrete hydrophobic patch formed by residues Ml 15 and LI 19 of BCL-2, with VI 33 and Fl 53 further reinforcing the hydrophobic network (FIGS. IOC and 10D). In addition, the hydrophobic network of the BH3-conserved leucine (LI 14) of BAD 4.2 with BCL-2 residues F104, Fl 12, Ml 15, V133, L137, A149, and F153 was also preserved (FIGS. IOC and 10D), as were the electrostatic and hydrophilic pairings (BAD 4.2-BCL-2) of DI 19-N143 / R146, E120-Y108, R115-E136 / D140, and R116-Y 108 / DI 11 (FIGS. 10E and 10F) Also of note, BAD 4.2 contained two C-terminal i, i+4 phenylalanine residues, F121 and F125, which participated in a network of aromatic contacts with BCL-2 residues Y108, Fl 04, Fl 98, and Y202, demonstrating edge-to-face interactions (T-stacking) that created a stabilized aromatic cage in this region of the binding interface (FIGS. 10G and 10H). Thus, BAD 4.2, with its three-times larger molecular weight and approximately double the binding surface of venetoclax, including the newfound hydrocarbon staple interaction, was unaffected by single point mutagenesis (FIG. 9D) Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0279] Differential structural impacts of venetoclax and BAD 4.2 engagement of WT and

[0280] G101 V-mutant BC -2

[0281] Differences in the disposition of residues between BCL-2 proteins bound to venetoclax and BAD 4.2 were identified using difference distance matrix plots (DDMPs). Comparing the DDMP derived from structures of venetoclax bound to GlOlV-mutant versus WT BCL-2, we observed (1) reductions between discrete residues adjacent the G101V mutation, tightening the protein core through VlOl’s increased hydrophobic interactions, and (2) increased distances in helices that underwent allosteric changes as a result of the mutation (FIG. 12A). In contrast, BAD 4.2 binding nullified the conformational consequences of G101V mutagenesis observed for venetoclax interaction, as similar perturbations were not observed in the DDMP for BAD 4.2 bound to G101 V- mutant versus WT BCL-2 (FIG. 12B). Comparing the relative impact of BAD 4.2 and venetoclax upon binding to WT BCL-2, the stapled peptide induced extensive conformation consequences throughout the protein (FIG. 12C). When compared in the context of GlOlV-mutant BCL-2 (FIG. 12D), the pattern of changes was largely similar although somewhat diminished, indicating that, while the mutant protein exhibited increased structural rigidity, BAD 4.2 induced an opening of the BH3 groove not observed upon interaction with venetoclax.

[0282] BAD 4.2 binding reverted the conformational consequences of G101 V mutagenesis to the wild-type state

[0283] Hydrogen deuterium exchange mass spectroscopy (HDX MS) was used to characterize the conformational consequences of BCL-2 protein point mutagenesis, in the presence and absence of BAD 4.2 interaction. The relative deuterium uptake of BCL-2 WT was first determined, comparing the apo and BAD 4.2-bound states. BAD 4.2 caused prominent protection from deuterium exchange at the very structural region of the canonical BH3-binding groove (FIG. 13A). BAD 4.2 similarly protected the canonical groove of BCL-2 G101V (FIG. 13B), which appeared less exposed or conformationally dynamic compared to BCL-2 WT (FIG. 13C). Notably, when the deuterium difference between BAD 4.2 / BCL-2 G101V and BAD 4.2 / BCL-2 WT was compared, there was Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO little to no conformational differences, reflecting both the capacity of BAD 4.2 to readily engage both targets and the ability of BAD 4.2 to revert conformational differences between BCL-2 G101V and BCL-2 WT, as demonstrated by HDX MS (FIG. 13D).

[0284] Structural basis for BAD 4.2 binding and increased affinity to R129L-nuitant BCL-2

[0285] The crystal structures of the complexes between BAD 4.2 and R129L-mutant BCL-2 were solved to further examine the structural basis for the largest enhancement of BAD 4.2 binding affinity for a clinical mutant. The high-resolution crystal structure of BCL-2 R129L with BAD 4.2 (PDB 9016, 1.73 A) demonstrated unique interactions between the all -hydrocarb on staple and the adjacent LI 29 residue (FIGS. 14A and 15). The R129L mutation extended the hydrophobic groove to a region previously occupied by the positively charged R129, forming a convergence of hydrophobic residues that expanded the hydrophobic binding interface and provided a larger surface to engage the hydrocarbon staple (FIG. 14B).

[0286] Consequences in BCL-2 conformation and peptide binding upon R129L mutagenesis

[0287] A DDMP comparing BAD 4.2 bound to R129L -mutant versus WT BCL-2 illustrated that the extension of the hydrophobic groove by an additional hydrophobic residue strengthened the interaction network of the all-hydrocarbon staple, as highlighted by the reduced distance between the helices comprising the BH3 groove and as mapped onto the structure of BAD 4.2 bound to R129L-mutant BCL-2 (FIGS. 16A and 16B). Evaluating comparative protein-peptide distances for the two complexes by DDMP likewise showed a reduction of the distances between the peptide N-terminus, which bore the staple, to this same region of mutagenesis bearing added hydrophobic density (FIG. 16C). A superposition of the two complexes revealed that the staple became further entrenched in the groove adjacent the R129L mutation, with associated downward displacement of the peptide backbone (FIG. 16D). The capacity of all-hydrocarbon stapling to exploit the expanded hydrophobic surface at the canonical groove showcased the utility of stapled BAD BH3 helices as tools for both dissecting structure-activity relationships and targeting BCL-2 mutants that confer resistance to small molecule inhibitors. Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0288] BAD 4.2 binding reverted the conformational consequences of R129L mutagenesis to the wild-type state

[0289] HDX MS analyses of the R129L mutant of BCL-2 were also performed, comparing its deuterium exchange pattern to that of BCL-2 WT and the influence of BAD 4.2 engagement. BAD 4.2 affected BCL-2 WT (FIG. 17A) and BCL-2 R129L (FIG. 17B) similarly, protecting the canonical groove regions of both proteins from deuterium exchange by nearly identical extents (FIGS. 17A, 17B). The influence of BAD 4.2 on the deuterium exchange profile of WT and mutant BCL-2 were very similar, underscoring the capacity of BAD 4.2 to effectively engage both proteins. Whereas the R129L mutation caused conformational protection from deuterium exchange in the canonical groove and in the a5-a6 hairpin that lay adjacent to the ot3-ot4 turn containing R129L (FIG. 17C), BAD 4.2 binding mitigated the differences observed in the canonical groove and fully eliminated the effects of mutagenesis on the oc5-ot,6 region (FIG. 17D). Taken together, these data highlight the influence of point mutagenesis on the conformational dynamics of BCL-2 WT, and the capacity of BAD 4.2 to not only bind BCL-2 WT and its clinical mutants, but also effectively revert the structural consequences of point mutagenesis toward the wild-type state.

[0290] To determine if BCL-2 targeting by BAD 4.2 could broadly combat the heterogeneous conformational impacts of individual mutants, HDX MS analysis of additional BCL-2 mutants (D103Y, Al 13G, V156D) was performed in the presence or absence of stapled peptide. Strikingly, irrespective of mutation site and resultant structural alteration, BAD 4.2 binding at the canonical groove was fully preserved (FIGS. 18A, 18B, and 18C), as reflected by the deuterium difference profiles that mirrored the protection of WT BCL-2 upon BAD 4.2 engagement (FIGS. 13A, 18D).

[0291] We also expanded the analysis to define the differences in deuterium exchange profiles of unbound mutant and WT BCL-2 proteins. Upon D103Y mutagenesis, we observed deprotection of both a portion of the ocl-a2 loop and the proximal half of ot.2 upstream of the point mutation, changes that localized to the vicinity of DI 03 Y in oc2 (FIG. 19A). Whereas Al 13G mutagenesis (located in ot.3 ) induced subtle deprotection of Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0292] 0.3 through a 4 starting just distal to the mutation (FIG. 19B), more prominent changes occurred in response to R129L (FIG. 17C) and V156D mutagenesis. V156D, which was located in the middle of oc5, caused circumferential deprotection of oc2, the proximal portion of 0.3, and of a7, which lay in direct proximity to the proximal portion of a 2 (FIG. 19C). In calculating the difference in deuterium exchange between BAD 4.2 when bound to mutant vs. WT BCL-2 proteins, we observed that the D103Y and Al 13G complexes were essentially indistinguishable from that of WT (FIGS. 20A, 20B), as observed with the G101V mutant (FIG. 13D) and illustrated with a heatmap of apo and bound comparisons with respect to each point mutation (FIGS. 21A, 21B, 21C). Whereas BAD SAHB 4.2 effectively protected the surface groove of BCL-2DC V156D (FIG. 18C), the relative difference in deuterium uptake compared to the other mutants was less negative (e.g., peptide 98-111 : D103Y, -5.79; A113G, -5.4; R129L, -5.88, V156D, - 4.81), consistent with V156D mutagenesis localizing to the oc5 core and substantially destabilizing apo BCL-2AC (FIG. 19C). Thus, by retaining effective surface groove interactions (FIG. 18D), BAD 4.2 achieved at least partial and in most cases complete reversal of the conformational alterations observed in the mutant apo proteins (FIGS. 13D, 17D, 20A-20C, 21A-21E) that otherwise conferred venetoclax resistance.

[0293] EXAMPLE 3. SELECT MUTATIONS OF STAPLED BAD BH3 HELICES

[0294] Relative WT and G101 V binding of mutated lead stapled BAD BH3 peptides

[0295] BAD 4.2 was subjected to mutagenesis at positions 10 and 17 (SEQ ID NO: 6, FIGS. 22 and 23), which were identified as residues that confer preferential binding to BCL-2 while mitigating binding to other anti-apoptotic BCL-2 family members, namely BCL-XL. We first examined the ability of mutant iterations of BAD 4.2 to maintain binding to GlOlV-mutant BCL-2 (FIG. 22) and then proceeded to examine the affinity of these BAD 4.2 mutants for BCL-XL to assess relative binding specificity (FIG. 23). To evaluate the importance of the conserved LI 14 residue of the BAD BH3 motif within the BAD BH3-only protein (SEQ ID NOs: 1 and 2), we mutated the corresponding LIO residue in BAD 4.2 (SEQ ID NO: 6) to an alanine (BAD 4.2-A) and observed complete Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO loss of binding to WT BCL-2, GlOlV-mutant BCL-2, and BCL-XL. We then examined the substitution of LIO with phenylalanine (BAD 4.2-B) and the non-natural amino acid 4-chloro-L -phenylalanine (BAD 4.2-C). BAD 4.2-B did not show improved binding affinity toward BCL-2 WT or BCL-XL, and lost affinity for GlOlV-mutant BCL-2, consistent with the increased steric hindrance of BCL-2’s mutant interface, as also seen with venetoclax at the same P2 pocket of the canonical BH3 groove. BAD 4.2-C introduced a chlorine moiety on the phenylalanine residue (similar to venetoclax’s moiety in this region) and, although it is subject to similar perturbation as venetoclax, BAD 4.2- C did not lose significant binding affinity for the G101V mutant, likely due to the peptide’s increased overall binding interface (FIG. 2D). BAD 4.2-C demonstrated an ~3- fold binding preference for BCL-2 over BCL-XL

[0296] Mutations of the phenylalanine residue of BAD BH3 at position 17 of BAD 4.2 (SEQ ID NO: 6) also demonstrated varying consequences for WT and GlOlV-mutant BCL-2 and BCL-XL binding affinity (FIGS. 22 and 23). BAD 4.2-D, which substituted a tryptophan for phenylalanine, showed significantly increased affinity toward BCL-2 and its G101V mutant, with no significant change in BCL-XL binding activity. Thus, relative to BAD 4.2, BAD 4.2-D exhibits an ~2-fold enhancement in BCL-2 protein binding affinity and an increased ~5-fold preference for BCL-2 over BCL-XL, as compared to BAD 4.2. BAD 4.2-E, which substituted the non-natural residue D-Ala(2-naphthyl)-OH at position 17, showed modestly decreased binding affinity to WT and GlOlV-mutant BCL-2 and BCL-XL. BAD 4.2-F, which substituted the non-natural residue beta-(4- biphenyl)-L-alanine at position 17, demonstrated modestly decreased binding affinity for BCL-2 and its G101V mutant as compared to BAD 4.2 but significantly perturbed binding to BCL-XL (~20-fold lower compared to BCL-2), a desirable attribute for the development of BCL-2-selective BAD BH3 stapled helices.

[0297] A double mutation, BAD 4.2-G (SEQ ID NO: 60), which replaced residues L and F at positions 10 and 17 of BAD 4.2 with F and W, respectively, demonstrated modestly decreased binding affinity for WT and GlOlV-mutant BCL-2 (FIG. 22). Notably, this Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO compound exhibited a 12-fold decrease in binding affinity for BCL-XL, a desirable attribute for the development of BCL-2-selective BAD BH3 stapled helices. (FIG. 23).

[0298] Methods used in Examples 1-3

[0299] Peptide Synthesis

[0300] Stapled BAD BH3 peptides were synthesized, derivatized with either biotin, acetyl or FITC at the N-terminus, purified, and quantified by amino acid analysis (See, e.g., Bird et al., Methods in Enzymol, 2008). Stapled peptides were synthesized by solid phase Fmoc chemistry using rink-amide resin. Elongation of the peptides was performed with hexafluorophosphate azabenzotriazole tetramethyl uranium (HATU) as the coupling reagent and diisopropylethylamine (DIEA). Two naturally-occurring amino acids at discrete i, i+4 or i, i+7 positions were replaced with the non-natural amino acid pairs S- pentenyl alanine and S-pentenyl alanine, or R-octenyl alanine and S-pentenyl alanine, respectively, at the indicated locations to enable stapling by olefin metathesis using the Grubb’s first-generation catalyst. Peptides were deprotected and cleaved from the resin by a solution of 95:2.5:2.5 (v / v) TFA / TIS / water and purified by C18 reverse phase HPLC in acetonitrile / water gradient supplemented with 0.1% TFA to >95% purity. The pure peptides were lyophilized and stored at -20 °C until use.

[0301] Recombinant Protein Expression and Purification

[0302] Recombinant BCL-2AC 1-34.92-207 containing a modified loop between al and 2 with a C-terminal alanine (Birkinshaw et al., 2019) was cloned into the pTYBl vector using Xbal and Kpnl restriction sites. Point mutations were performed via PCR-based mutagenesis (Q5 Site-Directed Mutagenesis KIT, New England BioLabs) and validated by DNA sequencing. BCL-2AC 1-207 and point mutations with a native loop between al and a2 were cloned into pGEX-4T-l expression vectors (GE Healthcare). BFL-IAC1-153, MCL-1ANAC no-327 and BCL-XLACI-212 in pET19b (BFL-1) or pGEX-4T-l (MCL-1 and BCL-XL) were produced as previously described (Huhn et al., 2016). Full-length BAX and BCL-WACi-193 in pTYBl was produced as previously described (Hauseman et al., 2020). Transformed E. coli BL21(DE3) were cultured in Luria Broth containing Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO ampicillin (0.1 g / L), grown to an OD of 0.6-0.8, and protein expression was induced by the addition of 0.5 mM (BCL-21-34, 92-207 and BCL-W) or 1 .0 mM (BCL-21-207, BFL-1, MCL-1, BCL-XL, and BAX) isopropyl 0-0-1 -thiogalactopyranoside (IPTG); expression was allowed to proceed overnight at 16°C with 0.5 mM IPTG or for 4 h at 37 °C with 1.0 mM IPTG. Bacterial pellets were collected by centrifugation. Pellets were then resuspended in lysis buffer (pTYBl vectors: 250 mM NaCl, 20 mM Tris, pH 7.2; pGEX vectors: PBS containing 0.1% Triton X-100; pET19b vector: 150 mM NaCl, 20 mM Tris, pH 8.0) containing complete protease-inhibitor tablets. The cell suspension was then lysed using a chilled microfluidizer, centrifuged to remove insoluble cellular debris, applied to resin (chitin for pTYBl vectors; glutathione for pGEX vectors; Ni-NTA for pET19b vectors) and washed with lysis buffer. Chitin-bound BCL-21-34, 92-207, BAX, and BCL-W were cleaved by overnight incubation with 50 mM dithiothreitol (DTT) at 4°C; to produce GST-cleaved BCL-2I-207 and BCL-XL, proteins were incubated with PBS containing thrombin overnight at 25 °C; to produce GST-BCL-2 and GST-MCL-1, protein was eluted from the glutathione column with elution buffer (50 mM Tris, pH 8.0, 10 mM GSH); nickel -bound BFL-1 was eluted with a gradient of imidazole ranging from 5 to 300 mM. Eluted proteins were concentrated and purified by size exclusion chromatography using a Superdex S-75 (GE Healthcare) gel filtration system (BCL-21. 34,92-207, BAX, and BCL-W: 150 mM KC1, 20 mM HEPES, pH 7.2; GST-MCL-1 and BCL-XL: 150 mM NaCl, 50 mM Tris, pH 7.4; BCL-21-207: 150 mM NaCl, 20 mM Tris, pH 7.4; BFL-1 : 150 mM NaCl, 20 mM Tris, pH 8.0). Protein purity and identity were verified by Coomassie staining and western blot analysis.

[0303] PISA (Proteins, Interfaces, Structures, and Assemblies) Analysis

[0304] To quantify the interfaces between WT BCL-2 and the BH3 -mimetic ligands venetoclax and BAD 4.2 (PDB IDs 6O0K and 9014, respectively), we used PDBe PISA (Proteins, Interfaces, Structures, and Assemblies; vl.52, 10 / 20 / 2014) to computationally identify interface residues and calculate: (i) the solvent-accessible surface area (ASA) for each ligand, (ii) the interface or buried surface area (A2) of BCL-2 upon complex formation, and (iii) the relative buried surface area (percentage of total ASA of BCL-2). Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0305] These parameters were reported as Total Surface Area, Surface Area of B CL -2 Interface, and Relative BCL-2 Coverage, respectively.

[0306] Fluorescence Polarization Binding Assays

[0307] Direct binding assays were first performed to determine IC90 concentrations with FITC BIM BH3 for standardized competition assays (See, e.g., Bernal et al, J Am Chem Soc, 2007). Recombinant BCL-2AC (aa 1-34, 92-207), its mutants, and other anti- apoptotic proteins (4 pM final maximal concentration) were serial diluted 1 :3 into binding assay buffer (5% DMSO, 0.01% Triton X-100 in lx PBS) in a 96 well plate. FITC-BIM BH3 peptide (3 nm final concentration) was then added and the mixture was incubated in the dark for 30 minutes at 25°C. Fluorescence readings (485 nm excitation, 525 nm emission) were performed on a SpectraMax M5 microplate reader (Molecular Devices). Data were analyzed by nonlinear regression analysis using Prism software (GraphPad) to calculate IC90 concentrations. Competitive binding assays were performed with the same buffer (5% DMSO, 0.01% Triton X-100 in lx PBS) using a 1 :3 serial dilution of acetylated BAD BH3 peptides. FITC-BIM BH3 and the specified proteins were then added for a final concentration of 3 nM and the previously calculated IC90 protein concentration, respectively. The mixture was incubated in the dark for 30 minutes at 25°C. Fluorescence readings (485 nm excitation, 525 nm emission) were performed on a SpectraMax M5 microplate reader (Molecular Devices). Data were analyzed by nonlinear regression analysis using Prism software (GraphPad) to calculate EC50 concentrations.

[0308] Biolayer Interferometry

[0309] BLI binding measurements were performed using an Octet Red384 System (ForteBio Inc ). Streptavidin (SA) sensors were prewetted in PBS and then assay buffer (PBS containing 1% [w / v] BSA) for 2 min prior to use, loaded with 100 nM Biotin- PEG2-BAD SAHB 4.2 for 3 min, and washed with assay buffer for 2 min. The sensors were then transferred into 2.33-fold serial dilutions (starting from 200 nM) of WT or G101V mutant GST-BCL-2 in assay buffer for 15 min (association step), followed by assay buffer alone for 10 min (dissociation step). Because less than 5% dissociation was Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO observed (precluding a kinetic fit), the association endpoints were fit to a concentrationwavelength shift curve (nm, Octet Software, version 9, ForteBio, Inc.) for two biological replicates and dissociation constants (K s) calculated by Prism software (Graphpad) using the binding saturation equation (one site, specific binding): [Y = Bmax * X / (Kd + X)].

[0310] Streptavidin Pull-Down - Cell Lysates

[0311] 293T cells were maintained in DMEM media supplemented with penicillin / streptomycin and 10% FBS. Cells at 70% confluence in a T175 Flask were transiently transfected with 3 pg of pCMV-Tag2B plasmid encoding either N-terminally FLAG-tagged, full-length BCL-2 WT or G101V mutant using Lipofectamine LTX & Plus reagent (ThermoFisher). 24 h following transfection, cells were harvested and lysed with CHAPS buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% CHAPS [v / v]). Protein concentration of the soluble fraction was measured by BSA assay following the manufacturer’s protocol (Thermo Scientific). 300 pg of lysate was subsequently incubated with biotinylated peptides (1 pM) in 200 pL volume overnight at 4 °C. Peptide-protein complexes were then captured by high-capacity streptavidin agarose beads (Thermo Scientific) for 2 h at RT. Beads were subsequently washed 3x with 200 pL PBS and 3x with 200 pL 1% NP-40 in PBS before elution in 2x LDS containing 300 mM DTT. Samples were then subjected to electrophoresis and western blotting using an HRP-conjugated anti -FLAG antibody to evaluate the relative affinity of the BAD peptides for BCL-2. For streptavidin pull-downs of cell lysates with venetoclax competition, 300 pg of lysate was incubated with biotinylated BAD SAHB 4.2 (1 pM) in 200 pL volume overnight at 4 °C in addition to varying venetoclax concentrations (0, 0.1, 1.0, and 10 pM). N-terminally FLAG-tagged, full-length BCL-2 WT or G101V mutant protein that still bound to biotinylated BAD SAHB 4.2 was captured, washed, and detected as described above.

[0312] Competitive Streptavidin Pull-Down of Recombinant Proteins

[0313] Recombinant BCL-2AC (aa 1-34, 92-207), BCL-XLAC (aa 1-212), and GST-

[0314] MCL-1ANAC (aa 170-327) (1 pM each) were combined and incubated with biotinylated

[0315] BAD peptide (1 pM) overnight at 4 °C. The following day, mixtures were added to high- Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO capacity streptavidin agarose beads (Thermo Scientific) for 2 h at RT, washed 3x with 200 pL PBS and 3x with 200 pL 1% NP-40 in PBS before elution in 2x LDS containing 300 mM DTT. Samples were then subjected to electrophoresis on a 12% Bis-Tris gel and visualized by silver staining following the manufacturer’s protocol (Pierce).

[0316] BAX Reactivation / Liposomal Release Assay

[0317] Large unilamellar vesicles (LUVs) were prepared with a lipid composition of 48 mol% phosphatidyl-choline, 28 mol% phosphatidylethanolamine, 10 mol% phosphatidylinositol, 10% di oleyl phosphatidyl-serine, and 4 mol% tetraoleoyl cardiolipoin (Avanti Polar Lipids). The lipid mixture was prepared from chloroform stocks, dried as a thin film by vacuum (1 mg total lipid), and stored under nitrogen at - 80°C until use. Fluorescent 8-aminonaphthalene-l,3,6-trisulfonic acid (ANTS) (6.3 mg) and fluorescence quencher p-xylene-bis-pyridinium bromide (DPX) (19.1 mg) were added to the dried film and rehydrated with 1 mL of liposome buffer (200 mM KC1, 5 mM MgCh, 10 mM Hepes, pH 7.0). Following 5 freeze-thaw cycles, the hydrated lipid solution was extruded through a 100-nm membrane (Whatman) using an Avanti mini extruder. LUVs were subsequently isolated from free ANTS / DPX by gravity flow SEC using Sepharose CL-2B resin (Sigma Aldrich) and liposome buffer. To assess BAX- mediated membrane poration, LUVs (5 pL) were mixed with the indicated combinations of stapled BAD BH3 peptide or venetoclax (3.6 pM final), BCL-2AC (aa 1-34, 92-207) (1.8 pM), tBID (100 nM), and BAX (300 nM). Following the addition of BAX (F0), the change in ANTS fluorescence (355 nm excitation and 520 nm emission) was monitored at one-minute intervals for 2 hours. At the completion of the time course, Triton X-100 was added to each well at a final concentration of 0.5% (vol / vol) to determine maximal release (F100). Fraction ANTS / DPX release was then calculated as [(F-F0) / (F100-F0)]. BAX reactivation as measured by AUC was calculated as [AUCF-AUCFI] / [AUCFIOO- AUCFI], where Fl represents the suppression of tB ID-triggered BAX by BCL-2 in the absence of peptide or venetoclax.

[0318] X-ray Crystallography Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO

[0319] Recombinant BCL-2AC (aa 1-34, 92-207) was preincubated at 1 mg / mL with 3x molar excess of stapled BAD BH3 peptide 4.2 (10 mM DMSO stock, 1.5% DMSO final) for 2 hours at 4°C and then concentrated to 15 mg / ml (773 pM) for co-crystallization. Proteins were mixed with precipitation solutions (200 nL each) and crystallized by hanging-drop vapor diffusion at 20°C. WT BCL2 complexed with BAD 4.2 crystallized in 15% PEG3350 and 150 mM CsCl. BCL-2 G101V complexed with BAD 4.2 crystallized in 1 .4 M INH4T artrate and 0.1 M Tris, pH 8. Crystals were transferred briefly into crystallization buffer containing 25% glycerol prior to flash-freezing in liquid nitrogen. Diffraction data were collected at beamline 17-ID-2 of the National Synchrotron Light Source II (Brookhaven National Laboratory). Datasets were integrated and scaled using xds or dials package (v.20200417, Ref). Structures were solved by molecular replacement using the program Phaser (v.2.8.3, McCoy et al., 2007) based on a series of search models. Iterative manual model building and refinement using Phenix (v.l.20.1_4487, Adams et al., 2010) and Coot (v.0.9.7, Emsley and Cowtan, 2004) led to the final models.

[0320] DDMP Analysis

[0321] For each crystal structure, we defined a distance map as D,,y= | | x, - x / 1 |, whereby x;, xywere the cartesian coordinates of the alpha carbons from residues i,j and | | X / - x;| | was the Euclidean norm, with each capturing the distance between the subscripted pair of residues. We further quantified relative shifts in structure using the distance difference map (DDM) A^ / = D4 / , / - D^ / y, where distance map D4, D';corresponded to structures A and B, respectively. Entries of A with a positive sign indicated that a pair of residues were further apart in structured, while a negative sign indicated that a pair of residues were closer together in structure A. All maps were calculated using Python-based analysis tools using the NumPy library for numerical analysis and Matplotlib for data visualization.

[0322] Hydrogen Deuterium Exchange Mass Spectrometry

[0323] Recombinant BCL-2I-207 (30 pM) was incubated individually or with BAD 4.2 (1 :5) for at least 1 hour at 23 °C in buffer (150 mM NaCl, 20 mM Tris, pH 7.4) prior to Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO deuterium labeling. Deuterium labeling of the mixture (2 pL sample) was then initiated with an 18-fold dilution into D2O labeling buffer (36 pL, 150 mM NaCl, 20 mM Tris, pH 7.4, 99.9% D2O). The labeling reaction was quenched over time (10 sec, 1 min, 10 min) by the addition of 38 pL of ice-cold quenching buffer (200 mM sodium phosphate, 4 M guanidinium chloride, 0.72 M TCEP pH 2.3, H2O) and analyzed immediately. Deuterated and control samples were digested online at 15°C using an AffiPro Nep-2 column (AffiPro, AP -PC-004). The cooling chamber of the HDX system, which housed all chromatographic elements, was held at 0.0 ± 0.1°C for the duration of measurements. Peptides were trapped and desalted on a VanGuard Pre-Column trap (2.1 mm x 5 mm, ACQUITY UPLC BEH C18, 1.7 pM [Waters, 186002346]) for 3 min at 100 pL / min. Peptides were eluted from the trap using a 5%-35% gradient of acetonitrile with 0.1% formic acid over 6 min at a flow rate of 100 pL / min, and separated using an ACQUITY UPLC HSS T3, 1.8 pm, 1.0 mm x 50 mm column (Waters, 186003535). The back pressure averaged -12,950 psi at 0 °C in 5% acetonitrile, 95% water, 0.1% formic acid buffer. To eliminate peptide carryover, a wash solution (1.5 M guanidinium chloride, 0.8% formic acid, and 4% acetonitrile) was injected over the protease column during each analytical run. Mass spectra were acquired using a Waters Synapt G2-Si HDMSE mass spectrometer in ion mobility mode. The mass spectrometer was calibrated with direct infusion of a glu-fibrinopeptide (Sigma, F3261) solution at 200 femtomole / pL at a flow rate of 5 pL / min prior to data collection. A conventional electrospray source was used and the instrument was scanned over the range 50 to 2000 m / z. The instrument was configured in accordance with the following parameters: capillary 3.2 kV, trap collision energy 4 V, sampling cone 40 V, source temperature 80°C and desolvation temperature 175 °C. The error of determining the average deuterium incorporation for each peptide was at or below ± 0.25 Da based on deuterated peptide standards. Peptides were identified from replicate HDMSE analyses of undeuterated control samples using PEGS 3.0.1 (Waters Corporation). Peptide masses were identified from searches using nonspecific cleavage of a custom database containing the sequence of BCL-2 (UniProt: Pl 0415), no missed cleavages, no post-translational modifications, low energy threshold of 135, elevated energy threshold of 35, and an intensity threshold of 500. No false Atorney Docket No: 00530-0425W01 / DFCI 3581.W01WO discovery rate (FDR) control was performed. The peptides identified in PLGS (excluding all neutral loss and in-source fragmentation identifications) were then filtered in DynamX 3.0 (Waters Corporation), implementing a minimum products per amino acid cut-off of 0.3 and at least 3 consecutive product ions. Peptides that met the filtering criteria were further processed by DynamX 3.0 (Waters Corporation). The relative amount of deuterium in each peptide was determined by the software, which subtracted the centroid mass of the undeuterated form of each peptide from the deuterated form at each time point and for each condition. Deuterium levels were not corrected for back exchange and thus reported as relative (See, e.g., Wales and Engen, 2006).

[0324] OTHER ASPECTS

[0325] 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.

[0326] 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

Attorney Docket No: 00530-0425W01 / DFCI 3581.W01WOWHAT IS CLAIMED IS:

1. A polypeptide comprising a stapled peptide, wherein the stapled peptide comprises the structure of formula (III):Formula (III) or a pharmaceutically acceptable salt thereof, wherein: each Ri and R2 is independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, any of which is substituted or unsubstituted, optionally wherein each Ri and R2 is a methyl; each R3 is independently alkylene, alkenylene, or alkynylene, any of which is substituted or unsubstituted, optionally wherein R3 is Cs alkylene, Cs alkenylene, or Cx alkynylene, optionally wherein R3 is (CH2)3-CH=CH-(CH2)3; and wherein the stapled peptide, or the pharmaceutically acceptable salt thereof, comprises 2 to 5 substitutions relative to the sequence WAAQRYGRELRRBSDEFVDSFK (SEQ ID NO:3), wherein B is norleucine, wherein the stapled peptide or the pharmaceutically acceptable salt thereof comprises two substitutions relative to the sequence of SEQ ID NO:3 at (i) positions 2 and 6 of the sequence of SEQ ID NO:3, or (ii) positions 6 and 10 of the sequence of SEQ ID N0:3; wherein the stapled peptide or the pharmaceutically acceptable salt thereof optionally comprises 1 to 3 additional substitutions relative to the sequence of SEQ IDAttorney Docket No: 00530-0425W01 / DFCI 3581.W01WONO: 3 on the BCL-2 non-interacting alpha helical face of the BAD BH3 peptide of SEQ ID NO:3, optionally wherein the 1 to 3 additional substitutions are at one or more of Wl, Q4, R5, R8, E9, R12, D15, E16, D19, or K22 (wherein the numbering is based on SEQ ID N0:3); and wherein [Xaa]x is (i) positions 3-5 of the sequence of SEQ ID NO:1, or (ii) positions 7-9 of the sequence of SEQ ID NO: 1; and wherein the polypeptide or the pharmaceutically acceptable salt thereof binds to both wild type BCL-2 and mutant BCL-2 proteins that are resistant to venetoclax, optionally wherein the mutant BCL-2 is G101V, GIOIA, D103Y, D103E, D103V, A 113 G, V 156D, R 129L, L 119 V, F 104C, F 104L, F 104S, or the in-frame insertion ArglO7_Argl lOdup, and further optionally wherein the polypeptide or the pharmaceutically acceptable salt thereof exhibits reduced or no binding to BCL-w, BCL- XL, MCL-1, and / or BFL-1 / A1.

2. The polypeptide or the pharmaceutically acceptable salt thereof, of claim 1, wherein:Ri is methyl,Rs is (CH2)3-CH=CH-(CH2)3, andR2is methyl.

3. The polypeptide or the pharmaceutically acceptable salt thereof, of claim 1 or 2, wherein:(a) [Xaa]wconsists of the sequence W,[Xaa]xconsists of the sequence AQR, and[Xaa]yconsists of the sequence GRELRRBSDEFVDSFK (SEQ ID NO: 41); and(b) [Xaa]wconsists of the sequence WAAQR (SEQ ID NO: 42), [Xaa]xconsists of the sequence GRE, and[Xaa]yconsists of the sequence RRBSDEFVDSFK (SEQ ID NO: 43).Attorney Docket No: 00530-0425W01 / DFCI 3581.W01WO4. A polypeptide comprising a stapled peptide, wherein the stapled peptide comprises the sequence of SEQ ID NOs.: 6 or 10, with 0, 1, 2 or 3 amino acid substitutions relative to the sequence of SEQ ID NOs.:6 or 10, respectively, or a pharmaceutically acceptable salt thereof, wherein B is norleucine and wherein each X in SEQ ID NOs.: 6 or 10 are a,a-disubstituted non-natural amino acids comprising olefinic side chains that are capable of being cross-linked to each other to form a hydrocarbon staple, optionally wherein each X is (S)-2-(4’-pentenyl)alanine, wherein the stapled peptide, or the pharmaceutically acceptable salt thereof, binds to both wild type BCL-2 and mutant BCL-2 proteins that are resistant to venetoclax, optionally wherein the 1, 2 or 3 amino acid substitutions relative to the sequence of SEQ ID NOs:6 or 10 are on the BCL-2 non-interacting face of SEQ ID NO: 1, optionally wherein the 1, 2 or 3 amino acid substitutions are at one or more of Wl, Q4, R5, R8, E9, R12, D15, E16, D19, or K22 (wherein the numbering is based on SEQ ID NO:6 or 10), and further optionally wherein the stapled peptide comprises the sequence of SEQ ID NO: 6.

5. A polypeptide comprising a stapled peptide, wherein the stapled peptide comprises the sequence of SEQ ID NOs: 6 or 10, optionally wherein the stapled peptide comprises the sequence of SEQ ID NO: 6, wherein B is norleucine and wherein each X in SEQ ID NOs.: 6 or 10 are a,a-disubstituted non-natural amino acids comprising olefinic side chains that are capable of being cross-linked to each other to form a hydrocarbon staple, optionally wherein each X is (S)-2-(4’-pentenyl)alanine.

6. A delivery vehicle comprising the comprising the polypeptide or the pharmaceutically acceptable salt thereof, of any one of claims 1 to 5, optionally wherein the delivery vehicle is a nanoparticle that encapsulates the polypeptide or the pharmaceutically acceptable salt thereof, optionally wherein the nanoparticle is a lipid nanoparticle.

7. A composition comprising: a nanoparticle, optionally a lipid nanoparticle, andAttorney Docket No: 00530-0425W01 / DFCI 3581.W01WO means for binding to both wild type BCL-2 and mutant BCL-2 proteins that are resistant to venetoclax or means for binding to both wild type BCL-2 and mutant BCL-2 proteins that are resistant to venetoclax and showing reduced or no binding to BCL-w, BCL-XL, MCL-1 and / or BFL-l / AL8. A pharmaceutical composition comprising the polypeptide or the pharmaceutically acceptable salt thereof, of any one of claims 1 to 5 or the delivery vehicle of claim 6; and a pharmaceutically acceptable carrier.

9. A pharmaceutical composition comprising:(i) means for binding to both wild type BCL-2 and mutant BCL-2 proteins that are resistant to venetoclax; and(ii) a pharmaceutically acceptable carrier.

10. A pharmaceutical composition comprising:(i) means for binding to both wild type BCL-2 and mutant BCL-2 proteins that are resistant to venetoclax and showing reduced or no binding to BCL-w, BCL-XL, MCL-1, and / or BFL-1 / A1; and(ii) a pharmaceutically acceptable carrier.

11. A method of making a stapled peptide, the method comprising:(a) providing a peptide comprising the sequence of SEQ ID NO:6 or 10, with0, 1, 2, or 3 amino acid substitutions, optionally wherein the 1, 2, or 3 amino acid substitutions are on the BCL-2 non-interacting face of the alpha helix of the sequence set forth in SEQ ID NO: 6 or 10; and(b) cross-linking the peptide thereby making the stapled peptide, optionally wherein the stapling amino acids (X) are (S)-2-(4’-pentenyl)alanine, optionally wherein the cross-linking is by a ruthenium catalyzed metathesis reaction, and optionally wherein the method further comprises formulating the stapled peptide as a sterile pharmaceutical composition.

12. A method of treating a cancer that expresses BCL-2 or is dependent on BCL-2 in a human subject in need thereof, the method comprising administering to the humanAttorney Docket No: 00530-0425W01 / DFCI 3581.W01WO subject a therapeutically effective amount of (i) the polypeptide or the pharmaceutically acceptable salt thereof, of any one of claims 1 to 5; (ii) the delivery vehicle of claim 6 or the composition of claim 7; or (iii) the pharmaceutical composition of any one of claims 8 to 10.

13. A method of treating a venetocl ax-resistant cancer in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of (i) the polypeptide or the pharmaceutically acceptable salt thereof, of any one of claims 1 to 5; (ii) the delivery vehicle of claim 6 or the composition of claim 7; or (iii) the pharmaceutical composition of any one of claims 8 to 10.

14. The method of claim 12 or 13, wherein the cancer is a hematological malignancy, optionally wherein the cancer is chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), small lymphocytic lymphoma (SLL) or multiple myeloma (MM) such as relapsed or refractor}- MM.

15. The method of claim 12 or 13, wherein the cancer is a solid tumor, optionally wherein the solid tumor is a breast cancer optionally ER+metastatic breast cancer, a nonsmall cell lung cancer (NSCLC), an ovarian cancer optionally a platinum-resistant ovarian cancer, or a pancreatic cancer.

16. The method of claim 12 or 13, wherein the method is for the treatment of patients with chronic lymphocytic leukemia (CLL) with 17p deletion, as detected by an FDA- approved test, who have received at least one prior therapy.

17. The method of claim 12 or 13, wherein the method is for the treatment of newly diagnosed acute myeloid leukemia (AML) in adults 75 years or older, or who have comorbidities that preclude use of intensive induction chemotherapy, in combination with azacitidine, or decitabine, or low-dose cytarabine.Attorney Docket No: 00530-0425W01 / DFCI 3581.W01WO18. The method of any one of claims 12 to 17, further comprising one or more of: chemotherapy, a targeted therapy, hormonal therapy, radiation therapy, immunotherapy, an antibody drug conjugate, an epigenetic modulator, and an anti-angiogenesis agent.

19. A method of inhibiting the interaction of BCL2 or a mutant BCL2 with a proapoptotic BCL2 family member protein in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of (i) the polypeptide or the pharmaceutically acceptable salt thereof, of any one of claims 1 to 5; (ii) the delivery vehicle of claim 6 or the composition of claim 7; or (iii) the pharmaceutical composition of any one of claims 8 to 10, optionally wherein the mutant BCL-2 is G101V, GIOIA, D103Y, D103E, D103V, Al 13G, V156D, R129L, LI 19V, F104C, F104L, F104S, or the in frame insertion ArglO7_Argl lOdup.

20. A polypeptide comprising a stapled peptide, wherein the stapled peptide comprises the structure of formula (III):Formula (III) or a pharmaceutically acceptable salt thereof, wherein: each Ri and R2 is independently H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl, any of which is substituted or unsubstituted, optionally wherein each Ri and R2 is a methyl; each R3 is independently alkylene, alkenylene, or alkynylene, any of which is substituted or unsubstituted, optionally wherein R3 is Cs alkylene, Cs alkenylene, or Cs alkynylene, optionally wherein R3 is (CH2)3-CH=CH-(CH2)3; andAttorney Docket No: 00530-0425W01 / DFCI 3581.W01WO wherein the stapled peptide, or the pharmaceutically acceptable salt thereof, comprises 2 to 5 substitutions relative to the sequence WAAQRYGRELRRBSDEFVDSFK (SEQ ID NO:3), wherein B is norleucine, wherein the stapled peptide or the pharmaceutically acceptable salt thereof comprises two substitutions relative to the sequence of SEQ ID NO:3 at (i) positions 2 and 6 of the sequence of SEQ ID NO:3, or (ii) positions 6 and 10 of the sequence of SEQ ID NO:3; wherein the stapled peptide or the pharmaceutically acceptable salt thereof optionally comprises 1 to 3 additional substitutions relative to the sequence of SEQ ID NO: 3 on the BCL-2 non-interacting alpha helical face of the BAD BH3 peptide of SEQ ID NO:3, optionally wherein the 1 to 3 additional substitutions are at one or more of Wl, Q4, R5, R8, E9, R12, D15, E16, D19, or K22 (wherein the numbering is based on SEQ ID N0:3); and wherein [Xaa]x is (i) positions 3-5 of the sequence of SEQ ID NO:1, or (ii) positions 7-9 of the sequence of SEQ ID NO: 1; and wherein the polypeptide or the pharmaceutically acceptable salt thereof binds to both wild type BCL-2 and mutant BCL-2 proteins that are resistant to venetoclax, optionally wherein the mutant BCL-2 is GIO IV, GIOIA, D103Y, D103E, DI 03V, Al 13 G, VI 56D, R129L, L 119V, F 104C, F 104L, F 104S, or the in-frame insertion ArglO7_Argl lOdup, and further optionally wherein the polypeptide or the pharmaceutically acceptable salt thereof exhibits reduced or no binding to BCL-w, BCL- XL, MCL-1, and / or BFL-l / AL21. A polypeptide comprising a stapled peptide, wherein the stapled peptide comprises the sequence of SEQ ID NOs.: 6 or 10, with 1, 2 or 3 amino acid substitutions relative to the sequence of SEQ ID NOs.:6 or 10, respectively, or a pharmaceutically acceptable salt thereof, wherein B is norleucine and wherein each X in SEQ ID NOs.: 6Attorney Docket No: 00530-0425W01 / DFCI 3581.W01WO or 10 are a,a-disubstituted non-natural amino acids comprising olefinic side chains that are capable of being cross-linked to each other to form a hydrocarbon staple, optionally wherein each X is (S)-2-(4’-pentenyl)alanine, wherein the stapled peptide, or the pharmaceutically acceptable salt thereof, binds to both wild type BCL-2 and mutant BCL-2 proteins that are resistant to venetoclax, optionally wherein the 1, 2 or 3 amino acid substitutions are at one or more of Wl, Q4, R5, R8, E9, R12, D15, E16, D19, or K22 (wherein the numbering is based on SEQ ID NO:6 or 10), optionally wherein the 1, 2, or 3 amino acid substitutions relative to SEQ ID NO:6 comprise (i) a substitution at position 10 (numbered according to SEQ ID NO:6) with phenylalanine further substituted with a chlorine moiety, optionally 4-C1-F, (ii) a substitution at position 17 (numbered according to SEQ ID NO:6) with tryptophan, (iii) a substitution at position 17 (numbered according to SEQ ID NO:6) with beta-(4-biphenyl)-L-alanine, or (iv) a substitution at position 10 and a substitution at position 17 (both numbered according to SEQ ID NO:6) with phenylalanine and tryptophan, respectively; optionally wherein the 1, 2, or 3 amino acid substitutions relative to SEQ ID NO: 10 comprise: (i) a substitution at position 17 (numbered according to SEQ ID NO: 10) with tryptophan, or (ii) a substitution at position 17 (numbered according to SEQ ID NO: 10) with beta-(4-biphenyl)-L-alanine, and further optionally wherein the stapled peptide comprises the sequence of SEQ ID NO: 6.

22. A delivery vehicle comprising the comprising the polypeptide or the pharmaceutically acceptable salt thereof, of claim 20 or 21, optionally wherein the delivery vehicle is a nanoparticle that encapsulates the polypeptide or the pharmaceutically acceptable salt thereof, optionally wherein the nanoparticle is a lipid nanoparticle.

23. A pharmaceutical composition comprising the polypeptide or the pharmaceutically acceptable salt thereof, of claim 20 or 21 or the delivery vehicle of claim 22; and a pharmaceutically acceptable carrier.

24. A method of treating a cancer that expresses BCL-2 or is dependent on BCL-2 in a human subject in need thereof, the method comprising administering to the humanAttorney Docket No: 00530-0425W01 / DFCI 3581.W01WO subject a therapeutically effective amount of (i) the polypeptide or the pharmaceutically acceptable salt thereof, of claim 20 or 21 ; (ii) the delivery vehicle of claim 22; or (iii) the pharmaceutical composition of claim 23.

25. A method of treating a venetoclax-resistant cancer in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of (i) the polypeptide or the pharmaceutically acceptable salt thereof, of claim 20 or 21; (ii) the delivery vehicle of claim 22; or (iii) the pharmaceutical composition of claim 23.

26. A method of inhibiting the interaction of BCL2 or a mutant BCL2 with a proapoptotic BCL2 family member protein in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of (i) the polypeptide or the pharmaceutically acceptable salt thereof, of claim 20 or 21; (ii) the delivery vehicle of claim 22; or (iii) the pharmaceutical composition of claim 23, optionally wherein the mutant BCL-2 is G101V, GIOIA, D103Y, D103E, D103V, A113G, V156D, R129L, LI 19V, F104C, F104L, F104S, or the in frame insertionArg 107_Arg 11 Odup.

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