# US 11,078,246 B2 · Peptides binding to Bfl-1 > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,078,246 B2 - Document ID: US11078246B2 - Status: Granted - Kind: B2 - Application number: 15/752,358 - Filed: 2016-08-26 - Published: 2019-01-03 - Granted: 2021-08-03 - Earliest claimed: 2015-08-28 - Assignees: Dana-Farber Cancer Institute, Inc. - Applicants: Dana-Farber Cancer Institute, Inc. - Inventors: Loren D. Walensky; Gregory H. Bird - Web page: https://patentgrep.com/patent/US11078246B2 ## CPC - C07K 14/4747: {Apoptosis related proteins} - A61K 38/1761: {Apoptosis related proteins, e.g. Apoptotic protease-activating factor-1 (APAF-1), Bax, Bax-inhibitory protein(s)(BI; bax-I), Myeloid cell leukemia associated protein (MCL-1), Inhibitor of apoptosis [IAP] or Bcl-2} - A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca - A61K 47/545: {Heterocyclic compounds (A61K47/558 takes precedence)} - A61P 35/00: Antineoplastic agents - C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575) - A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) - C12Q 2600/106: Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## Abstract This disclosure features stapled peptide inhibitors (e.g., cysteine-reactive stapled peptides) of the anti-apoptotic protein, BFL-1, and methods of using same in the treatment of BFL-1 expressing cancers. ## Claims ### Claim 1 A polypeptide that binds to Bfl-1, the polypeptide comprising: (a) the amino acid sequence set forth in SEQ ID NO: 22-36, 41-119, 184, 186, 195, 197, or 208, wherein the polypeptide comprises a non-natural amino acid bearing an electrophilic group or an electrophilic warhead at the N-terminus of the amino acid sequence; (b) an amino acid sequence that is at least 50% identical to the Bfl-1 interacting alpha-helical face of ATQLRRFGDKLNFRQ (SEQ ID NO:121) and that selectively binds Bfl-1 over MCL-1, wherein at least two amino acids in the amino acid sequence of SEQ ID NO:121 are substituted by non-natural amino acids with olefinic side chains, wherein F at position 7 of the amino acid sequence of SEQ ID NO:121 is substituted with an amino acid with a bulkier side chain, and wherein the polypeptide comprises a non-natural amino acid bearing an electrophilic group or an electrophilic warhead at the N-terminus of the amino acid sequence; (c) an amino acid sequence that is at least 50% identical to the Bfl-1 interacting alpha-helical face of an amino acid sequence set forth below: (i) (SEQ ID NO: 122) AELEVECATQLRRFGDKLNFRQKLLN; (ii) (SEQ ID NO: 123) EIWIAQELRRIGDEFNAYYARR; (iii) (SEQ ID NO: 124) DIIRNIARHLAQVGDSMDRSI; (iv) (SEQ ID NO: 125) SSTMGQVGRQLAIIGDDINRRY; (v) (SEQ ID NO: 126) QDASTKKLSESLKRIGDELDSNMEL; or (vi) (SEQ ID NO: 127) RLAEVCAVLLRLGDELEMIR, wherein the polypeptide selectively binds Bfl-1 over MCL-1; wherein at least two amino acids in each amino acid sequence are substituted by non-natural amino acids with olefinic side chains, wherein at least one cysteine if present in the polypeptide is optionally replaced by serine, and wherein at least one methionine if present in the polypeptide is optionally replaced by norleucine; and wherein the polypeptide comprises a non-natural amino acid bearing an electrophilic group or a non-amino acid warhead at the N-terminus of the amino acid sequence; (d) an amino acid sequence that is at least 50% identical to the Bfl-1 interacting alpha-helical face of JATQLRRFGDKLNFRQKLL (SEQ ID NO: 128) and that selectively binds Bfl-1 over MCL-1; wherein the polypeptide forms a covalent bond with Cys 55 of Bfl-1; wherein at least two amino acids in the amino acid sequence of SEQ ID NO:128 are substituted by non-natural amino acids with olefinic side chains, and wherein J is a non-natural amino acid bearing an electrophilic group, or an electrophilic warhead that does not comprise an amino acid; (e) an amino acid sequence that is at least 50% identical to the Bfl-1 interacting alpha-helical face of JEVESATQLRRFGDKLNFRQKLL (SEQ ID NO:129) and that selectively binds Bfl-1 over MCL-1; wherein the polypeptide forms a covalent bond with Cys 55 of Bfl-1; wherein at least two amino acids in the amino acid sequence of SEQ ID NO:129 are substituted by non-natural amino acids with olefinic side chains, and wherein J is a non-natural amino acid bearing an electrophilic group, or an electrophilic warhead that does not comprise an amino acid; (f) an amino acid sequence that is at least 50% identical to the Bfl-1 interacting alpha-helical face of JIAQELRRIGDEFNAYYARR (SEQ ID NO: 130) and that selectively binds Bfl-1 over MCL-1; wherein the polypeptide forms a covalent bond with Cys 55 of Bfl-1; wherein at least two amino acids in the amino acid sequence of SEQ ID NO:130 are substituted by non-natural amino acids with olefinic side chains, and wherein J is a non-natural amino acid bearing an electrophilic group, or an electrophilic warhead that does not comprise an amino acid; (g) an amino acid sequence with 1 to 5 amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 22-36, 41-119, 184, 186, 195, 197, or 208, wherein the polypeptide selectively binds Bfl-1 over MCL-1 and forms a covalent bond with Cys 55 of Bfl-1, and wherein the polypeptide comprises a non-natural amino acid bearings an electrophilic group or an electrophilic warhead at the N-terminus of the amino acid sequence; or (h) an amino acid sequence with 1 to 5 amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 128, wherein the polypeptide selectively binds Bfl-1 over MCL-1 and forms a covalent bond with Cys 55 of Bfl-1, and wherein the polypeptide comprises a non-natural amino acid bearing an electrophilic group or an electrophilic warhead at the N-terminus of the amino acid sequence. ### Claim 2 (depends on 1) A method for treating a human subject suffering from a cancer that exhibits expression of BFL-1 or dependency on BFL-1, the method comprising administering a therapeutically effective amount of the polypeptide of claim 1 to the human subject. ### Claim 3 (depends on 2) The method of claim 2, wherein a biological sample comprising a tumor from the human subject is or has previously been determined to express Bfl-1. ### Claim 4 (depends on 1) A pharmaceutical composition comprising the polypeptide of claim 1, and a pharmaceutically acceptable carrier. ### Claim 5 (depends on 4) A method for treating a human subject suffering from a cancer that exhibits expression of BFL-1 or dependency on BFL-1, the method comprising administering to the human subject a therapeutically effective amount of the pharmaceutical composition of claim 4. ### Claim 6 (depends on 1) The polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NQ: 22-36, 41-119, 184, 186, 195, 197, or 208, and wherein the polypeptide comprises a non-natural amino acid bearing an electrophilic group or an electrophilic warhead at the N-terminus of the amino acid sequence. ### Claim 7 (depends on 1) The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence with 1 to 5 amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 22-36, 41-119, 184, 186, 195, 197, or 208, wherein the polypeptide comprises a non-natural amino acid bearing an electrophilic group or an electrophilic warhead at the N-terminus of the amino acid sequence, and wherein the polypeptide selectively binds Bfl-1 over MCL-1 and forms a covalent bond with Cys 55 of Bfl-1. ### Claim 8 (depends on 1) The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence with 1 or 2 amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NQ: 22-36, 41-119, 184, 186, 195, 197, or 208, wherein the polypeptide comprises a non-natural amino acid bearing an electrophilic group or an electrophilic warhead at the N-terminus of the amino acid sequence, and wherein the polypeptide selectively binds Bfl-1 over MCL-1 and forms a covalent bond with Cys 55 of Bfl-1. ### Claim 9 (depends on 1) The polypeptide of claim 1, wherein the polypeptide is stapled. ### Claim 10 (depends on 9) A pharmaceutical composition comprising the polypeptide of claim 9 and a pharmaceutically acceptable carrier. ### Claim 11 (depends on 9) A method for treating a human subject suffering from a cancer that exhibits expression of BFL-1 or dependency on BFL-1, the method comprising administering a therapeutically effective amount of the polypeptide of claim 9 to the human subject. ### Claim 12 (depends on 1) The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence with 1 to 5 amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 128, wherein the polypeptide comprises a non-natural amino acid bearing an electrophilic group or an electrophilic warhead at the N-terminus of the amino acid sequence, and wherein the polypeptide selectively binds Bfl-1 over MCL-1 and forms a covalent bond with Cys 55 of Bfl-1. ### Claim 13 (depends on 1) The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence with 2 to 3 amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 128, wherein the polypeptide comprises a non-natural amino acid bearing an electrophilic group or an electrophilic warhead at the N-terminus of the amino acid sequence, and wherein the polypeptide selectively binds Bfl-1 over MCL-1 and forms a covalent bond with Cys 55 of Bfl-1. ### Claim 14 (depends on 1) The polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 22-25, 49-67, 184, or 186, and wherein the polypeptide comprises a non-natural amino acid bearing an electrophilic group or an electrophilic warhead at the N-terminus of the amino acid sequence. ### Claim 15 (depends on 1) The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 50% identical to the Bfl-1 interacting alpha-helical face of ATQLRRFGDKLNFRQ (SEQ ID NO:121) and that selectively binds Bfl-1 over MCL-1, wherein at least two amino acids in the amino acid sequence of SEQ ID NO:121 are substituted by non-natural amino acids with olefinic side chains, wherein F at position 7 of the amino acid sequence of SEQ ID NO:121 is substituted with an amino acid with a bulkier side chain, and wherein the polypeptide comprises a non-natural amino acid bearing an electrophilic group or an electrophilic warhead at the N-terminus of the amino acid sequence. ### Claim 16 (depends on 1) The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 90% identical to the Bfl-1 interacting alpha-helical face of ATQLRRFGDKLNFRQ (SEQ ID NO:121) and that selectively binds Bfl-1 over MCL-1, wherein at least two amino acids in the amino acid sequence of SEQ ID NO:121 are substituted by non-natural amino acids with olefinic side chains, wherein F at position 7 of the amino acid sequence of SEQ ID NO:121 is substituted with an amino acid with a bulkier side chain, and wherein the polypeptide comprises a non-natural amino acid bearing an electrophilic group or an electrophilic warhead at the N-terminus of the amino acid sequence. ### Claim 17 (depends on 1) The polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence that is at least 50% identical to the Bfl-1 interacting alpha-helical face of JATQLRRFGDKLNFRQKLL (SEQ ID NO: 128) and that selectively binds Bfl-1 over MCL-1; wherein the polypeptide forms a covalent bond with Cys 55 of Bfl-1; wherein at least two amino acids in the amino acid sequence of SEQ ID NO:128 are substituted by non-natural amino acids with olefinic side chains, and wherein J is a non-natural amino acid bearing an electrophilic group, or an electrophilic warhead that does not comprise an amino acid. ### Claim 18 (depends on 1) The polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence that is at least 90% identical to the Bfl-1 interacting alpha-helical face of JATQLRRFGDKLNFRQKLL (SEQ ID NO: 128) and that selectively binds Bfl-1 over MCL-1; wherein the polypeptide forms a covalent bond with Cys 55 of Bfl-1; wherein at least two amino acids in the amino acid sequence of SEQ ID NO:128 are substituted by non-natural amino acids with olefinic side chains, and wherein J is a non-natural amino acid bearing an electrophilic group, or an electrophilic warhead that does not comprise an amino acid. ### Claim 19 (depends on 1) The polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence that is at least 50% identical to the Bfl-1 interacting alpha-helical face of JEVESATQLRRFGDKLNFRQKLL (SEQ ID NO:129) and that selectively binds Bfl-1 over MCL-1; wherein the polypeptide forms a covalent bond with Cys 55 of Bfl-1; wherein at least two amino acids in the amino acid sequence of SEQ ID NO:129 are substituted by non-natural amino acids with olefinic side chains, and wherein J is a non-natural amino acid bearing an electrophilic group, or an electrophilic warhead that does not comprise an amino acid. ### Claim 20 (depends on 1) The polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence that is at least 90% identical to the Bfl-1 interacting alpha-helical face of JEVESATQLRRFGDKLNFRQKLL (SEQ ID NO:129) and that selectively binds Bfl-1 over MCL-1; wherein the polypeptide forms a covalent bond with Cys 55 of Bfl-1; wherein at least two amino acids in the amino acid sequence of SEQ ID NO:129 are substituted by non-natural amino acids with olefinic side chains, and wherein J is a non-natural amino acid bearing an electrophilic group, or an electrophilic warhead that does not comprise an amino acid. ### Claim 21 (depends on 1) The polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence that is at least 50% identical to the Bfl-1 interacting alpha-helical face of JIAQELRRIGDEFNAYYARR (SEQ ID NO: 130) and that selectively binds Bfl-1 over MCL-1; wherein the polypeptide forms a covalent bond with Cys 55 of Bfl-1; wherein at least two amino acids in the amino acid sequence of SEQ ID NO:130 are substituted by non-natural amino acids with olefinic side chains, and wherein J is a non-natural amino acid bearing an electrophilic group, or an electrophilic warhead that does not comprise an amino acid. ### Claim 22 (depends on 1) The polypeptide of claim 1, wherein the polypeptide comprises the amino acid sequence that is at least 90% identical to the Bfl-1 interacting alpha-helical face of JIAQELRRIGDEFNAYYARR (SEQ ID NO: 130) and that selectively binds Bfl-1 over MCL-1; wherein the polypeptide forms a covalent bond with Cys 55 of Bfl-1; wherein at least two amino acids in the amino acid sequence of SEQ ID NO:130 are substituted by non-natural amino acids with olefinic side chains, and wherein J is a non-natural amino acid bearing an electrophilic group, or an electrophilic warhead that does not comprise an amino acid. ### Claim 23 (depends on 1) The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence with 1 to 5 amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 22-25, 49-67, 184, or 186, wherein the polypeptide comprises a non-natural amino acid bearing an electrophilic group or an electrophilic warhead at the N-terminus of the amino acid sequence, and wherein the polypeptide selectively binds Bfl-1 over MCL-1 and forms a covalent bond with Cys 55 of Bfl-1. ### Claim 24 (depends on 1) The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence with 1 or 2 amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 22-25, 49-67, 184, or 186, wherein the polypeptide comprises a non-natural amino acid bearing an electrophilic group or an electrophilic warhead at the N-terminus of the amino acid sequence, and wherein the polypeptide selectively binds Bfl-1 over MCL-1 and forms a covalent bond with Cys 55 of Bfl-1. ### Claim 25 (depends on 1) The polypeptide of claim 1, wherein the non-natural amino acid bearing an electrophilic group or the electrophilic warhead at the N-terminus of the amino acid sequence is (S)-1-acryloylpyrrolidine-3-carboxamide, 1-acryloylpiperidine-4-carboxamide, (R)-1-acryloylpiperidine-3-carboxamide, (S)-1-acryloylpiperidine-3-carboxamide, (S)-1-acryloylpyrrolidine-2-carboxamide, (R)-1-acryloylpyrrolidine-2-carboxamide, (E)-4-(dimethylamino)but-2-enamide, or acrylamide. ### Claim 26 (depends on 1) The polypeptide of claim 1, wherein the non-natural amino acid bearing an electrophilic group or the electrophilic warhead at the N-terminus of the amino acid sequence is (R)-1-acryloylpiperidine-3-carboxamide. ### Claim 27 (depends on 1) The polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence that is at least 90% identical to the Bfl-1 interacting alpha-helical face of an amino acid sequence set forth below: (i) (SEQ ID NO: 122) AELEVECATQLRRFGDKLNFRQKLLN; (ii) (SEQ ID NO: 123) EIWIAQELRRIGDEFNAYYARR; (iii) (SEQ ID NO: 124) DIIRNIARHLAQVGDSMDRSI; (iv) (SEQ ID NO: 125) SSTMGQVGRQLAIIGDDINRRY; (v) (SEQ ID NO: 126) QDASTKKLSESLKRIGDELDSNMEL; or (vi) (SEQ ID NO: 127) RLAEVCAVLLRLGDELEMIR, wherein the polypeptide selectively binds Bfl-1 over MCL-1; wherein at least two amino acids in each amino acid sequence are substituted by non-natural amino acids with olefinic side chains, wherein at least one cysteine if present in the polypeptide is optionally replaced by serine, and wherein at least one methionine if present in the polypeptide is optionally replaced by norleucine; and wherein the polypeptide comprises a non-natural amino acid bearing an electrophilic group or a non-amino acid warhead at the N-terminus-of the amino acid sequence. ### Claim 28 A polypeptide that binds to Bfl-1, the polypeptide comprising an amino acid sequence that is at least 70% identical to the Bfl-1 interacting alpha-helical face of the amino acid sequence set forth in SEQ ID N: 22-36, 41-119, 184, 186, 195, 197, or 208, wherein the polypeptide comprises a non-natural amino acid bearing an electrophilic group or a non-amino acid warhead at the N-terminus in of the amino acid sequence. ### Claim 29 (depends on 28) A pharmaceutical composition comprising the polypeptide of claim 28, and a pharmaceutically acceptable carrier. ### Claim 30 (depends on 29) A method for treating a human subject suffering from a cancer that exhibits expression of BFL-1 or dependency on BFL-1, the method comprising administering to human subject therapeutically effective amount of the pharmaceutical composition of claim 29. ### Claim 31 (depends on 28) A method for treating a human subject suffering from a cancer that exhibits expression of BFL-1 or dependency on BFL-1, the method comprising administering to the human subject a therapeutically effective amount of the polypeptide of claim 28. ### Claim 32 (depends on 31) The method of claim 31, wherein a biological sample comprising a tumor from the human subject is or has previously been determined to express Bfl-1. ### Claim 33 (depends on 28) The polypeptide of claim 28, wherein the poly comprises an amino acid sequence that is at least 90% identical to the Bfl-1 interacting alpha-helical face of the amino acid sequence set forth in SEQ ID NO: 62, wherein the polypeptide comprises a non-natural amino acid bearing an electrophilic group or a non-amino acid warhead at the N-terminus of the amino acid sequence. ### Claim 34 (depends on 33) The polypeptide of claim 33, which is stapled. ### Claim 35 (depends on 34) A pharmaceutical composition comprising the polypeptide of claim 34 and a pharmaceutically acceptable carrier. ### Claim 36 (depends on 34) A method for treating a human subject suffering from a cancer that exhibits expression of BFL-1 or dependency on BFL-1, the method comprising administering a therapeutically effective amount of the polypeptide of claim 34 to the human subject. ### Claim 37 (depends on 28) The polypeptide of claim 28, which is stapled. ### Claim 38 (depends on 37) A pharmaceutical composition comprising the polypeptide of claim 37 and a pharmaceutically acceptable carrier. ### Claim 39 (depends on 37) A method for treating a human subject suffering from a cancer that exhibits expression of BFL-1 or dependency on BFL-1, the method comprising administering a therapeutically effective amount of the polypeptide of claim 37 to the human subject. ### Claim 40 (depends on 28) The polypeptide of claim 28, wherein the polypeptide comprises the amino acid sequence of: (i) (SEQ ID NO: 24) JEVESATQLRXFGDXLNFRQKLL; or (ii) (SEQ ID NO: 30) JIAQELRXIGDXFNAYYARR; wherein J is a non-natural electrophile containing amino acid or an electrophilic warhead that does not comprise an amino acid, and each X is a non-natural amino acid with an olefinic side chain. ### Claim 41 (depends on 40) The polypeptide of claim 40, wherein the non-natural amino acid bearing an electrophilic group or the electrophilic warhead at the N-terminus of the amino acid sequence is (R)-1-acryloylpiperidine-3-carboxamide. ### Claim 42 (depends on 41) A method for treating a human subject suffering from a cancer that exhibits expression of BFL-1 or dependency on BFL-1, the method comprising administering a therapeutically effective amount of the polypeptide of claim 41 to the human subject. ### Claim 43 (depends on 41) A pharmaceutical composition comprising the polypeptide of claim 41, and a pharmaceutically acceptable carrier. ### Claim 44 (depends on 28) The polypeptide of claim 28, wherein the polypeptide comprises: (S)-1-acryloylpyrrolidine-3-carboxamide-EVESATQLRXFGDXLNFRQKLLK (SEQ ID NO: 184), 1-acryloylpiperidine-4-carboxamide-EVESATQLRXFGDXLNFRQKLLK (SEQ ID NO: 184), (R)-1-acryloylpiperidine-3-carboxamide-EVESATQLRXFGDXLNFRQKLL (SEQ ID NO: 186), (R)-1-acryloylpiperidine-3-carboxamide-EVESATQLRXFGDXLNFRQKLL (SEQ ID NO: 186)-Lys(biotin), (S)-1-acryloylpiperidine-3-carboxamide-EVESATQLRXFGDXLNFRQKLLK (SEQ ID NO: 184), (S)-1-acryloylpyrrolidine-2-carboxamide-EVESATQLRXFGDXLNFRQKLLK (SEQ ID NO: 184), (R)-1-acryloylpyrrolidine-2-carboxamide-EVESATQLRXFGDXLNFRQKLLK (SEQ ID NO: 184), (E)-4-(dimethylamino)but-2-enamide-EVES ATQLRXFGDXLNFRQKLLK (SEQ ID NO: 184), or acrylamide-EVESATQLRXFGDXLNFRQKLLK (SEQ ID NO: 184), wherein X indicates non-natural amino acids that are joined by an intramolecular cross-link. ### Claim 45 (depends on 28) The polypeptide of claim 28, wherein polypeptide comprises an amino acid sequence that is at least 90% identical to the Bfl-1 interacting alpha-helical face of the amino acid sequence set forth in SEQ ID NO: 22-36, 41-119, 184, 186, 195, 197, or 208, wherein the polypeptide comprises a non-natural amino acid bearing an electrophilic group or a non-amino acid warhead at the N-terminus of the amino acid sequence. ### Claim 46 (depends on 28) The polypeptide of claim 28, wherein polypeptide comprises an amino acid sequence that is at least 90% identical to the Bfl-1 interacting alpha-helical face of the amino acid sequence set forth in SEQ ID NO: 62, wherein the polypeptide comprises a non-natural amino acid bearing an electrophilic group or a non-amino acid warhead at the N-terminus of the amino acid sequence. ### Claim 47 (depends on 28) The polypeptide of claim 28, wherein the polypeptide comprises the amino acid sequence of: (i) JGRQLAXIGDXINR (SEQ ID NO:72) (ii) JGR8LAIIGDXINR (SEQ ID NO:79) (iii) JVGRQLAXIGDXINR (SEQ ID NO:89) (iv) JVGR8LAIIGDXINR (SEQ ID NO:96) (v) JLSESLKXIGDXLDS (SEQ ID NO:107); or (vi) JLSE8LKRIGDXLDS (SEQ ID NO:115); wherein J is a non-natural electrophile containing amino acid or an electrophilic warhead that does not comprise an amino acid, each X is a non-natural amino acid with olefinic side chain, and 8 is R-octenyl alanine. ### Claim 48 (depends on 47) The polypeptide of claim 47, wherein the polypeptide is stapled and each X is S-pentenyl-alanine. ### Claim 49 A polypeptide that binds to Bfl-1, the polypeptide comprising the amino acid sequence JAT8LRRFGDXLNFRQ (SEQ ID NO:62) with 0 to 5 amino acid substitutions relative to the amino acid sequence of SEQ ID NO:62, wherein J is a non-natural electrophile containing amino acid or an electrophilic warhead that does not comprise an amino acid at the N-terminus of the amino acid sequence, 8 is R-octenyl alanine, and X is a non-natural amino acid with an olefinic side chain. ### Claim 50 (depends on 49) The polypeptide of claim 49, wherein the polypeptide is stapled. ### Claim 51 (depends on 50) A pharmaceutical composition comprising the polypeptide of claim 50 and a pharmaceutically acceptable carrier. ### Claim 52 (depends on 50) A method for treating a human subject suffering from a cancer that exhibits expression of BFL-1 or dependency on BFL-1, the method comprising administering a therapeutically effective amount of the polypeptide of claim 50 to the human subject. ### Claim 53 (depends on 49) The polypeptide of claim 49, wherein X is S-pentenyl alanine. ### Claim 54 (depends on 53) The polypeptide of claim 53, wherein the polypeptide is stapled, and wherein the R-octenyl alanine is linked to the S-pentenyl alanine. ### Claim 55 (depends on 49) The polypeptide of claim 49, wherein the polypeptide comprises the amino acid sequence JAT8LRRFGDXLNFRQ (SEQ ID NO:62) with 1 to 5 amino acid substitutions relative to the amino acid sequence of SEQ ID NO:62, wherein J is a non-natural electrophile containing amino acid or an electrophilic warhead that does not comprise an amino acid at the N-terminus of the amino acid sequence, 8 is R-octenyl alanine, and X is a non-natural amino acid with an olefinic side chain. ### Claim 56 (depends on 55) The polypeptide of claim 55, wherein the polypeptide is stapled, wherein X is S-pentenyl alanine, and wherein the R-octenyl alanine is linked to the S-pentenyl alanine. ### Claim 57 (depends on 56) A pharmaceutical composition comprising the polypeptide of claim 56 and a pharmaceutically acceptable carrier. ### Claim 58 (depends on 56) A method for treating a human subject suffering from a cancer that exhibits expression of BFL-1 or dependency on BFL-1, the method comprising administering a therapeutically effective amount of the polypeptide of claim 56 to the human subject. ### Claim 59 (depends on 56) The polypeptide of claim 56, wherein J is (S)-1-acryloylpyrrolidine-3-carboxamide, 1-acryloylpiperidine-4-carboxamide, (R)-1-acryloylpiperidine-3-carboxamide, (S)-1-acryloylpiperidine-3-carboxamide, (S)-1-acryloylpyrrolidine-2-carboxamide, (R)-1-acryloylpyrrolidine-2-carboxamide, (E)-4-(dimethylamino)but-2-enamide, or acrylamide. ### Claim 60 (depends on 56) The polypeptide of claim 56, wherein J is (R)-1-acryloylpiperidine-3-carboxamide. ### Claim 61 (depends on 60) A method for treating a human subject suffering from a cancer that exhibits expression of BFL-1 or dependency on BFL-1, the method comprising administering a therapeutically effective amount of the polypeptide of claim 60 to the human subject. ### Claim 62 (depends on 60) A pharmaceutical composition comprising the polypeptide of claim 60, and a pharmaceutically acceptable carrier. ### Claim 63 (depends on 49) The polypeptide of claim 49, wherein the polypeptide comprises the amino acid sequence JAT8LRRFGDXLNFRQ (SEQ ID NO:62) with 1 or 2 amino acid substitutions relative to the amino acid sequence of SEQ ID NO:62, wherein J is a non-natural electrophile containing amino acid or an electrophilic warhead that does not comprise an amino acid at the N-terminus of the amino acid sequence, 8 is R-octenyl alanine, and X is a non-natural amino acid with an olefinic side chain. ### Claim 64 (depends on 63) The polypeptide of claim 63, wherein the polypeptide is stapled, wherein X is S-pentenyl alanine, and wherein the R-octenyl alanine is linked to the S-pentenyl alanine. ### Claim 65 (depends on 64) A pharmaceutical composition comprising the polypeptide of claim 64 and a pharmaceutically acceptable carrier. ### Claim 66 (depends on 64) A method for treating a human subject suffering from a cancer that exhibits expression of BFL-1 or dependency on BFL-1, the method comprising administering a therapeutically effective amount of the polypeptide of claim 64 to the human subject. ### Claim 67 (depends on 64) The polypeptide of claim 64, wherein J is (S)-1-acryloylpyrrolidine-3-carboxamide, 1-acryloylpiperidine-4-carboxamide, (R)-1-acryloylpiperidine-3-carboxamide, (S)-1-acryloylpiperidine-3-carboxamide, (S)-1-acryloylpyrrolidine-2-carboxamide, (R)-1-acryloylpyrrolidine-2-carboxamide, (E)-4-(dimethylamino)but-2-enamide, or acrylamide. ### Claim 68 (depends on 64) The polypeptide of claim 64, wherein J is (R)-1-acryloylpiperidine-3-carboxamide. ### Claim 69 (depends on 68) A method for treating a human subject suffering from a cancer that exhibits expression of BFL-1 or dependency on BFL-1, the method comprising administering a therapeutically effective amount of the polypeptide of claim 68 to the human subject. ### Claim 70 (depends on 68) A pharmaceutical composition comprising the polypeptide of claim 68, and a pharmaceutically acceptable carrier. ### Claim 71 (depends on 49) The polypeptide of claim 49, wherein the polypeptide comprises the amino acid sequence JAT8LRRFGDXLNFRQ (SEQ ID NO:62), wherein J is a non-natural electrophile containing amino acid or an electrophilic warhead that does not comprise an amino acid at the N-terminus of the amino acid sequence, 8 is R-octenyl alanine, and X is a non-natural amino acid with an olefinic side chain. ### Claim 72 A polypeptide that binds to Bfl-1, the polypeptide comprising an amino acid sequence that is at least 70% identical to the Bfl-1 interacting alpha-helical face of the amino acid sequence set forth in SEQ ID NO: 131, wherein the polypeptide selectively binds Bfl-1 over MCL-1; wherein at least two amino acids in the amino acid sequence are substituted by non-natural amino acids with olefinic side chains, wherein the at least two amino acids are separated by three or six amino acids, and wherein the polypeptide comprises a non-natural amino acid bearing an electrophilic group at the N-terminus of the amino acid sequence. ### Claim 73 (depends on 72) The polypeptide of claim 72, which is stapled. ### Claim 74 (depends on 73) A pharmaceutical composition comprising the polypeptide of claim 73 and a pharmaceutically acceptable carrier. ### Claim 75 (depends on 73) A method for treating a human subject suffering from a cancer that exhibits expression of BFL-1 or dependency on BFL-1, the method comprising administering a therapeutically effective amount of the polypeptide of claim 73 to the human subject. ### Claim 76 (depends on 72) The polypeptide of claim 72, wherein the polypeptide comprises an amino acid sequence that is at least 90% identical to the Bfl-1 interacting alpha-helical face of the amino acid sequence set forth in SEQ ID NO: 131, wherein the polypeptide selectively binds Bfl-1 over MCL-1; wherein at least two amino acids in the amino acid sequence are substituted by non-natural amino acids with olefinic side chains, wherein the at least two amino acids are separated by three or six amino acids, and wherein the polypeptide comprises a non-natural amino acid bearing an electrophilic group at the N-terminus of the amino acid sequence.