Structural stabilization and / or selective targeting of apoptotic proteins by cysteine-reactive NOXA peptides

Abstract

Provides a structurally stabilized and / or cysteine-reactive NOXA peptide. [Solution] This disclosure relates to a structurally stabilizing and / or cysteine-reactive NOXA BH3 peptide that can covalently bind to BFL-1 and can also bind to BFL-1 and / or MCL-1 by non-covalent interactions. This disclosure also features methods for using such peptides alone or in combination with other therapeutic agents (e.g., inhibitors of DNA damage response pathway members (e.g., ATM kinase inhibitors, ATR kinase inhibitors, CHK1 / 2 inhibitors, PARP inhibitors), selective inhibitors of MCL-1, selective inhibitors of BCL-2, inhibitors of BCL-2 / BCL-XL) in the treatment of BFL-1 and / or MCL-1-dependent or MCL-1-expressing cancers (e.g., hematopoietic malignancies, melanoma).

Description

[Technical Field]

[0001] Cross-reference of related applications This application is U.S. Provisional Application No. 62 / 599,229, filed on December 15, 2017. Priority is claimed for the aforementioned, which is incorporated herein by reference in its entirety.

[0002] Description of research funded by the federal government. This invention was awarded by the National Institutes of Health. Under the assigned numbers 5R35CA197583 and R50CA211399, This was done with the support of the prefectural government. The government has certain rights to this invention.

[0003] Technical field This disclosure relates to the structural stabilization and / or cysteine-reactive NOXA peptide, as well as its Peptides such as these, either alone or as inhibitors of DNA damage response pathway members (e.g., ATM kinase inhibitors, ATR kinase inhibitors, CHK1 / 2 inhibitors, PARP inhibitors) , or MCL-1 inhibitors, selective BCL-2 inhibitors, or BCL-2 / BCL - When combined with XL inhibitors, cancer and cellular excess diseases, such as autoimmune conditions This relates to a method for use in the treatment of inflammatory conditions. [Background technology]

[0004] background BCL- The two protein families are involved in the complex network of checks and balances that determine cell fate. It forms twerks. This family has up to four conserved "BCL-2 homologs". The structure is defined by the presence of (BH) domains, and these domains are all ALF Includes the ahelix portion. Anti-apoptotic proteins such as BFL-1 and MCL-1. It exhibits sequence conservation in all BH domains, but on the other hand, it promotes apoptosis. The protein consists of "multi-BH domain" members (e.g., BAX and BAK), "BH3 only" - showing sequence similarity only in the alpha-helix BH3 domain. Members are divided into groups (e.g., BIM and NOXA). BH3 only subgroup. The chain is diverse, and the death-promoting signals from heterogeneous stimuli are located in the mitochondria. It transmits signals to the apoptotic mechanism. The death signal of BH3-only proteins is an anti-apoptotic signal. Neutralized by cis proteins, or directly or indirectly, mitochondria It is delivered to the executioners BAX and BAK. Once activated, BA X / BAK induces permeabilization of the mitochondrial outer membrane, releasing mitochondrial factors. However, this makes it possible to induce caspases that irreversibly execute the death program.

[0005] Human cancers resist treatment and ensure cell survival by utilizing mitochondrial apex receptors. The systole is constantly suppressed. The most common strategy is to use one or more Anti-apoptotic BCL-2 family proteins, e.g., BCL-2, BCL-X L This involves overexpression of MCL-1 or BFL-1. These proteins are multido Main proteins BAX and BAK, as well as BH3-only stress sensor protein Pro-apoptotic substances, such as BIM, BID, BAD, and NOXA. Suppression of mitochondrial apoptosis, including capture of the BH3 helix of members share a common mechanism. Considering the overlap of binding specificities among anti-apoptotic proteins, molecular targeting of one member can also result in upregulation of another, leading to a tumorological version of "whack-a-mole." For example, small molecule targeting of BCL-2 with the selective small molecule inhibitor ABT-737 has been shown to trigger upregulation of MCL-1 or BFL-1 in lymphoma cell lines. Selective small molecule inhibitors of BCL-2 are currently approved by the FDA, and selective compounds for MCL-1 have advanced to clinical trials in recent years, leaving BFL-1 as a high-priority, druggable target in cancer. In fact, in addition to its ability to confer resistance to BCL-2 and MCL-1 selective inhibitors, BFL-1 is an independent oncogenic driver in the human cancer host, including melanoma, leukemia, and lymphoma.

[0006] BFL-1 / A1, a BCL-2-related protein, is involved in the suppression of the mitochondrial apoptosis pathway in a wide range of liquid and solid tumors. For example, BFL-1 induces chemoresistance in individual lymphomas, including germinal center lymphomas and diffuse large B-cell lymphomas of the BCR-dependent / activated NFκB subclass, when overexpressed or mutated to be resistant to ubiquitin-mediated degradation. BFL-1 has also been identified as a pathological survival factor in approximately 30% of human melanomas, including those with clinically relevant BRAF V600E resistance mutations.

[0007] ​​​​​​​​​​​​​​Myeloid cell leukemia-1, an anti-apoptotic BCL-2 family survival protein (MCL-1) is involved in the development, maintenance, and chemotherapy resistance of a wide range of cancers, and in humans It is one of the top 10 most widely expressed pathological factors in cancer. For example, see Beroukhim, Nature, 463(7283):899-905 (2010). When highly overexpressed in cancer, MCL-1 becomes a pro-apoptotic BCL-2 Binding to the essential BH3 domain helix of a family member and sealing it off. This results in extremely high resistance to apoptosis.

[0008] Therefore, compounds that interfere with BFL-1 and / or MCL-1 activity are diverse. It is useful in treating diseases of excess cells, such as autoimmune and inflammatory conditions. It is possible. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Beroukhim, Nature, 463(7283):899-905 (2010) [Overview of the project] [Means for solving the problem]

[0010] Abstract This disclosure can be covalently bonded to BFL-1, and through non-covalent interactions It can also bind to BFL-1 and / or MCL-1, for structural stabilization and / or This relates to the cysteine-reactive NOXA BH3 peptide. This disclosure also relates to such peptides Tide, alone or with other therapeutic agents (e.g., inhibitors of DNA damage response pathway members) For example, ATM kinase inhibitors, ATR kinase inhibitors, CHK1 / 2 inhibitors, PAR P inhibitors), selective inhibitors of MCL-1, selective inhibitors of BCL-2, BCL-2 / BC In combination with L-XL inhibitors, BFL-1 and / or MCL-1 dependent or It is used in the treatment of cancers that manifest it (e.g., hematopoietic malignancies, melanoma). This method is also a distinctive feature.

[0011] In a first aspect, the present disclosure relates to an electrophilic warhead and the modification of the sequence described in Sequence ID No. 39. The invention provides peptides containing a modified amino acid sequence. The modified amino acid sequence contains at least Each is 5 amino acid long and contains at least one modification that stabilizes the peptide structure, and the sequence number One or more of item 39 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1) 1, 12, 13, or 14 amino acids are substituted by other amino acids. The drug binds to BFL-1 / A1, or to both BFL-1 / A1 and MCL-1. It binds to at least five amino acids, for example, ATQLR (SEQ ID NO: 110), RFGDK (SEQ ID NO: 143), LNFRQ (SEQ ID NO: 124), TQLRR (SEQ ID NO: 143) 145), FGDKL (Sequence ID 146), QLRRF (Sequence ID 147), GDKLN (SEQ ID NO: 148), LRRFG (SEQ ID NO: 149), DKLNF (SEQ ID NO: 150), Variants of RRFGD (sequence code 142) and KLNFR (sequence code 144) It is possible. For example, at least two of the five amino acids may be modified for structural stabilization. (For example, non-natural amino acids that can form hydrocarbon staples) By substitution, one or more (for example, 1, 2, or 3) amino acids can be replaced. It is substituted with other amino acids. In certain cases, the substitution is a non-interaction of the helix. It lies on the surface. In certain cases, the substitution lies on the non-interacting surface of the helix. In some cases In this case, the substitution is a conservative amino acid substitution. In a particular embodiment, at least five One or more amino acids are L-alanine, D-alanine, and α-alanine. It consists of noisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives. It is substituted with an amino acid selected from the group.

[0012] In some embodiments, the modified amino acid sequence consists of 5 to 35 amino acids (for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 32, The length is 35. In some embodiments, the modified amino acid sequence is 8-18. For example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) amino acid length That is the case.

[0013] In some embodiments, modifications to stabilize the peptide structure include staples and / or It is a staple. In some cases, the staples are i-th and i+3-th, i-th and i+4-th. It is located in the i-th position, or in one or more of the i-th and i+7th positions. In some embodiments, Modifications to stabilize the ptyde structure include at least two of sequence number 39 (for example, 2, 3, 4) This includes substitution of 5 or 6 amino acids with unnatural amino acids having olefin side chains. In some cases, unnatural amino acids with olefin side chains are S-pentenylalanine. , R-octenylalanine; R-propenylalanine, S-pentenylalanine; R-pe Pentenylalanine, S-pentenylalanine; bis-pentenylglycine, S-pentenyl Lalanine, R-octenylalanine; and bis-pentenylglycine, S-octenyl Selected from the group consisting of rualanine and R-octenylalanine. In a particular embodiment, The staples and / or stitches are 1st and 5th, 2nd and 6th, 3rd and 7th, 6th and 10th, 10th and 14th, 11th and 15th, 12th and 1st 6th place, 1st and 8th place, 3rd and 10th place, 6th and 13th place, 7th and 14th place, and This includes positions 8 and 15 (where the position is based on sequence number 39).

[0014] In certain embodiments, modifications to stabilize the peptide structure involve hydrocarbon staples / s Stitch, lactam staples / stitch, UV-cyclization staples / stitch, oxy Staples / stitches, thioether staples / stitches, double-click staples Stitching, vis-lactum staples, vis-aryl staples Tetch, or any combination of two or more of these.

[0015] In certain embodiments, one of the amino acids in SEQ ID NO: 39 is substituted with another amino acid. Multiple amino acids (for example, 1, 2, 3, 4, 5, 6) form the helix of SEQ ID NO: 39. It is located on the BFL-1 / A1 non-interaction surface. In some embodiments, 0 in SEQ ID NO: 39 ~5 amino acids are removed from the C-terminus, or removed, resulting in alanine, D-alanine. N, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine It is replaced by 1 to 6 amino acids from the group consisting of derivatives. In some embodiments, The 0-6 amino acids on the non-interaction plane of row number 39 are alanine, D-alanine, and α- Aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives It is substituted with an amino acid selected from the following group. In some embodiments, SEQ ID NO: 39 Of the following amino acids A1, L4, R5, F7, G8, L11, N12, and Q15 One or more of these are alpha-methylated or alpha-ethylated natural amino acids. It is substituted with an acid. In some embodiments, the following amino acids A1, L4 of SEQ ID NO: 39 One or more of R5, F7, G8, L11, N12, and Q15 are L-Ara Nin, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine It is substituted with an amino acid selected from the group consisting of , and glycine derivatives. In the application form, the following amino acids of SEQ ID NO: 39, T2, Q3, R6, K10, F13, and One or more of the R14s are alpha-methylated or alpha-ethylated. It is substituted with natural amino acids. In certain embodiments, the following amino acid of SEQ ID NO: 39 One or more of the following no acids are L- Alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine It is substituted with an amino acid selected from the group consisting of nin and glycine derivatives. In certain embodiments, the following amino acids A1, T2, Q3, R5, R6, F of SEQ ID NO: 39 7, G8, K10, L11, N12, F13, R14, and Q15 or Multiple compounds are modified with alpha-methyl or ethyl, or with L-alanine, D- Alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine It is substituted with an amino acid selected from the group consisting of lysine derivatives. One or more amino acids in sequence number 39 that are substituted by another amino acid are sequence numbers It is located on the BFL-1 / A1 interaction plane of helix 39. In other cases, a different amino acid One or more amino acids in SEQ ID NO: 39 that are substituted by the helical of SEQ ID NO: 39 It is located on the non-interacting and interacting surfaces of the BFL-1 / A1 of the cus. In certain embodiments, One or more amino acids in Sequence ID No. 39 are L-alanine, D-alanine, and α-alanine. It consists of noisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives. It is substituted with an amino acid selected from the group.

[0016] In some embodiments, one or more of the following (numbering is based on Sequence ID No. 39) (k) is not replaced, stapled, or stitched: L4 and G8 together; R5 and D 9 together; F7 and L11 together; G8 and N12 together; D9 and F13 together Together; T2 and D9 together; L4 and L11 together; R5 and N12 together; A1 ;L4;G8;or D9.

[0017] In some embodiments, the overall hydrophobicity of the peptide is as shown in SEQ ID NO: 39 or 16. It is reduced compared to [the previous version]. In some embodiments, the overall positive charge of the peptide is [the sequence number]. It is reduced compared to peptides 39 or 16. In certain embodiments, the peptide The overall hydrophobicity and overall positive charge of the peptide are compared to the peptides of SEQ ID NO: 39 or 16. It has been reduced.

[0018] In some embodiments, the electrophilic warhead is a non-natural amino acid having an electrophilic group. In certain cases, unnatural amino acids with electrophilic groups are linked to the polypeptide backbone. It has a bonded electrophilic acrylamide or substituted acrylamide. In some cases, The unnatural amino acid with an electrophilic group is (S)-1-acryloylpyrrolidine-3-ca Ruboxamide, 1-Acrylopiperidine-4-carboxamide, (R)-1 Acryloy Lupiperidine-3-carboxamide, (S)-1-acryloylpiperidine-3-carbo Xamide, (S)-1-acryloylpyrrolidine-2-carboxamide, (R)-1-A Cryloylpyrrolidine-2-carboxamide, (E)-4-(dimethylamino)buta-2 - Enamide, acrylamide, aziridine, diaziridine, azetidine, pyrrolidine, i Midazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, Sothiazolidinedione, piperidine, piperazine, morpholine, thiomorpholine, azepaneazi Phosphorus (azepaneazirine), diazirine, azeto, pyrrole, imidazole, pyrazole, Oxazole, isoxazole, thiazole, isothiazole, pyridine, diazine, Oxazine, thiazine, azepinephenyl (aniline), naphthalene, anthracene, f Enanthrene, indole, isoindole, indoridine, quinolone, isoquinoline, Quinoxaline, phthalidine, quinazoline, purine, carbazole, indazole, benz Imidazole, azaindole, α-cyanoacrylamide, propioluamide, tran 4-dimethylamino-2-butenamide, trans-4-piperidinyl-2-butenamide Selected from the group consisting of ion, substituted acrylamide, and vinyl sulfonamide. In one embodiment, the electrophilic warhead is a cysteine-reactive D-nipecotinic acid moiety. In the embodiment, the electrophilic warhead is a cysteine-reactive portion. In a particular case, The electrophilic warhead is located at the N-terminus of the peptide. In other cases, the electrophilic warhead is located at the N-terminus of the peptide. It's not at the N-terminus of the do.

[0019] In a second aspect, the present disclosure relates to an electrophilic warhead and A0B0C0D0E0A1B1C1D Five or more amino acids of sequence 1E1A2B2C2D2E2 (Sequence ID 93) Inside, (a) A0 is not present, A, D-alanine, α-aminoisobutyric acid, or staple / Stitch position, (b) B0 does not exist, T, A, D-alanine, α-aminoisobutyric acid, I, M, B( Norleucine), or staple / stitch position, (c) C0 is not present, Q, A, D-alanine, α-aminoisobutyric acid, or stay This is the pull / stitch position. (d) D0 is not present, L, A, D-alanine, α-aminoisobutyric acid, I, or F And, (e) E0 is R, A, D-alanine, α-aminoisobutyric acid, K, T, or staple / Stitch position, (f) A1 is R, A, D-alanine, α-aminoisobutyric acid, E, or staple / s This is the touch position, (g)B1 is F, A, D-alanine, α-aminoisobutyric acid, I, L, V, or ste This is the pull / stitch position. (h)C1 is G, A, D-alanine, α-aminoisobutyric acid, or staple / step It is the position, (i) D1 is D, (j)E1 is absent, K, A, D-alanine, α-aminoisobutyric acid, or stay This is the pull / stitch position. (k)A2 is absent, L, A, D-alanine, α-aminoisobutyric acid, W, V, also This is the staple / stitch position. (l) B2 is absent, N, A, D-alanine, α-aminoisobutyric acid, S, D, also This is the staple / stitch position. (m)C2 is not present, F, A, L, D-alanine, α-aminoisobutyric acid, or S Table / stitch position, (n)D2 is absent, R, A, D-alanine, α-aminoisobutyric acid, E, or S Table / stitch position, (o)E2 is not present, Q, A, D-alanine, α-aminoisobutyric acid, L, or S It is characterized by peptides containing (table / stitch sites).

[0020] The peptide binds to BFL-1 / A1 (covalently and / or noncovalently). Alternatively, the peptide binds to BFL-1 / A1 (covalently) and to MCL-1 (non To join (by covalent bonding).

[0021] In certain embodiments, the peptides are 5-35 (for example, 5, 6, 7, 8, 9, 1) 0, 11, 12, 13, 14, 15, 20, 22, 25, 30, 35) are amino acid lengths. In certain embodiments, the peptides are 8-18 (for example, 8, 9, 10, 11, 1) 2, 13, 14, 15, 16, 17, 18) These are amino acid lengths.

[0022] In some embodiments, the overall hydrophobicity of the peptide is as shown in SEQ ID NO: 39 or 16. It is reduced compared to [the previous version]. In some embodiments, the overall positive charge of the peptide is [the sequence number]. It is reduced compared to peptides 39 or 16. In certain embodiments, the peptide The overall hydrophobicity and overall positive charge of the peptide are compared to the peptides of SEQ ID NO: 39 or 16. It has been reduced.

[0023] In some embodiments, the electrophilic warhead is a non-natural amino acid having an electrophilic group. In certain cases, unnatural amino acids with electrophilic groups are linked to the polypeptide backbone. It has a bonded electrophilic acrylamide or substituted acrylamide. In some cases, The unnatural amino acid with an electrophilic group is (S)-1-acryloylpyrrolidine-3-ca Ruboxamide, 1-Acrylopiperidine-4-carboxamide, (R)-1 Acryloy Lupiperidine-3-carboxamide, (S)-1-acryloylpiperidine-3-carbo Xamide, (S)-1-acryloylpyrrolidine-2-carboxamide, (R)-1-A Cryloylpyrrolidine-2-carboxamide, (E)-4-(dimethylamino)buta-2 - Enamide, acrylamide, aziridine, diaziridine, azetidine, pyrrolidine, i Midazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, Sothiazolidinedione, piperidine, piperazine, morpholine, thiomorpholine, azepaneazi Phosphorus, diaziline, azeto, pyrrole, imidazole, pyrazole, oxazole, iso Oxazole, thiazole, isothiazole, pyridine, diazine, oxazine, thiazole Azepinephenyl (aniline), naphthalene, anthracene, phenanthrene, yn Dole, isoindole, indoridine, quinolone, isoquinoline, quinoxaline, lid Rudin, quinazoline, purine, carbazole, indazole, benzimidazole, bruise Indole, α-cyanoacrylamide, propioluamide, trans-4-dimethylamide No-2-butenamide, trans-4-piperidinyl-2-butenamide, substituted acrylamide Selected from the group consisting of mids and vinyl sulfonamides. In one embodiment, the electrophoresis is performed. The electrophilic warhead is a cysteine-reactive D-nipecotinic acid moiety. In another embodiment, The electrophilic warhead is the cysteine-reactive portion. In certain cases, the electrophilic warhead is, It is located at the N-terminus of the peptide. In other cases, the electrophilic warhead is not located at the N-terminus of the peptide.

[0024] In some embodiments, the peptide includes staples and / or stitches. In this example, if staples exist, they are located at position i and position i+3, position i and position i+4, and so on. or are located at one or more of the i-position and i+7-position. In some embodiments, the peptide Modifications to stabilize the structure include at least two of sequence numbers 93 (for example, 2, 3, 4, 5, Includes substitution of 6 amino acids with unnatural amino acids having olefin side chains. In this example, the unnatural amino acids with olefin side chains are S-pentenylalanine, R- Octenylalanine; R-propenylalanine, S-pentenylalanine; R-pentenyl Lalanine, S-pentenylalanine; bis-pentenylglycine, S-pentenylalanine R-octenylalanine; and bis-pentenylglycine, S-octenylalanine Selected from the group consisting of nin and R-octenylalanine. In a particular embodiment, Petitdo is a hydrocarbon staple / stitch, lactam staple / stitch, UV-cyclized Additional staples / stitches, oxime staples / stitches, thioether staples / Stitching, double-click staples / stitching, vis-lactum staples / stitching , swivel-arylated staples / stitches, or any two of these or more This includes combinations. In a particular embodiment, any of the following positions in sequence number 93 Two or more staples are involved in the stapling or stitching: A0, B0, C0 E0, A1, B1, C1, E1, A2, B2, C2, D2, E2. In a particular case... Staple positions may be introduced after E2.

[0025] In a third aspect, the disclosure is covalently bonded to BFL-1 / A1 (for example, to C55). Provides a peptide that combines with an electrophilic warhead and one of the sequence numbers 94-109. Peptide containing a sequence that is at least 14% identical to the amino acid sequence described in one document. The peptide selectively binds to BFL-1 / A1 rather than MCL-1. Morphologically, peptides are 8-18 (for example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) These are amino acid lengths.

[0026] In certain embodiments, the electrophilic warhead is located at the N-terminus of the peptide. Other embodiments Therefore, the electrophilic warhead is not at the N-terminus of the peptide. In some embodiments, the electrophilic warhead The part is a non-natural amino acid that has an electrophilic group. In certain cases, the electrophilic group The unnatural amino acids it contains are electrophilic acrylamide linked to the polypeptide backbone. It has a substituted acrylamide. In some cases, the linker is a nitrogen-containing heterocycle, nitrogen-containing It is a heterocyclic amino acid, an amino-functionalized benzene ring, a carbocyclic, polycyclic, or heterocyclic compound. In the case of the part, the unnatural amino acid having an electrophilic group is (S)-1-acryloylpyrro Lysine-3-carboxamide, 1-acrylopiperidine-4-carboxamide, (R)- 1 Acryloylpiperidine-3-carboxamide, (S)-1-acryloylpiperidine n-3-carboxamide, (S)-1-acryloylpyrrolidine-2-carboxamide; (R)-1-Acryloylpyrrolidine-2-carboxamide, (E)-4-(Dimethyl Minobuta-2-enamide, acrylamide, aziridine, diaziridine, azetidine, Pyrrolidine, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, th Azolidine, isothiazolidine, piperidine, piperazine, morpholine, thiomorpholine Azepane, Aziline, Diaziline, Azeto, Pyrrole, Imidazole, Pyrazole, Oxy Sazole, isoxazole, thiazole, isothiazole, pyridine, diazine, oxy Sazidine, thiazine, azepinephenyl (aniline), naphthalene, anthracene, phena Indole, isoindole, indoridine, quinolone, isoquinoline, quinolone Xaline, phthalidine, quinazoline, purine, carbazole, indazole, benzimi Dazole, azaindole, α-cyanoacrylamide, propioluamide, trans-4 -dimethylamino-2-butenamide, trans-4-piperidinyl-2-butenamide, Selected from the group consisting of substituted acrylamides and vinyl sulfonamides. Morphologically, the electrophilic warhead is the cysteine-reactive D-nipecotinic acid portion.

[0027] In certain cases, peptides are hydrocarbon staples / stitches, lactam tapes. Staples / stitching, UV-cyclization addition staples / stitching, oxime staples / stitching, Thioether staples / stitches, double-click staples / stitches, Vis-Lak Tam staples / stitching, screw-aryl staples / stitching, or any of these. Includes combinations of two or more of the above.

[0028] In some cases, peptides make up at least 15%, 20%, or 27% of the amino acid sequence. %, 34%, 40%, 47%, 50%, 53%, 60%, 65%, 70%, 75%, 80 Includes sequences that are identical by %, 85%, 90%, 95%, 97%, or 100%. In some cases, the variation from the amino acid sequence is due to the BFL-1 non-interaction of the amino acid sequence. It is located on the phahelic plane. In certain cases, the amino acid sequence is also alpha-methyl. or modified to contain alpha-ethyl natural amino acids, and / or Lanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine Substituted with one or more amino acids selected from the group consisting of , and glycine derivatives. It is being done.

[0029] In a fourth aspect, the Disclosure applies to BFL-1 / A1 (for example, C5 of BFL-1 / A1). 5) A peptide that is covalently bonded, and the amino acids listed in SEQ ID NOs. 60-75 The peptide is characterized by containing a sequence that is at least 14% identical to the given sequence. In this case, the peptide selectively binds to BFL-1 / A1 rather than MCL-1. In this case, the peptide was covalently bound to BFL-1 / A1 and noncovalently bound to MCL-1. They are joined together.

[0030] In certain embodiments, the electrophilic warhead is made of a non-natural amino acid having an electrophilic group. be.

[0031] In some embodiments, the peptide is at least 15%, 20%, relative to the amino acid sequence. 27%, 34%, 40%, 47%, 50%, 53%, 60%, 65%, 70%, 75%, Contains sequences that are 80%, 85%, 90%, 95%, 97%, or 100% identical. In certain cases, variations from the amino acid sequence are BFL-1 non-interacting amino acids. It is located on the phahelic plane. In some cases, it is located at positions 2, 3, 6, 7, and 8 of the aforementioned sequence. , 9th, 12th, 13th, 14th, 15th, or 16th (1st place is "J") One or more amino acids in may be substituted with other amino acids. In this example, one or more of the amino acids at these positions are alanine, D-alanine. It is substituted with one or more of the following: ranin or α-aminoisobutyric acid. In the example, the amino acids at positions 7 and / or 15 are substituted with glutamic acid. The amino acid at position 13 is substituted with aspartic acid. The amino acid at position 16 is substituted with leucine, and also This is one of the following: 2nd, 3rd, 6th, 8th, 9th, 12th, 14th, and 15th place or In multiple cases, the amino acids are substituted with alanine, D-alanine, or α-aminoisobutyric acid. (The number one is "J"). In some cases, the 8th and / or 12th place is The amino acids are substituted with alanine, D-alanine, or α-aminoisobutyric acid. (The number one spot goes to "J").

[0032] In some cases, the peptide has the sequence JATX1LREFGDX2LNFRQ(Sequence ID). 62) containing or consisting of, where the peptide is BFL-1 rather than MCL-1 The peptide selectively binds to / A1, and covalently binds to BFL-1 / A1, and MCL- 1 is bonded to J by a non-covalent bond, J is an electrophilic warhead, and X1 and X2 are non-natural It is a amino acid.

[0033] In some cases, the peptide has the sequence JATX1LRRAGDX2LNFRQ (Sequence ID). 60) containing or consisting of, where the peptide is BFL-1 rather than MCL-1 The peptide selectively binds to / A1, and covalently binds to BFL-1 / A1, and MCL- 1 is bonded to J by a non-covalent bond, J is an electrophilic warhead, and X1 and X2 are non-natural It is a amino acid.

[0034] In some cases, electrophilic warheads have electrophilic portions linked to a polypeptide skeleton. In some cases, the linker is a nitrogen-containing heterocyclic amino acid, an amino acid. The functionalized benzene ring, carbocyclic ring, polycyclic ring, or heterocyclic ring is used. In some embodiments, the electrophile is used. The ferrous parts are electrophilic acrylamide, substituted acrylamide, vinyl sulfonamide, and or α,β unsaturated amide. In some embodiments, the electrophilic warhead has an electrophilic group It is a non-natural amino acid that possesses an electrophilic group. In some cases, it is a non-natural amino acid that has an electrophilic group. (S)-1-acryloylpyrrolidine-3-carboxamide, 1-acrylopiperidine n-4-carboxamide, (R)-1 acryloylpiperidine-3-carboxamide, (S)-1-Acryloylpiperidine-3-carboxamide, (S)-1-Acryloyl Pyrrolidine-2-carboxamide; (R)-1-acryloylpyrrolidine-2-carboxamide Thamide, (E)-4-(dimethylamino)buta-2-enamide, acrylamide, azili Zin, diaziridine, azetidine, pyrrolidine, imidazolidine, pyrazolidine, oxa Zolidine, isoxazolidine, thiazolidine, isothiazolidine, piperidine, pipera Zin, morpholine, thiomorpholine, azepane, azirine, diazirine, azeto, pyrrole Imidazole, pyrazole, oxazole, isoxazole, thiazole, isothia Zole, pyridine, diazine, oxazine, thiazine, azepine phenyl (aniline), Naphthalene, anthracene, phenanthrene, indole, isoindole, indride N, quinolone, isoquinoline, quinoxaline, phthalidine, quinazoline, purine, carba Zole, indazole, benzimidazole, azaindole, α-cyanoacrylamine D, propioamide, trans-4-dimethylamino-2-butenamide, trans-4- Piperidinyl-2-butenamide, substituted acrylamide, and vinyl sulfonamide Selected from the group consisting of . In one embodiment, the electrophilic warhead is cysteine-reactive D- This is the nipecotinic acid portion.

[0035] In certain embodiments, the peptide is 15-35 (for example, 15, 16, 17, 1) 8, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35 ) Amino acid length. In some embodiments, the modified amino acid sequence is 8-18 (for example For example, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18) by amino acid length be.

[0036] In a fifth aspect, the Disclosure applies to BFL-1 / A1 (for example, C5 of BFL-1 / A1). 5) A peptide that is covalently bonded, and one of the sequence numbers 60-75 The BFL-1 / A1 interaction surface of the alpha helix of the amino acid sequence described is small Peptides each contain two amino acids (for example, 2, 3, 4, 5, 6, 7, 8, or 9). To provide.

[0037] In certain embodiments, the peptide is 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, They are 27, 28, 29, or 30 amino acids long.

[0038] In a particular embodiment, the peptide is at least five of the sequence numbers 60-75 (even if ba, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 1 It contains 9, 20, 21, 22, 23, 24, and 25 amino acids, of which 3, 2, and One amino acid is substituted. In some cases, the substitution is alanine, D-alanine. It is either α-aminoisobutyric acid or α-aminoisobutyric acid.

[0039] In some embodiments, the electrophilic warhead is a cysteine-reactive D-nipecotinic acid moiety. In other embodiments, the electrophilic warhead is a cysteine-reactive portion.

[0040] In certain cases, unnatural amino acids are unnatural amino acids that have olefin side chains. In some cases, unnatural amino acids with olefin side chains are S-pentenyl It is alanine and / or R-octenylalanine.

[0041] In a sixth aspect, the disclosure relates to a peptide that is covalently bound to BFL-1 / A1. a peptide containing the amino acid sequence described in any one of sequence numbers 60-75. It is characterized by the following. In some embodiments, the peptide is an amino acid combination as described in SEQ ID NO: 62. It includes or consists of columns. In some embodiments, the peptide is described in SEQ ID NO: 60. It contains or consists of the following amino acid sequence.

[0042] In some embodiments, X1 is R-octenylalanine and X2 is S-pentenylalanine. It is rualanine. In some embodiments, X1 is S-pentenylalanine and X2 is R-octenylalanine. In certain embodiments, X1 is S-pentenyl X2 is alanine, and X2 is S-pentenylalanine. In other embodiments, X1 is R-propenylalanine and X2 are S-pentenylalanine. In the application form, X1 is R-pentenylalanine and X2 is S-pentenylalanine. That is the case.

[0043] In a particular embodiment, the electrophilic warhead is an electrophile linked to a polypeptide backbone. Includes a portion. In some embodiments, the electrophilic portion is a non-natural amino having an electrophilic group. It is an acid. In certain cases, unnatural amino acids that have electrophilic groups are electrophilic acids. lylamides, substituted acrylamides, vinyl sulfonamides, or α,β unsaturated amides This includes. In some cases, unnatural amino acids with electrophilic groups are (S)-1-acryl. Roylpyrrolidine-3-carboxamide, 1-acrylopiperidine-4-carboxamide (R)-1 Acryloylpiperidine-3-carboxamide, (S)-1-Acryloy Lupiperidine-3-carboxamide, (S)-1-acryloylpyrrolidine-2-carbo Xamide; (R)-1-Acryloylpyrrolidine-2-carboxamide, (E)-4-( Dimethylaminobuta-2-enamide, acrylamide, aziridine, diaziridine, A Zethidine, pyrrolidine, imidazolidine, pyrazolidine, oxazolidine, isoxazo Lysine, thiazolidinediones, isothiazolidines, piperidines, piperazines, morpholine, thio Morpholine, azepane, aziline, azeto, pyrrole, imidazole, pyrazo ol, oxazole, isoxazole, thiazole, isothiazole, pyridine, diazepam Zin, oxazine, thiazine, azepinephenyl (aniline), naphthalene, anthrace Phenanthrene, indole, isoindole, indoridine, quinolone, isoquinolone Phosphorus, quinoxaline, phthalidine, quinazoline, purine, carbazole, indazole, Benzimidazole, azaindole, α-cyanoacrylamide, propioluamide, trans-4-dimethylamino-2-butenamide, trans-4-piperidinyl-2-butenamide Selected from the group consisting of ionamides, substituted acrylamides, and vinyl sulfonamides. In certain cases, the electrophilic warhead is reacted to the cysteine-reactive D-nipecotinic acid moiety. Yes, in other cases, the electrophilic warhead is the cysteine-reactive portion.

[0044] In some embodiments, the peptide is 15-35 (for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35) A This is the amino acid length. In some embodiments, the peptide is 8-18 (for example, 8, 9, 10) ,11,12,13,14,15,16,17,18) are amino acid lengths.

[0045] In a seventh aspect, the present disclosure relates to the NOXA peptide described herein and pharmaceutically acceptable This relates to a pharmaceutical composition comprising a carrier.

[0046] In the eighth aspect, the disclosure describes a person who expresses BFL-1 / A1 or who is dependent on BFL-1 / A1. This method is characterized by its ability to treat persistent diseases in human subjects who require such treatment. This refers to a therapeutically effective dose of the NOXA peptide described herein or specified herein in human subjects. This includes administering the pharmaceutical composition described.

[0047] In some embodiments, BFL-1 / A1 is expressed or BFL-1 / A1-dependent Diseases are selected from the group consisting of hematopoietic malignancies, solid tumors, autoimmune diseases, and inflammatory diseases. It is selected. In certain cases, hematopoietic malignancies are leukemia or lymphoma. In certain cases, solid tumors such as melanoma, breast cancer, or lung cancer. Autoimmune colitis, thyroiditis, arthritis, nephritis, dermatitis, vasculitis, systemic lupus erythematosus Autoimmune diseases such as diabetes or Sjögren's disease. In certain cases, asthma. Inflammatory diseases such as psoriasis, inflammatory bowel disease, thyroiditis, arthritis, nephritis, dermatitis, or vasculitis. Suffering.

[0048] In some embodiments, BFL-1 / A1 is expressed or BFL-1 / A1-dependent The disease is cancer. In some embodiments, cancer is a solid tumor or a humoral tumor. In the embodiment of this part, cancers include breast cancer, autonomic ganglion cancer, pancreatic cancer, skin cancer, and CNS cancer. hematopoietic or lymphatic cancer, lung cancer, colorectal cancer, stomach cancer, soft tissue sarcoma, or bone cancer In some embodiments, hematopoietic malignancies include acute myeloid leukemia and acute lymphoblastic disease. Myelodysplastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome, or multiple leukemia It is multiple myeloma.

[0049] In some embodiments, NOXA peptide is used in SEQ ID NOs. 60-79, 151, and 15 Selected from 3. In some embodiments, the NOXA peptide is SEQ ID NO: 62. In this embodiment, the NOXA peptide is SEQ ID NO: 60.

[0050] In some embodiments, this method involves administering an inhibitor of a DNA damage response pathway member. The following further includes: In some embodiments, inhibitors of DNA damage response pathway members cause vasodilation. Ataxia mutation (ATM) kinase inhibitors, vasodilatory ataxia and rad3-related (ATR) ) Kinase inhibitors, checkpoint kinase 1 and / or checkpoint kinase -ze 2 (CHK1 / 2) inhibitors, and / or poly(ADP-ribose) polymers It is a PARP inhibitor. In some cases, ATM inhibitors are used, such as KU-559403. KU-55933, KU-60019, CP-466722, Chloroquine Phosphate, CGK The group is selected from 733 and AZD0156. In certain cases, ATR The inhibitors are cisandrin B, NU6027, NVP-BEZ235, VE-821, VE Selected from the group consisting of -822 / VX-970, AZ20, and AZD6738. In certain cases, CHK1 / 2 inhibitors include AZD7762, CCT 241533, LY 2603618, LY 2606368, NSC 109555 ditosylate, P D 407824, PF47736, SB 218078, TCS 2312, and S The group consisting of RA737 is selected. In certain cases, PARP inhibitors are used in olapa Rib, A-966492, Beriparib, Lucaparib, AG-14361, Iniparib, I NO-1001, Niraparib, Thalassoparib, AZD2461, 2X-121, BMN The group is selected from 673 and E7449.

[0051] In the ninth aspect, the disclosure relates to a disease that expresses MCL-1 or is MCL-1 dependent. This method is characterized by performing the procedure on human subjects who require it. in a therapeutically effective amount of the NOXA peptide described herein or described herein This includes administering a pharmaceutical composition.

[0052] In some embodiments, NOXA peptide is used in SEQ ID NOs. 60-79, 151, and 15 Select from 3, or the structural stabilization bars of the sequences listed in sequence numbers 126-141. It's John.

[0053] In some embodiments, this method involves administering an inhibitor of a DNA damage response pathway member. The following further includes: In some embodiments, inhibitors of DNA damage response pathway members are ATMK ATR kinase inhibitors, CHK1 / 2 inhibitors, and / or PARP inhibitors It is a harmful drug. In some cases, ATM inhibitors, such as KU-559403 and KU-55933, are harmful. KU-60019, CP-466722, chloroquine phosphate, CGK733, and A The group consisting of ZD0156 is selected. In certain cases, the ATR inhibitor is Cissane Dorin B, NU6027, NVP-BEZ235, VE-821, VE-822 / VX- The group is selected from 970, AZ20, and AZD6738. In a particular case, CHK1 / 2 inhibitors include AZD7762, CCT 241533, and LY 26036. 18, LY 2606368, NSC 109555 ditosylate, PD 407824 From PF47736, SB 218078, TCS 2312, and SRA737 The group is selected from the following. In certain cases, the PARP inhibitor is olaparib, A-966 492, beriparib, lucaparib, AG-14361, iniparib, INO-1001, Niraparib, thalazoparib, AZD2461, 2X-121, BMN 673, and E It is selected from a group consisting of 7449.

[0054] In the tenth aspect, the disclosure expresses BFL-1 / A1 or BFL-1 / A1 Treatment of MCL-1-dependent diseases and MCL-1-expressing or MCL-1-dependent diseases, This method is characterized by being performed on human subjects who require it. An effective amount of the NOXA peptide or the pharmaceutical composition described herein. This includes administering a substance.

[0055] In some embodiments, NOXA peptide is used in SEQ ID NOs. 60-79, 151, and 15 Select from 3, or the structural stabilization bars of the sequences listed in sequence numbers 126-141. This is John. In some embodiments, the NOXA peptide is derived from SEQ ID NOs. 60 and 62. Selected or structurally stabilized versions of sequences described in sequence numbers 126 and 128 It is.

[0056] In some embodiments, this method involves administering an inhibitor of a DNA damage response pathway member. The following further includes: In some embodiments, inhibitors of DNA damage response pathway members are ATMK ATR kinase inhibitors, CHK1 / 2 inhibitors, and / or PARP inhibitors It is a harmful drug. In some cases, ATM inhibitors, such as KU-559403 and KU-55933, are harmful. KU-60019, CP-466722, chloroquine phosphate, CGK733, and A The group consisting of ZD0156 is selected. In certain cases, the ATR inhibitor is Cissane Dorin B, NU6027, NVP-BEZ235, VE-821, VE-822 / VX- The group is selected from 970, AZ20, and AZD6738. In a particular case, CHK1 / 2 inhibitors include AZD7762, CCT 241533, and LY 26036. 18, LY 2606368, NSC 109555 ditosylate, PD 407824 From PF47736, SB 218078, TCS 2312, and SRA737 The group is selected from the following. In certain cases, the PARP inhibitor is olaparib, A-966 492, beriparib, lucaparib, AG-14361, iniparib, INO-1001, Niraparib, thalazoparib, AZD2461, 2X-121, BMN 673, and E It is selected from a group consisting of 7449.

[0057] In the eleventh aspect, the disclosure expresses BFL-1 / A1 or BFL-1 / A1 Dependent diseases (e.g., hematopoietic malignancies, solid tumors, autoimmune diseases, and inflammatory diseases) A combination therapeutic agent for treating ) comprising the NOXA peptide described herein, The present invention provides combination therapeutic agents, including inhibitors of members of the DNA damage response pathway. In this context, inhibitors of DNA damage response pathway members include ATM kinase inhibitors and ATR kinase inhibitors. These are enzyme inhibitors, CHK1 / 2 inhibitors, and / or PARP inhibitors.

[0058] In a twelfth aspect, the disclosure relates to diseases that express MCL-1 or are MCL-1 dependent. Treating (for example, hematopoietic malignancies, solid tumors, autoimmune diseases, and inflammatory diseases) A combination therapeutic agent for DNA damage, comprising the NOXA peptide described herein and The present invention provides combination therapeutic agents, including inhibitors of response pathway members. In some embodiments, DN Inhibitors of A-damage response pathway members include ATM kinase inhibitors, ATR kinase inhibitors, and C These are HK1 / 2 inhibitors and / or PARP inhibitors.

[0059] In the 13th aspect, the Disclosure expresses BFL-1 / A1 or BFL-1 / A1 Diseases that are dependent on MCL-1 and diseases that express MCL-1 or are MCL-1 dependent (for example, Combinations for treating hematopoietic malignancies, solid tumors, autoimmune diseases, and inflammatory diseases A therapeutic agent comprising the NOXA peptide described herein and a DNA damage response pathway member The present invention provides a combination therapeutic agent comprising an inhibitor of DNA damage response. Inhibitors of the pathway members include ATM kinase inhibitors, ATR kinase inhibitors, and CHK1 / 2 inhibitors. It is a harmful agent and / or a PARP inhibitor.

[0060] These embodiments apply to the 11th to 13th aspects. In some embodiments, AT M inhibitors include KU-559403, KU-55933, KU-60019, and CP-466. Selected from the group consisting of 722, chloroquine phosphate, CGK733, and AZD0156. In certain embodiments, the ATR inhibitor is cisandrin B, NU6027, NV P-BEZ235, VE-821, VE-822 / VX-970, AZ20, and AZ The group consisting of D6738 is selected. In certain cases, CHK1 / 2 inhibitors are A ZD7762, CCT 241533, LY 2603618, LY 2606368, NSC 109555 ditosylate, PD 407824, PF47736, SB 21 Selected from the group consisting of 8078, TCS 2312, and SRA737. In this case, the PARP inhibitors were olaparib, A-966492, veliparib, and lucaparib. Bu, AG-14361, Iniparib, INO-1001, Niraparib, Thalazoparib, A Selected from the group consisting of ZD2461, 2X-121, BMN 673, and E7449. In some embodiments, the NOXA peptide is represented by SEQ ID NOs. 60-79, 151, and Structural stabilization of sequences selected from 153 or listed in sequence numbers 126-141 This is the version. In some embodiments, the NOXA peptide is used in SEQ ID NOs. 60 and 62. Select from or the structural stabilization b of the sequence described in sequence numbers 126 and 128 It is a version.

[0061] In the fourteenth aspect, the present disclosure relates to the amino acid sequence described in SEQ ID NO: 44 with respect to less They are both 50% identical (for example, at least 55%, 60%, 65%, 70%, 75%) , containing amino acid sequences that are 80%, 85%, 90%, 95%, or 100% identical, Provides staple peptides. In certain cases, staple peptides are sequence numbers. It contains the amino acid sequence described in 44, but only 1 to 10 (for example, 1, 2, 3, Except for having 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In this case, the substitutions were L-alanine, D-alanine, α-aminoisobutyric acid, and N-methylglycol. amino acids selected from the group consisting of lysine, serine, substituted alanine, and glycine derivatives. It is acid-resistant. The staple peptide is bound to BFL-1 and / or MCL-1. do.

[0062] In the fourteenth aspect, the present disclosure relates to the amino acid sequence described in SEQ ID NO: 46 with respect to less They are both 50% identical (for example, at least 55%, 60%, 65%, 70%, 75%) , containing amino acid sequences that are 80%, 85%, 90%, 95%, or 100% identical, Provides staple peptides. In certain cases, staple peptides are sequence numbers. It contains the amino acid sequence described in 46, but only 1 to 10 (for example, 1, 2, 3, Except for having 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In this case, the substitutions were L-alanine, D-alanine, α-aminoisobutyric acid, and N-methylglycol. amino acids selected from the group consisting of lysine, serine, substituted alanine, and glycine derivatives. It is acid-resistant. The staple peptide is bound to BFL-1 and / or MCL-1. do.

[0063] These embodiments apply to a 14th embodiment. In some embodiments, staples The peptide further comprises a cysteine-reactive moiety. In some embodiments, staple peptide D contains the cysteine-reactive D-nipecotinic acid moiety. These moieties contain staple peptides. It may be bound to or included in the N-terminus of the cydo. In some embodiments, staples Butide is combined with a DNA damage response pathway inhibitor. In some embodiments, DNA damage Damage response pathway inhibitors are ATM kinase inhibitors. In some embodiments, DNA damage response The response pathway inhibitor is an ATR kinase inhibitor. In some embodiments, the DNA damage response pathway The pathway inhibitor is a CHK1 / 2 inhibitor. In some embodiments, it inhibits the DNA damage response pathway. The agent is a PARP inhibitor. In some embodiments, the ATM inhibitor is KU-55940 3. KU-55933, KU-60019, CP-466722, Chloroquine phosphate, C Selected from the group consisting of GK733 and AZD0156. In a particular embodiment, ATR inhibitors include cisandrin B, NU6027, NVP-BEZ235, and VE-82. 1. Select from the group consisting of VE-822 / VX-970, AZ20, and AZD6738. In a particular embodiment, the CHK1 / 2 inhibitor is AZD7762, CCT 2 41533, LY 2603618, LY 2606368, NSC 109555 Jito Silate, PD 407824, PF47736, SB 218078, TCS 231 Selected from the group consisting of 2, and SRA737. In a particular embodiment, PARP The inhibitors are olaparib, A-966492, veliparib, lucaparib, and AG-14361. Iniparib, INO-1001, Niraparib, Thalazoparib, AZD2461, 2X- Selected from the group consisting of 121, BMN 673, and E7449. Disclosed herein The staple peptides used express BFL-1 or are in a state dependent on it (for example) For example, it can be used to treat cancer, autoimmune diseases, and inflammatory diseases. The staple peptides disclosed in this document express MCL-1 or are dependent on it. It can be used to treat conditions (for example, cancer, autoimmune diseases, inflammatory diseases) Cut.

[0064] In a 15th aspect, the present disclosure relates to a disease that expresses MCL-1 and BFL-1 / A1. Or treatment of MCL-1-dependent and BFL-1 / A1-dependent diseases, as needed. A combination therapeutic agent for use in human subjects, wherein NOXA stabilizer as described herein We provide a combination therapeutic agent comprising a compounded peptide and an MCL-1 inhibitor. (Specific example) So, the MCL-1 inhibitors are S64315, S63845, BAY 1000394, M The model is selected from the group consisting of IK665, AMG397, and AMG176.

[0065] In the sixteenth aspect, the disclosure relates to a disease that expresses BCL-2 and BFL-1 / A1. Or treatment of BCL-2-dependent and BFL-1 / A1-dependent diseases, as needed. A combination therapeutic agent for use in human subjects, wherein NOXA stabilizer as described herein We provide a combination therapeutic agent comprising a peptide and a BCL-2 inhibitor. (Specific example) Therefore, the BCL-2 inhibitor is venetoclax.

[0066] In the 17th aspect, the disclosure relates to a disease expressing BCL-XL and BFL-1 / A1. , or treatment of BCL-XL-dependent and BFL-1 / A1-dependent diseases, if necessary A combination therapeutic agent for use in human subjects, wherein NOXA as described herein The present invention provides a combination therapeutic agent comprising a stabilized peptide and a BCL-XL inhibitor. In this case, the BCL-XL inhibitor is Navitoclax.

[0067] In the 18th aspect, the Disclosure relates to BCL-XL, BCL-2, and / or MCL- Diseases that express 1 and BFL-1 / A1, or BCL-XL, BCL-2, and Treatment of MCL-1-dependent and BFL-1 / A1-dependent diseases for the people who need it A combination therapeutic agent for use in elephants, comprising NOXA stabilized peptide as described herein. A combination comprising tide and an inhibitor of BCL-XL, BCL-2, and / or MCL-1. We provide treatments for this condition. In certain cases, MCL-1 inhibitors are used in S64315, S63 845, BAY 1000394, MIK665, AMG397, and AMG176 A selection is made from the following group. In certain cases, BCL-2 inhibitors are venetoclax In certain cases, the BCL-XL inhibitor is Navitoclax.

[0068] These embodiments apply to each of the 15th to 18th embodiments. In some embodiments... The NOXA peptide is selected from SEQ ID NOs. 60-79, 151, and 153. or structurally stabilized versions of the sequences described in sequence numbers 126-141. In this embodiment, the NOXA peptide is selected from SEQ ID NOs. 60 and 62, or This is a structurally stabilized version of the sequence described in sequence numbers 126 and 128.

[0069] Unless otherwise defined, all technical and scientific terms used herein are defined in accordance with the present invention. It has the same meaning as it would normally be understood by those skilled in the art. Methods and materials similar to or equivalent to those used in the implementation or testing of the present invention shall be used. This can be done, but exemplary methods and materials are described below. All publications, patent applications, patents, and other references are incorporated in their entirety by reference. In case of any inconsistencies, this application, including the definitions, will take control. The laws and examples are illustrative and not intended to limit the scope of the law.

[0070] Other features and advantages of the present invention will become apparent from the following detailed description and claims. It will happen. [Brief explanation of the drawing]

[0071] [Figure 1] Figure 1 shows the structure of the NOXA BH3 / BFL-1ΔC complex (PDB:3MQP), with selected NOXA residues highlighted in dark gray in the shortened NOXA peptide staple scan library. [Figure 2] Figure 2 provides sequences of the NOXA BH3 staple scan peptide library, in which all hydrocarbon (i, i+4) or (i, i+7) staples are arranged consecutively over the length of a peptide template having an N-terminal cysteine-reactive D-nipecotinic acid moiety. Sequences (i.e., those having a D-nipecotinic acid derivatization located at the N-terminus of the staple peptide) are assigned, from top to bottom, SEQ ID NOs: 1–21. [Figure 3] Figure 3 shows the reactivity of NOXA SAHB containing D-nipecotinic acid (D-NA) with BFL-1ΔC C4S / C19S, which retains only the native Cys55. [Figure 4] Figure 4 shows the cell permeability and responsiveness of D-NA-NOXA SAHB to BFL-1 in 293T cells expressing HA-BFL-1ΔC C4S / C19S. [Figure 5]Figure 5 shows the quantification of LDH release when A375P cells were treated with NOXA-15 for 30 minutes, demonstrating the induction of nonspecific cell lysis by NOXA-15. [Figure 6] Figure 6 provides the sequences of the NOXA-15 SAHB alanine scan library, which has an N-terminal cysteine-reactive D-nipecotinic acid moiety. The sequences (i.e., those with a D-nipecotinic acid derivatization located at the N-terminus of the staple peptide) are assigned sequence numbers 22-34 from top to bottom. [Figure 7] Figure 7 is a bar graph of LDH release from the D-NA-NOXA-15 SAHB alanine scan library, showing three peptides that lacked lytic activity as a result of alanine mutations. [Figure 8] Figure 8 shows a Coomassie-stained gel of the D-NA-NOXA-15 SAHB alanine scan library, illustrating its reactivity with recombinant BFL-1ΔC C4S / C19S, highlighting the broad tolerance of alanine mutations, excluding selective sites such as L29A and D34A, which show reduced or partial reactivity due to localized alanine mutagenesis. [Figure 9] Figure 9 is a Western blot showing the cell permeability and responsiveness to BFL-1 of the non-soluble D-NA-NOXA-15 alanine mutant SAHB in 293T cells expressing HA-BFL-1ΔC C4S / C19S. [Figure 10] Figure 10 lists the sequences of the NOXA-15 SAHB mutant having a cysteine-reactive D-nipecotinic acid moiety at the N-terminus. Sequences (i.e., those with a D-nipecotinic acid derivatization located at the N-terminus of the staple peptide) are assigned the sequence numbers 35-38 from top to bottom. [Figure 11] Figure 11 is a bar graph showing LDH release from the D-NA-NOXA-15 SAHB Arg-to-Glu charge reversal mutant peptide, indicating a significant reduction or elimination of solubility activity. [Figure 12]Figure 12 shows a Coomaschy-stained gel illustrating the reactivity of the D-NA-NOXA-15 SAHB Arg-to-Glu charge reversal mutant with recombinant BFL-1ΔC C4S / C19S. [Figure 13] Figure 13 is a blot showing the cell permeability and responsiveness to BFL-1 of a non-soluble D-NA-NOXA-15 SAHB Arg-to-Glu charge reversal mutant in 293T cells expressing HA-BFL-1ΔC C4S / C19S. [Figure 14] Figure 14 is a silver-stained gel showing that D-NA-NOXA-15 F32A exhibits a significant competitive advantage over its acetylated counterpart with respect to BFL-1 targeting, as indicated by streptavidin pull-down of an equimolar mixture of recombinant GST-MCL-ΔNΔC, BCL-XLΔC (untagged), His-BFL-1ΔC, and biotinylated SAHB. [Figure 15] Figure 15 is a silver-stained gel showing that D-NA-NOXA-15 R31E exhibits a significant competitive advantage over its acetylated counterpart with respect to BFL-1 targeting, as indicated by streptavidin pull-down of an equimolar mixture of recombinant GST-MCL-ΔNΔC, BCL-XLΔC (untagged), His-BFL-1ΔC, and biotinylated SAHB. [Figure 16] Figure 16 is a blot showing that D-NA-NOXA-15 F32A exhibits enhanced native BFL-1 targeting in the A375P lysate compared to the corresponding acetylated construct, as monitored by streptavidin pulldown and BFL-1 Western blotting. Both constructs bind similarly to MCL-1, as shown by streptavidin pulldown and MCL-1 Western blotting. [Figure 17]Figure 17 is a blot showing that D-NA-NOXA-15 R31E exhibits enhanced native BFL-1 targeting in the A375P lysate compared to the corresponding acetylated construct, as monitored by streptavidin pulldown and BFL-1 Western blotting. Both constructs bind similarly to MCL-1, as shown by streptavidin pulldown and MCL-1 Western blotting. [Figure 18] Figure 18 includes a blot (left) showing that D-NA-NOXA15 F32A effectively competes with tBID for the HA-BFL-1ΔC C4S / C19S interaction in the 293T lysate, achieving a strong covalent conjugation, as measured by immunoprecipitation and Western blotting, but its acetylated counterpart does not. In contrast, neither D-NA-NOXA15 F32A nor Ac-NOXA15 F32A can dissociate the FLAG-MCL-1 and tBID interaction (right). [Figure 19] Figure 19 shows a blot (left) indicating that D-NA-NOXA15 R31E effectively competes with tBID for the HA-BFL-1ΔC C4S / C19S interaction in the 293T lysate, achieving a strong covalent conjugation, as measured by immunoprecipitation and Western blotting, but its acetylated counterpart does not. In contrast, neither D-NA-NOXA15 R31E nor Ac-NOXA15 R31E can dissociate the FLAG-MCL-1 and tBID interaction (right). [Figure 20] Figure 20 is a graph showing that D-NA-NOXA-15 R31E has the ability to disrupt the interaction between BFL-1ΔC C4S / C19S and FITC-BID BH3 in a fluorescence polarization competitive binding assay, but its acetylated counterpart does not. [Figure 21]Figure 21 is a graph showing that neither D-NA-NOXA15 R31E nor Ac-NOXA15 R31E compete for the binding of FITC-BID BH3 to BFL-1ΔC C4S / C19S / C55S, indicating that C55 is required in the BH3 binding pocket of BFL-1 for selective covalent targeting. [Figure 22] Figure 22 is a graph showing that both Ac-NOXA-15 R31E and D-NA-NOXA-15 R31E exhibit similar and relatively limited to moderate ability to compete with the binding of FITC-BID BH3 to MCL-1ΔNΔC. [Figure 23] Figure 23 is a graph showing that neither Ac-NOXA-15 R31E nor D-NA-NOXA-15 R31E can compete for binding of FITC-BID BH3 to BCL-XLΔC. [Figure 24] Figure 24 is a plot of AML cell lines showing varying levels of BFL-1 dependence for cell survival, as determined by whole-genome CRISPR screening. [Figure 25] Figure 25 is a table showing gene dependencies that positively correlate with BFL-1 dependence in AML cell lines, as determined by whole-genome CRISPR screening. [Figure 26] Figure 26 is a graph showing that treatment of BFL-1-dependent U937 cells with a combination of cysteine-reactive NOXA-15 R31E and ATM inhibitor KU-55933 resulted in a synergistic decrease in cell viability (left), as shown by the Calcusyn analysis and plotted dose-effect curve (right). [Figure 27] Figure 27 is a bar graph showing that U937 cells treated with cysteine-reactive NOXA-15 R31E and KU-55933 alone or in combination exhibited significantly increased caspase-3 / 7 activation over time when treated with combination agents compared to monotherapy. [Figure 28]Figure 28 is a graph showing that co-treatment of BFL-1-independent MV4-11 cell lines resulted in little to no cytotoxic synergy with cysteine-reactive NOXA-15 R31E and ATM inhibitors. [Figure 29] Figure 29 is a bar graph showing that MV4-11 cells treated with a combination of cysteine-reactive NOXA-15 R31E and KU-55933 did not show increased caspase-3 / 7 activation compared to monotherapy. [Figure 30] Figure 30 is a table providing crystallization conditions, data collection, and refinement statistics for the crystal structure of apo BFL-1 and its complex with a cysteine-reactive NOXA SAHB inhibitor. [Figure 31A] Figure 31A provides the amino acid sequence of BFL-1ΔC (SEQ ID NO: 83), where the shaded α-helix corresponds to the structure shown in Figure 31B. The BH1-3 domain is underlined. [Figure 31B] Figure 31B shows the ribbon diagram and surface diagram of apo BFL-1ΔC (PDB identifier: 5WHI), illustrating the relative arrangement of BH3 bonded surface grooves formed by α-helices and α-helices 2-4. Surface-accessible cysteine ​​is observed within the grooves. [Figure 31C] Figure 31C shows a ribbon diagram overlay illustrating the similarities (top) and differences (bottom) between the α-helix orientations of the apo structures of BFL-1 (PDB identifier: 5WHI), MCL-1 (PDB identifier: 4WMS), and BCL-XL (PDB identifier: 1R2D). [Figure 31D] Figure 31D is a table comparing the α-carbon distance measurements between the top and bottom residues of the BH3 binding groove in BFL-1, MCL-1, and BCL-XL. [Figure 31E]Figure 31E shows an alignment of the amino acid sequences of the α2-α5 regions of BCL-2 family proteins, with shading corresponding to the structure of Figure 31D. BFL-1 is the only protein in which cysteine is located at the indicated position within the BH3 binding groove (see C of α3). The sequences are assigned SEQ ID NOs: 84-91 from top to bottom. [Figure 31F] Figure 31F shows a structural comparison of the BH3 binding grooves of BCL-2 family proteins, showing that only BFL-1 contains a surface-accessible cysteine residue. [Figure 32A] Figure 32A provides the results of an in vitro cross-linking analysis showing the selective derivatization of a BFL-1 construct having C55 with an i, i+7 stapled NOXA BH3 peptide (amino acids 26-40) containing an N-terminal cysteine-reactive D-nicotine acid (D-NA). *AT8LRRFGDXLNFRQ, where *=Ac or D-nicotine acid, 8=R-octenylalanine, and X=S-pentenylalanine. (SEQ ID NO: 92) [Figure 32B] Figure 32B provides intact mass spectrometry showing an increase in the molecular weight of BFL-1ΔC C4S / C19S due to a covalent reaction with D-NA-NOXA SAHB. [Figure 32C] Figure 32C shows the results of incubation of the indicated C-terminal biotinylated NOXA SAHB with a mixture of recombinant MCL-1, BCL-XL, and BFL-1 proteins, showing selective covalent modification of BFL-1 by D-NA-NOXA SAHB (lane 3). Streptavidin pull-down revealed no interaction between NOXA SAHB and BCL-XL and relatively minimal equivalent binding of MCL-1 (lanes 5-6). In contrast, D-NA-NOXA SAHB showed a strong BFL-1 pull-down as shown by silver staining, while Ac-NOXA SAHB did not (lane 6). [Figure 32D]Figure 32D shows a blot of lysate derived from A375P melanoma cells expressing BFL-1, obtained by subjecting cells to the same conditions as described in Figure 32C, followed by BFL-1 Western blotting of the electrophoretic eluate obtained by streptavidin pull-down. Incubation with D-NA-NOXA SAHB resulted in significantly enhanced BFL-1 pull-down compared to Ac-NOXA SAHB. [Figure 33A] Figure 33A shows the crystal structure of the D-NA-NOXA SAHB / BFL-1ΔC complex (PDB identifier: 5WHH), which exhibits complementary hydrophilic interactions between the amphiphilic BH3 helix and BFL-1, with i and i+7 staples observed in cis orientation and located on the non-interacting surface. [Figure 33B] Figure 33B shows a three-dimensional view of the BFL-1 / D-NA-NOXA SAHB covalent complex. [Figure 33C] Figure 33C shows the crystal structure of the D-NA-NOXA SAHB / BFL-1ΔC complex, which exhibits complementary hydrophobic interactions between the amphiphilic BH3 helix and BFL-1, with i and i+7 staples observed in cis orientation and located on the non-interacting surface. [Figure 33D] Figure 33D identifies the covalent bond between the C55 and D-NA moieties of BFL-1. [Figure 33E] Figure 33E shows the non-covalent NOXA BH3 (PDB identifier: 3MQP) complex with BFL-1 / A1. [Figure 33F] Figure 33F shows the covalent D-NA-NOXA SAHB complex with BFL-1. [Figure 33G] Figure 33G shows how the D-NA moiety makes auxiliary contact with the hydrophobic pore residue located below C55. [Figure 34A] Figure 34A shows an overlay of the apo structure of BFL-1 and a ribbon of the D-NA-NOXA SAHB-bound structure, illustrating the relative upward displacement of α2 and α3, and the downward displacement of α4, due to covalent ligand interactions. [Figure 34B]Figure 34B is a table comparing α-carbon distance measurements between the top and bottom residues of the grooves in the ligand-unbound and NOXA BH3-bound forms of BFL-1 and MCL-1. [Figure 34C] Figure 34C shows the localized changes evident in the corresponding distance difference matrix plot (see the black arrows). [Figure 34D] Figure 34D shows a plot of the α-carbon distance difference during covalent bonding of BFL-1 by D-NA-NOXA SAHB, quantitatively indicating the displacement of residues within the α2-α4 region of the BH3 binding groove. [Figure 34E] Figure 34E shows the apo morphology of BFL-1ΔC. [Figure 34F] Figure 34F shows that the ligand-binding configuration of BFL-1ΔC results in a larger interaction surface, including the formation of a separate hydrophobic pore tangent to C55 (see white arrow). [Figure 35A] Figure 35A shows a distance difference matrix plot of the non-covalent bond between NOXA and BH3 with BFL-1, revealing localized changes in the α2-α4 region similar to those observed with covalent BH3 targeting (see black arrows). [Figure 35B] Figure 35B shows a distance difference matrix plot of the non-covalent bond between mouse NOXA BH3 and MCL-1. Notably, it lacks the localized changes in the α2-α4 region observed in the interaction of NOXA BH3 with BFL-1 (both covalent and non-covalent). [Figure 36] Figure 36 shows the chemical structures of exemplary non-natural amino acids used to generate various types of staples (top). The middle panel illustrates peptides with staples of varying lengths. The bottom panel illustrates staple walks along the peptide sequence. [Figure 37] Figure 37 is a schematic diagram illustrating various types of double and triple stapling strategies along an exemplary staple walk. [Figure 38]Figure 38 is a schematic diagram showing an exemplary staple walk using branched double-staple sections of various lengths. [Figure 39] Figure 39 is a schematic diagram illustrating an exemplary chemical alternative method used to produce staple peptide derivatives. [Figure 40] Figure 40 is a graph showing that treatment of BFL-1-dependent U937 cells with a combination of cysteine-reactive NOXA-15 R31E and ATM inhibitor AZD0156 resulted in a synergistic decrease in cell viability (left), as shown by the Calcusyn analysis dose-effect plot (right). Left panel: For each concentration in the left panel, the bars (from left to right) are (i) D-NA-NOXA-15 R31E, (ii) AZD0156, and (iii) 8:1 D-NA-NOXA-15 R31E:AZD0156. Right panel: Each dot in the plot is classified as “strong synergy”. [Figure 41] Figure 41 is a bar graph showing that U937 cells treated with cysteine-reactive NOXA-15 R31E and AZD0156 alone and in combination exhibited significantly increased caspase-3 / 7 activation over time when treated with combination compared to monotherapy. At each time point, the bars (from left to right) represent (i) 40 μM D-NA-NOXA-15 R31E, (ii) 5 μM AZD0156, and (ii) 40 μM D-NA-NOXA-15 R31E + 5 μM AZD0156. [Figure 42]Figure 42 is a graph showing that co-treatment of BFL-1-independent MV4;11 cell lines resulted in little to no cytotoxic synergy between cysteine-reactive NOXA-15 R31E and ATM inhibitor AZD0156. Left panel: For each concentration in the left panel, the bars (from left to right) are (i) D-NA-NOXA-15 R31E, (ii) AZD0156, and (iii) 8:1 D-NA-NOXA-15 R31E:AZD0156. Right panel: The dots (from left to right) are (i) strong antagonistism, (ii) strong synergy, (iii) synergy, (iv) additive effect, (v) additive effect, and (vi) additive effect. [Figure 43] Figure 43 is a bar graph showing that MV4;11 cells treated with cysteine-reactive NOXA-15 R31E and AZD0156 alone and in combination did not show increased caspase-3 / 7 activation compared to monotherapy. For each time point, the bars (from left to right) represent (i) 40 μM D-NA-NOXA-15 R31E, (ii) 5 μM AZD0156, and (ii) 40 μM D-NA-NOXA-15 R31E + 5 μM AZD0156. [Figure 44] Figure 44 is a graph showing that treatment of BFL-1-dependent OCI-AML-3 cells with a combination of cysteine-reactive NOXA-15 R31E and ATM inhibitor AZD0156 resulted in a synergistic decrease in cell viability (left), as shown by the Calcusyn analysis dose-effect plot (right). Left panel: For each concentration in the left panel, the bars (from left to right) are (i) D-NA-NOXA-15 R31E, (ii) the indicated drug, and (iii) the NOXA-drug combination. Right panel: Each dot in the plot is classified as “strong synergistic effect”. [Figure 45]Figure 45 is a graph showing that treatment of BFL-1-dependent Jurkat cells with a combination of cysteine-reactive NOXA-15 R31E and ATM inhibitor AZD0156 resulted in a synergistic decrease in cell viability (left), as shown by the Calcusyn analysis dose-effect plot (right). Left panel: For each concentration in the left panel, the bars (from left to right) are (i) D-NA-NOXA-15 R31E, (ii) the indicated drug, and (iii) the NOXA:drug combination. Right panel: The dots (from left to right) are (i) strong antagonistism, (ii) antagonistism, (iii) strong synergy, (iv) strong synergy, (v) strong synergy, and (vi) strong synergy. [Figure 46] Figure 46 is a graph showing that treatment of BFL-1-dependent U937 cells with a combination of cysteine-reactive NOXA-15 R31E and ATM inhibitor Ku-60019 resulted in a synergistic decrease in cell viability (left), as shown by the Calcusyn analysis dose-effect plot (right). Left panel: For each concentration in the left panel, the bars (from left to right) are (i) D-NA-NOXA-15 R31E, (ii) the indicated drug, and (iii) the NOXA:drug combination. Right panel: The dots (from left to right) are (i) additive effect, (ii) strong synergistic effect, (iii) strong synergistic effect, (iv) strong synergistic effect, (v) strong synergistic effect, and (vi) strong synergistic effect. [Figure 47] Figure 47 is a graph showing that treatment of BFL-1-dependent U937 cells with a combination of cysteine-reactive NOXA-15 R31E and ATM inhibitor Ku-55933 resulted in a synergistic decrease in cell viability (left), as shown by the Calcusyn analysis dose-effect plot (right). Left panel: For each concentration in the left panel, the bars (from left to right) are (i) D-NA-NOXA-15 R31E, (ii) the indicated drug, and (iii) the NOXA:drug combination. Right panel: The dots (from left to right) are (i) strong antagonistism, (ii) strong synergy, (iii) strong synergy, (iv) strong synergy, and (v) strong synergy. [Figure 48] Figure 48 is a graph showing that co-treatment of the BFL-1-independent MV4;11 cell line produced little or no cytotoxic synergy between cysteine-reactive NOXA-15 R31E and the ATM inhibitor Ku-60019. Left panel: For each concentration in the left panel, the bars are (left to right), (i) D-NA-NOXA-15 R31E, (ii) the indicated drug, and (iii) NOXA: drug combinations. Right panel: The dots are (left to right), (i) additive effect, (ii) strong synergy, (iii) additive effect, (iv) synergy, (v) additive effect, and (vi) additive effect. [Figure 49] Figure 49 is a graph showing that co-treatment of the BFL-1-independent MV4;11 cell line produced little or no cytotoxic synergy between cysteine-reactive NOXA-15 R31E and the ATM inhibitor Ku-55933. Left panel: For each concentration in the left panel, the bars are (left to right), (i) D-NA-NOXA-15 R31E, (ii) the indicated drug, and (iii) NOXA: drug combinations. Right panel: The dots are (left to right), (i) synergy, (ii) synergy, (iii) additive effect, (iv) antagonism, (v) antagonism, and (vi) strong synergy. [Figure 50] Figure 50 is a graph showing that treatment of BFL-1-dependent U937 cells with the combination of cysteine-reactive NOXA-15 R31E and the CHK1 / 2 inhibitor AZD7762 caused a synergistic decrease in cell viability, as shown by the Calcusyn analysis dose-effect plot (right) (left). Left panel: For each concentration in the left panel, the bars are (left to right), (i) D-NA-NOXA-15 R31E, (ii) the indicated drug, and (iii) NOXA: drug combinations. Right panel: The dots are (left to right), (i) additive effect, (ii) antagonism, (iii) strong synergy, (iv) strong synergy, (v) strong synergy, and (vi) strong synergy. [Figure 51]Figure 51 is a graph showing that treatment of BFL-1-dependent U937 cells with a combination of cysteine-reactive NOXA-15 R31E and the PARP inhibitor olaparib resulted in a synergistic decrease in cell viability (left), as shown by the Calcusyn analysis dose-effect plot (right). Left panel: For each concentration in the left panel, the bars (from left to right) are (i) D-NA-NOXA-15 R31E, (ii) the indicated drug, and (iii) the NOXA:drug combination. Right panel: The dots (from left to right) are (i) strong antagonistism, (ii) strong synergy, (iii) strong synergy, (iv) strong synergy, (v) strong synergy, and (vi) synergy. [Figure 52] Figure 52 is a graph showing that co-treatment of BFL-1-independent MV4;11 cell lines resulted in little to no cytotoxic synergy between cysteine-reactive NOXA-15 R31E and the CHK1 / 2 inhibitor AZD7762. Left panel: For each concentration in the left panel, the bars (from left to right) represent (i) D-NA-NOXA-15 R31E, (ii) indicated drug, and (iii) NOXA:drug combinations. Right panel: The dots (from left to right) represent (i) strong synergy, (ii) synergy, (iii) additive effect, (iv) antagonistism, (v) antagonistism, and (vi) additive effect. [Figure 53] Figure 53 is a graph showing that co-treatment of BFL-1-independent MV4;11 cell lines did not result in any cytotoxic synergy between cysteine-reactive NOXA-15 R31E and the PARP inhibitor olaparib. Left panel: For each concentration in the left panel, the bars (from left to right) represent (i) D-NA-NOXA-15 R31E, (ii) the indicated drug, and (iii) the NOXA:drug combination. Right panel: Each of the six dots represents a strong antagonist. [Figure 54]Figure 54 shows the results for cell viability (A), Calcusyn dose-effect plot (B), and caspase-3 / 7 activation (C) of KG-1 cells treated with the indicated doses of D-NA-NOXA SAHB-15 R31E, the ATM inhibitor AZD0156, or a combination thereof. A: For each concentration, the bars (from left to right) represent (i) D-NA-NOXA-15 R31E, (ii) the indicated drug, and (iii) the NOXA:drug combination. B: The dots (from left to right) represent (i) strong antagonistism, and (ii)-(vi) additive effects. C: For each time point, the bars (from left to right) are (i) 40 μM D-NA-NOXA-15 R31E, (ii) 5 μM AZD0156, and (ii) 40 μM D-NA-NOXA-15 R31E + 5 μM AZD0156. [Figure 55] Figure 55 shows the cell viability of MV4;11 cells (A) and U937 cells (B) treated with the indicated doses of the selective MCL-1 inhibitor S63845, the ATM inhibitor AZD0156, or a combination thereof. For both panels, for each concentration, the bars (from left to right) are: (i) 0 nM S63845, (ii) 1.25 nM S63845, (iii) 2.5 nM S63845, (iv) 5 nM S63845, and (v) 10 nM S63845. [Figure 56]Figure 56 shows (A) the covalent reactivity of D-NA-NOXA SAHB-15 F32A and R31E peptides with recombinant BFL-1ΔC (upper) and full-length BFL-1 (lower), indicated by the shift in BFL-1 molecular weight (indicated by *) and detected by reduction and denaturation gel electrophoresis; (B) the covalent reactivity of D-NA-NOXA SAHB-15 F32A and R31E peptides with HA-BFL-1ΔC (upper) and full-length HA-BFL1 (lower) expressed in 293T cells, indicated by the shift in HA-BFL-1 molecular weight (indicated by *) after treatment with NOXA SAHB (20 μM) for 12 hours and detected by reduction and denaturation gel electrophoresis of the lysate and anti-HA Western analysis; and (C) the cysteine-reactive NOXA SAHB-15, evaluated by streptavidin pull-down and anti-BFL-1 Western analysis of U937 lysate. This demonstrates the targeting of native BFL-1 by R31E and NOXA SAHB-15 F32A peptides. Notably, native BFL-1 migrates at a higher molecular weight than recombinant or expressed BFL-1. [Figure 57] Figure 57 shows the loss of mitochondrial membrane potential in response to 24-hour treatment of U937 cells with the indicated doses of D-NA-NOXA SAHB-15 R31E and AZD0156, both as monotherapy and in combination. FCCP is a mitochondrial uncoupler that serves as a positive control for loss of mitochondrial membrane potential. [Figure 58] Figure 58 shows the effect of treatment of U937 cells with the indicated dose of AZD0156 for 2 hours on the phosphorylation status of ATM S1981 and CHK2 S33 / 35, as monitored by Western blotting. [Figure 59] Figure 59 shows the effect of AZD0156 treatment (5 μM) on BAX protein levels in U937 cells, as determined by BAX Western blotting of the prepared lysates at the indicated time points. [Figure 60]Figure 60 shows that treatment of U937 cells with the indicated doses of D-NA-NOXA SAHB-15 R31E for 30 minutes (A) and 4 hours (B) does not induce LDH release. [Figure 61] Figure 61 demonstrates the ability of a selective cysteine-reactive NOXA SAHB construct to induce undesirable nonspecific cytotoxicity via membrane perturbation. Sequence IDs 78 and 150-153 (top to bottom). [Figure 62] Figure 62 shows the BFL-1-dependent Pearson and Spearman correlations in AML cell lines, identified from the genome-scale CRISPR-Cas9 screen and based on the following criteria: (A) BFL-1-dependent Z-scores and BFL-1 scaling ranks across AML cell lines as assessed by the genome-scale CRISPR-Cas9 screen; (B) BFL-1-dependent Z-scores and BFL-1 scaling ranks of the top-scoring BFL-1-dependent human cancer cell lines assessed by the genome-scale CRISPR-Cas9 screen; and (C) BFL-1-dependent Pearson and Spearman correlations in AML cell lines identified from the genome-scale CRISPR-Cas9 screen and based on the following criteria: (1) the gene is scored as a potential dependency in at least two AML cell lines (score below -0.1); (2) the gene is expressed in at least two AML cell lines (TPM greater than 1); (3) the gene is not predicted to be a total lethal gene; and (4) both Pearson and Spearman correlations are greater than 0.9. [Modes for carrying out the invention]

[0072] Detailed explanation Currently, the target is apoptosis resistance activated by MCL-1 and BFL-1 / A1. To do so, the Food and Drug Administration has the following available: No drugs have been approved. In particular, there are no BFL-1 selective targeting agents, nor any BFL-1 / MCL-1 targeting agents. -1 Both dual-targeting agents and combinations of covalent and non-covalent dual inhibitors are moving to clinical trials. Progress is slow. This disclosure relates to a covalent BFL-1 selective targeting agent and to BFL-1. BFL-1 / MCL- can bind covalently to MCL-1 and noncovalently to MCL-1. 1. Exhibits the characteristics of a dual-targeting agent, with structural stabilization and cysteine ​​reactivity. NOXA BH3 The disclosure also provides such stabilized peptides, alone or In combination with other therapeutic agents (e.g., ATM kinase / ATR kinase inhibitors), BF L-1 and / or MCL-1-dependent cancers and / or cancers that express them (For example, hematopoietic malignancies, melanoma or other solid tumors, cytotoxic diseases, for example) Treatment of autoimmune diseases or inflammatory diseases, or other cancers as described below in this specification. The method for use in the setting is also a feature. Such staple peptides are MCL Cancers that express BFL-1 and / or BFL-1, and / or are dependent on them. Or, in cellular excess diseases, alone or in combination, to reactivate apoptosis It is useful.

[0073] protein The amino acid sequence of human MCL-1 is provided below (UniProtKB-Q0782) 0). [ka]

[0074] The amino acid sequence of human BFL-1 is provided below (UniProtKB-Q1654) 8). [ka]

[0075] The amino acid sequence of human NOXA is provided below (UniProtKB-Q13794) ). [ka]

[0076] NOXA peptide The exemplary NOXA peptide of this disclosure begins at amino acid 26 of SEQ ID NO: 82, This is a BH3 peptide up to 40 amino acids. The amino acid sequence of this peptide is: ATQLRRF It possesses GDKLNFRQ (SEQ ID NO: 39) and interacts with BFL-1 / A1. This pept The numbering used for Chido is provided below. [Table 1]

[0077] NOXA's BFL-1 interaction residue (i.e., NOXA that interacts with BFL-1) The residues of the BH3 alpha helix are Leu-21, Glu-22, Val-23, G lu-24, Cys-25, Ala-26, Leu-29, Arg-30, Phe-32 , Gly-33, Asp-34, Leu-36, Asn-37, and Gln-40 (The amino acid numbering is based on SEQ ID NO: 82). The position of SEQ ID NO: 39 provided above In relation to the numbering, the BFL-1 interacting residues are Ala-1, Leu-4, Arg- 5, Phe-7, Gly-8, Asp-9, Leu-11, Asn-12, and Gln It is -15. Alanine-1, leucine-4, phenylalanine-7, glycine-8, Leucine-11 is involved in hydrophobic interactions, while arginine-5 and asparagine are involved. Acid-9 and asparagine-12 are involved in hydrogen bonding interactions.

[0078] NOXA residues that do not interact with BFL-1 (i.e., NOXA BH3 alpha) The residues in the non-interacting side chains of LIXIL are Thr-27, Gln-28, Arg- These are 31, Lys-35, Phe-38, and Arg-39 (amino acid numbering is, (Based on Sequence ID 82). In relation to the position numbering of Sequence ID 39 provided above, N The residues not located on the interaction plane of the alpha-helix of the OXA peptide are residues Thr-2 and Gl. These are n-3, Arg-6, Lys-10, Phe-13, and Arg-14.

[0079] NOXA alpha helix MCL-1 interaction residue (i.e., interaction with MCL-1) The NOXA residues that act on the BH3 alpha-helix are Gln-28 and Leu-29. , Arg-30, Phe-32, Gly-33, Leu-36, Asn-37, Gln- The number is 40 (amino acid numbering is based on SEQ ID NO: 82).

[0080] NOXA residues that do not interact with MCL-1 (i.e., NOXA BH3 alpha) The helical residues in the non-interacting side chain of LIX are Ala-26, Thr-27, These are Arg-31, Asp-34, Lys-35, Phe-38, and Arg-39. (Amino acid numbering is based on Sequence ID No. 82).

[0081] In certain embodiments, the NOXA BH3 peptide of this disclosure is expressed in SEQ ID NO 39. Then, place 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acids. It may have a replacement (for example, 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, or (13 amino acids are substituted either conservatively or nonconservatively). In some cases, the sequence 1 to 3 amino acids in sequence number 39 are substituted. Amino acid substitution in sequence number 39 This is in one or both of the interacting and non-interacting side chains of the alpha helix. It may be. It does not interact with BFL-1 in the alpha helix of the NOXA peptide. In non-interacting side chains, higher variability is tolerated than in interacting side chains. In fact, this Almost all of these amino acids (for example, 5, 4, 3, 2, and the non-interacting surface of the helix) (or one amino acid) may be substituted (for example, a conservative or non-conservative amino acid) (mino acid substitution, or alanine). In certain embodiments, these peptides are helical The interaction surfaces of the s are 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, and 1 It has ~3, 1-2, or 1 amino acid substitutions. In some cases, the substitutions are conserved. This is a conserved amino acid substitution. In other cases, the substitution is a non-conservative amino acid substitution. In the example, if there is more than one amino acid substitution, the substitutions are considered conservative amino acid substitutions and This includes both non-conservative amino acid substitutions. In some cases, it involves one to three amino acids in SEQ ID NO: 39. If an acid (for example, 1, 2, or 3) is substituted, then all substitutions are to NOXA. It is located on the non-interaction surface of Lix. In some cases, 1 to 3 amino acids of SEQ ID NO: 39 ( For example, if 1, 2, or 3 are substituted, the substitution is that all NOXA helices It is located on the interaction surface of the cus. In some cases, 1 to 3 amino acids of SEQ ID NO: 39 (even If (1, 2, or 3) is substituted, the substitution is on the interaction surface of the NOXA helix. It is present on both the and non-interacting surfaces. In certain cases, the amino acid to be substituted is LA la, D-Ala, Aib, Sar, Ser, substituted alanine, or substituted glycine derivatives The group is selected from the following: A26, T27, Q28, R30, R 31, F32, G33, K35, L36, N37, F38, R39, or Q40 (number) One or more of the following (as indicated by sequence number 82) are replaced. In a particular case, The amino acids that are substituted are L-Ala, D-Ala, Aib, Sar, Ser, and the substituted amino acids. Selected from the group consisting of ranine or substituted glycine derivatives. In certain cases, Either L29 or D34 of column number 39 (numbering according to sequence number 82) or Neither is replaced. In some cases, L29 of sequence number 39 (numbering is sequence number 8) According to 2), isoleucine, phenylalanine, L-Ala, D-Ala, Aib, S It is substituted with ar, ser, substituted alanine, or substituted glycine derivatives.

[0082] In certain embodiments, the NOXA BH3 peptide of this disclosure is 1, 2, 3, 4, 5 Even if one amino acid is removed / deleted from the C-terminus of the sequence described in SEQ ID NO: 39 i. In certain embodiments, the NOXA BH3 peptide of this disclosure is 1, 2, 3, 4, Even if five amino acids are removed / deleted from the N-terminus of the sequence described in SEQ ID NO: 39 Good. In certain embodiments, the NOXA BH3 peptide of this disclosure is 1, 2, 3, 4 , 5 amino acids are removed from both the N-terminus and C-terminus of the sequence described in SEQ ID NO: 39. They may be removed / deleted. In certain cases, these removed amino acids are L- Ala, D-Ala, Aib, Sar, Ser, substituted alanine, or substituted glycine derivatives One to six (one, two, three, four, five, or six) amino acids selected from a group consisting of bodies and It can be replaced.

[0083] Non-exclusive examples of modifications to the sequence of sequence number 39 are provided below. [Table 2]

[0084] This disclosure also relates to Sequence ID No. 39, at least 14% (for example, at least 14 ~50%, at least 14-45%, at least 14-40%, at least 14-35% , at least 14-30%, at least 14-25%, at least 14-20%, less Both 20% to 50%, at least 20% to 45%, at least 20% to 40%, at least Also 20% to 35%, at least 20% to 30%, at least 20% to 25%, at least 15%, at least 20%, at least 27%, at least 34%, at least 40%, At least 45%, at least 50%, at least 55%, at least 60%, and 65%, at least 70%, at least 75%, at least 80%, at least 85% , at least 90%, at least 95%, at least 98%, at least 99%, or It also includes the NOXA peptide, which is 100% identical. Amino acid sequence in SEQ ID NO: 39 The variability is due to one or both of the interacting and non-interacting side chains of the alpha helix. It could be in the vicinity of the amino acids on the non-interacting surface of the NOXA BH3 helix. Everything is subject to change. The amino acids on the interaction surfaces of a helix are also subject to change.

[0085] In some embodiments, the present disclosure is characterized by the variant of Sequence ID No. 39, where NO The XA peptide variant binds to BFL-1 noncovalently. This disclosure also relates to sequence number Characterized by variant no. 39, in which the NOXA peptide variant is present in MCL-1. The bonds are non-covalent. In some cases, the Varian bond is used as shown in Sequence ID No. 39 described herein. The molecule binds to both BFL-1 and MCL-1 via non-covalent bonds.

[0086] In certain cases, the NOXA peptide has the amino acid sequence described below. [ka]

[0087] The NOXA peptides described herein can be optimized for therapeutic use. For example, one of the NOXA peptides mentioned above may trigger membrane disruption (cell lysis). When this occurs, this peptide reduces the overall hydrophobicity of the peptide, This can be optimized. For example, by using hydrophobic residues to have lower hydrophobicity. This can be achieved by substituting it with an amino acid (for example, alanine). Destruction can also be reduced by decreasing the overall positive charge of the peptide. This can be achieved by substituting basic residues with uncharged or acidic residues. This is possible. In certain cases, the overall hydrophobicity of the peptide and the overall Both positive charges are reduced.

[0088] In certain embodiments, the NOXA peptide described herein is 5-35 amino acids. It is acidic. In certain embodiments, the NOXA peptide described herein is 5- It is 25 amino acid length. In certain embodiments, the NOXA peptide described herein The length of the 'do' is 5 to 20 amino acids. In certain embodiments, NO as described herein The XA peptide is 5 to 18 amino acids long. In certain embodiments, as described herein The NOXA peptide is 10 to 35 amino acids long. In a particular embodiment, The NOXA peptide described in the specification is 10 to 25 amino acids long. Morphologically, the NOXA peptides described herein are 10 to 20 amino acid long. In certain embodiments, the NOXA peptide described herein comprises 10 to 18 amino acids. It is long. In certain embodiments, the NOXA peptide described herein is 15~ It has a length of 26 amino acids. In certain embodiments, the NOXA peptide described herein Each molecule is 15-18 amino acids long.

[0089] In certain cases, NOXA peptides are structurally stabilized. To stabilize peptides, they can be stapled and / or stitched. For example, peptides include the amino acids of NOXA peptide, and non-natural amino acids. This may include two or more substitutions.

[0090] Stabilized peptides Peptide helices regulate numerous important biological processes, such as apoptosis. It is a key mediator of protein-protein interactions, but Then, such helices are extracted from their environment within the protein and isolated. When prepared, it typically adopts a random coil three-dimensional structure, resulting in a dramatic reduction in biological activity. , and therefore a decrease in therapeutic capacity occurs. This disclosure relates to NOXA's structural stabilizing peptide. This disclosure provides a small number of connections formed by internal (intramolecular) crosslinking (or stapling). It contains at least two modified amino acids, and in certain cases, the structurally stabilizing peptide is It shares a cysteine ​​(Cys) residue within the target protein it binds to (e.g., BFL-1). A reactive group ("warhead") capable of forming a bonding bond, for example, an electrophilic group Structurally stabilized NOXA peptides (for example, those mentioned above) that have non-natural amino acids. ) contains. The stabilized peptides described herein include staple peptides and Stitch peptides, as well as multiple stitches, multiple staples, staples and stitches Peptides that are mixed with or contain other chemical strategies for structural enhancement (e.g.) , Balaram P. Cur. Opin. Struct. Biol. 1992;2:845, Kemp DS, et al., J. Am. Chem. Soc. 1996;118:4240, Orner BP, et al., J. Am. Chem. Soc. 2 001;123:5382, Chin JW, et al., Int. Ed. 2001;40:3806, Chapman RN, et al ., J. Am. Chem. Soc. 2004;126:12252, Horne WS, et al., Chem., Int. Ed. 2008;47:2853, Madden et al., Chem Commun (Camb). 2009 Oct 7; (37): 5588-5590, Lau et al., Chem. Soc. Rev., 2015, 44:91-102, and Gunnoo et al. Please refer to al., Org. Biomol. Chem., 2016, 14:8002-8013, all of which are: (The entire text is incorporated herein by reference.)

[0091] In certain embodiments, one of the NOXA polypeptides described herein Alternatively, multiple peptides can be stabilized by peptide stapling (for example, Wale Please refer to nsky, J. Med. Chem., 57:6275-6288 (2014), for its contents. (The whole is more fully incorporated herein). The peptide maintains its native secondary structure. It is "stabilized" in the sense that it holds. For example, stapling is α-helix 2 A polypeptide that has been modified to have the following structure has its native α-helix stereostructure This allows for maintenance. This secondary structure is resistant to protein degradation and heat. It can increase cytotoxicity, as well as increase target binding affinity, hydrophobicity, and cell permeability. Therefore, the stapled (crosslinked) polypeptides described herein are Compared to the corresponding non-stapled (non-crosslinked) polypeptide, improved raw It possesses physical activity.

[0092] "Peptide stapling" is a term derived from synthetic methodology, and refers to the stapling of polypeptides. Two olefin-containing side chains (e.g., crosslinkable side chains) present in the do-chain form ring-closing metacesium Covalent bonding (e.g., "stapled together") is achieved using the RCM reaction, and crosslinking is performed. It forms a ring (for example, Blackwell et al., J. Org. Chem., 66: 5291-5302, See Angew et al., Chem. Int. Ed. 37:3281, 1994 (2001). When used herein, the term "stapling of cytoside" refers to facilitating such a reaction. and any number of reaction conditions and / or contacts that provide a single "staple" polypeptide. Two (e.g., at least one pair) that may be present in a polypeptide chain using a medium ) Double bond-containing side chain, triple bond-containing side chain, or double bond-containing and triple bond-containing side chain Includes joining. The term "multiply staple" polypeptide refers to one Includes individual staples exceeding 2, 3, or more, at various intervals. This refers to polypeptides that may contain standing staples. When used herein, the term "compounding" refers to two staples, for example, a common residue. The "stitches" (e.g., in series or multiply staples) that connect to the polypept Multiple, serial "stapling" events in a single polypeptide chain that provide cytoplasm. This refers to the stitching of peptides, for example, WO2008 / 121767 and WO20 Disclosed in 10 / 068684, both of which are included herein by reference in their entirety. It is incorporated. In some cases, staples are unsaturated bonds, as used herein. It can be retained or reduced.

[0093] In certain embodiments, one or more polypeptides described herein may be used. It can be stabilized, for example, by hydrocarbon stapling. Some implementations Morphologically, staple peptides contain amino acids 26-40 of NOXA or similar. It consists of, or has 1 to 13 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9) (10, 11, 12, or 13 amino acid substitutions, deletions, and / or insertions) It is a polypeptide that includes. In certain cases, the staple peptide is at least It contains two (for example, 2, 3, 4, 5, or 6) amino acid substitutions, where the substituted amino acids Mino acids are separated by 2, 3, or 6 amino acids, and the substituted amino acids are olefins. It is a non-natural amino acid that has a side chain. Numerous known non-natural or non-natural amino acids Non-natural amino acids exist, and any of them can be included in the peptides of this disclosure. Some examples include 4-hydroxyproline, desmosine, gamma-aminobutyric acid, and beta-hydroxyproline. Anoalanine, norvaline, 4-(E)-butenyl-4(R)-methyl-N-methyl-L -Threonine, N-methyl-L-leucine, 1-aminocyclopropanecarboxylic acid, 1 -amino-2-phenyl-cyclopropanecarboxylic acid, 1-amino-cyclobutanecarboxylic acid nic acid, 4-amino-cyclopentenecarboxylic acid, 3-amino-cyclohexanecarboxylic acid , 4-piperidylacetic acid, 4-amino-l-methylpyrrole-2-carboxylic acid, 2,4-di Aminobutyric acid, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2-aminohepta 4-(aminomethyl)benzoic acid, 4-aminobenzoic acid, ortho, meta, and / Para-substituted phenylalanine (e.g., -C(=O)C6H5, -CF3, -CN, -H) (substituted with -NO2, CH3), disubstituted phenylalanine, substituted tyrosine ( For example, -C=O)C6H5, -CF3, -CN, -Halo, -NO2, CH3 and further... It is a statin (which has been converted). In addition, amino acids are hydroxylated, phosphate The derivatives include amino acid residues that have been decomposed, sulfonated, acylated, or glycosylated. It's okay if it's been transformed.

[0094] Hydrocarbon staple polypeptides have one or more tethers between two non-natural amino acids. Including the (linking) tether, this tether significantly enhances the α-helix secondary structure of the polypeptide. Generally, a tether consists of one or two spiral turns (i.e., about 3.4 or about 3.4 turns). It extends over the length of 7 amino acids. Therefore, i and i+3; i and The i+4th or i and i+7th amino acids undergo chemical modification and cross-linking. It is an ideal candidate for this. Therefore, for example, the peptide has the sequence...X1,X2 If X3, X4, X5, X6, X7, X8, X9... are present, then between X1 and X4, Alternatively, the crosslinking between X1 and X5, or between X1 and X8, is useful for the carbonization of the peptide. This is the basic stapling configuration, between X2 and X5, or between X2 and X6, or X2 The same applies to bridges between X9 and other points. Multiple bridges (for example, 2, 3, 4, or more) The use of crosslinks (exceeding ) is also being considered. The use of multiple crosslinks is beneficial for stabilizing and optimizing peptides. It is highly effective in this regard, especially with an increase in peptide length. Therefore, this disclosure relates to sequence Perhaps to further stabilize it, or to stabilize the structure of longer polypeptide elongations, protein Promotes enhanced resistance to degradation, acid stability, thermal stability, cell permeability, and / or biological activity. To achieve this, the polypeptide sequence includes the incorporation of more than one crosslink. Additional descriptions regarding the preparation and use of the single polypeptide can be found, for example, in the published U.S. patent application. Issues 2012 / 0172285, 2010 / 0286057, and 2005 This can be found in issue / 0250680, and all of this content is referenced in its entirety. This specification is incorporated herein.

[0095] In a particular embodiment, if the staples are at residues i and i+3, R-P Lopenylalanine and S-pentenylalanine; or R-pentenylalanine and S-pentenylalanine is substituted with amino acids at these positions. In this embodiment, when the staples are at residues i and i+4, S-pentenyl Lanine is substituted with amino acids at these positions. In a particular embodiment, If the staples are at the i and i+7th residues, then S-pentenylalanine and R - Octenylalanine is substituted with amino acids at these positions in some cases. So, if peptides are stitched together, what are the amino acids of the peptides involved in the "stitching"? The acids are bis-pentenylglycine, S-pentenylalanine, and R-octenylalanine. Nin; or bis-pentenylglycine, S-octenylalanine, and R-octenyl It is substituted with rualanine.

[0096] In a particular embodiment, the staples and / or stitches are 1st and 5th, 2 1st and 6th, 3rd and 7th, 6th and 10th, 10th and 14th, 11th and 15th, 12th and 16th, 1st and 8th, 3rd and 10th, 6th and 13th, They are made in positions 7 and 14, or 8 and 15, where the provided positions are distributed Column number 39 or a modified version of the above sequence number 39 (for example, sequence number 94 to 1 Based on any one of 09. The position of the staples differs in the staple walk. The location of the staples can be varied by testing them.

[0097] The upper panel of Figure 36 shows non-crosslinking compounds that can be used to generate various crosslinking compounds. Figure 36 shows exemplary chemical structures of natural amino acids. The central panel of Figure 36 shows the i-position and i+ position. It has hydrocarbon bridges between the residues at positions 3, i, and i+4, and between positions i and i+7. The peptide is illustrated. The lower panel of Figure 36 shows stapled walls along the peptide sequence. The diagram is illustrated. Figure 37 shows various peptides using double and triple stapling strategies. The arrangement and an exemplary staple walk are shown. Figure 38 shows branching staples of various lengths. An exemplary staple walk using the ch section is shown.

[0098] In one embodiment, the NOXA SAHB polypeptide has formula (I), [ka] During the ceremony, Each of R1 and R2 is independently H, or C1-C 10 Alkyl, Alkenyl , alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, Alternatively, it is a heterocyclylalkyl, R3 is alkyl, alkenyl, alkynyl, [R4-K-R4] n And these Each is replaced by 0 to 6 R5s. R4 is an alkyl, alkenyl, or alkynyl. R5 is a halo, alkyl, OR6, N(R6)2, SR6, SOR6, SO2R6, CO 2R6, R6, the fluorescent moiety, or a radioactive isotope, K is O, S, SO, SO2, CO, CO2, CONR6, or [ka] And, R6 is H, alkyl, or therapeutic agent. n is an integer between 1 and 4. x is an integer between 2 and 10. Each y is an independent integer between 0 and 100. z is an integer from 1 to 10 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) , Each Xaa is an amino acid independently. In some embodiments, the N-terminus of formula (I) End [Xaa] y It does not exist, A, AT, ATQLR (sequence number 110), ATQLR R (sequence code 111), ATQLRRF (sequence code 112), ATQLRRFGD (sequence code) Number 113), ATQLRRFGDK (Sequence ID 114), or ATQLRRFGDK L (sequence number 115). In some embodiments, [Xaa] x TQL, QLR, LRR, FGD, LNF, NFR, FRQ, TQLRRF (Sequence ID 116), LRRF GD (SEQ ID NO: 117), FGDKLN (SEQ ID NO: 118), GDKLNF (SEQ ID NO: 117) 19), or DKLNFR (Sequence ID 120). In some embodiments, formula (I) C-terminus [Xaa] y This is RFGDKLNFRQ (Sequence ID 121), FGDKLNFR Q (sequence number 122), GDKLNFRQ (sequence number 123), LNFRQ (sequence number 1 24) Q, DKLNFRQ (sequence number 125), or RQ. In certain cases The sequences described above and in sequence numbers 110-125 must be at least one (for example) may have amino acid substitutions or deletions of 1, 2, 3, 4, 5, or 6 amino acids. NOXA SAH The B polypeptide may comprise any amino acid sequence described herein.

[0099] The tether may include an alkyl, alkenyl, or alkynyl moiety (e.g., C5, C8, or or C 11 alkyl, C5, C8, or C 11 alkenyl, or C5, C8, or or C 11 alkynyl). The tethered amino acid can be alpha-disubstituted (e.g., C1-C3 or methyl).

[0100] In some cases, x is 2, 3, or 6. In some cases, each y is, independently, an integer from 1 to 15 or from 3 to 15. In some cases, R1 and R2 are each independently, H or C1-C6 alkyl. In some cases, R1 and R2 are each independently, C1-C3 alkyl. In some cases, at least one of R1 and R2 is methyl. For example, R1 and R2 can both be methyl. In some cases, R3 is alkyl (e.g., C8 alkyl) and x is 3 and x is 6. In some cases 11 alkyl and x is 6. In some cases R3 is alkenyl (e.g., C8 alkenyl) and x is 3. In some cases x is 6 and R3 is C 11 alkenyl. In some cases, R3 is a straight-chain al kyl, alkenyl, or alkynyl. In some cases, R3 is -CH2-CH2 -CH2-CH=CH-CH2-CH2-CH2-.

[0101] In another embodiment, the two alpha,alpha disubstitution stereocenters are both R-configured or S-configured. Is it in the configuration (for example, a bridge between i and i+4), or is one of the solid centers R and the other S is (for example, a bridge between i and i+7). Therefore, equation I is, [ka] It was shown as, C' and C'' disubstitution stereocenters can both be in the R configuration, or they can be For example, if x is 3, both can be in the S configuration. If x is 6, C' bi-substitution configuration. The body center is in an R configuration, and the C'' disubstituted stereocenter is in an S configuration. The R3 double bond is E Or it could be a Z-stereochemical configuration.

[0102] In some cases, R3 is [R4-K-R4] n R4 is a linear alkyl, aldehyde It is Kenil, or Alkinil.

[0103] In some embodiments, this disclosure refers to the amino acid sequence ATQLRRFGDKLNFRQ (sequence). Internally crosslinked ("stapled" or "stitched") peptides, including number 39. And, the side chains of two amino acids separated by 2, 3, or 6 amino acids are the internal st Either replaced by a chain, or the side chain of three amino acids is replaced by an internal stitch. Either they are replaced, or the side chains of four amino acids are replaced by two internal staples. Either the side chain of five amino acids is obtained, or the internal staple and internal stitch It is characterized by peptides that are replaced by combinations. In certain cases, the sequence The amino acids at position 4, 8, and 9 of number 39 are ste Neither pull nor stitch replaces it. In a specific case, position 4 of sequence number 39. and / or the amino acid at position 9 is replaced by either staple or stitch. No. Staple / stitch peptides are 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 4 The amino acid length is 2, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids. In one embodiment, the staple / stitch peptide is 15, 16, 17, 18, 19, 2 The amino acid length is 0, 21, 22, 23, 24, or 25. In a specific embodiment, Tablet / stitch peptides are 8, 9, 10, 11, 12, 13, 14, 15, 16, It is 17 or 18 amino acids long. An example NOXA staple peptide is shown in Figure 2. In certain embodiments, staple polypeptides are as shown in SEQ ID NOs: 2-21. It contains or consists of any one of the amino acid sequences listed in one of them. Morphologically, the NOXA staple peptide has the amino acid sequence described in SEQ ID NO. 16. In a particular embodiment, the staple polypeptide is one of the sequence numbers 22-34. It contains or consists of an amino acid sequence described in any one of the following: a certain implementation In terms of form, staple polypeptides are described in one of sequence numbers 35-38. It contains or consists of an amino acid sequence that is included. In a particular embodiment, staples A polypeptide is defined by the amino acid sequence described in one of sequence numbers 94-109. It contains or consists of, where at least 2, 3, or 6 amino acids are separated. Also, two amino acids have been modified to stabilize the peptide structure (for example, (These are replaced with unnatural amino acids that allow hydrocarbon stapling.)

[0104] Hydrocarbon tethers are common, but other tethers are also described herein. It can be used in NOXA BH3 peptide. For example, the tether is ether , thioether, ester, amine, or amide, or triazole moiety It may contain one or more. In some cases, naturally occurring amino acid side chains are used as tethers. It can be incorporated. For example, a tether can be a functional group, such as hydro in serine. Xyl, thiols in cysteine, primary amines in lysine, and aspartic acid. Or, the acid in glutamic acid, the amide in asparagine or glutamine, and It can be coupled. Therefore, two naturally occurring amino acids can be coupled. Instead of using tethers created by mixing, use naturally occurring amino acids. It is possible to create a tether using a single amino acid that does not exist naturally. It can also be used with naturally occurring amino acids. Triazole-containing (for example) Crosslinking (1,4-triazole or 1,5-triazole) can be used (for example) For example, Kawamoto et al. 2012 Journal of Medicinal Chemistry 55:1137, International (See Public Issue No. WO 2010 / 060112). In addition, different types of stays Other methods for performing pulling are well known in the art and are described herein. Can be used with OXA BH3 peptide (for example, lactam stem cells) Shepherd et al., J. Am. Chem. Soc., 127:2974-2983 (2005), UV-cyclization Additive stapling: Madden et al., Bioorg. Med. Chem. Lett., 21:1472-1475 (2011), Disulfide stapling: Jackson et al., Am. Chem. Soc., 113:93 91-9392 (1991), Oximes Strapping: Haney et al., Chem. Commun., 47:10 915-10917 (2011), Thioether Stapling: Brunel and Dawson, Chem. Comm un., 552-2554 (2005), Optically switchable stapling: JR Kumita et al. ., Proc. Natl. Acad. Sci. USA, 97:3803-3808 (2000), DoubleClick Stapling: Lau et al., Chem. Sci., 5:1804-1809 (2014), bis-lactam Stapling: JC Phelan et al., J. Am. Chem. Soc., 119:455-460 ( 1997), and bis-arylated stapling: AM Spokoyny et al., J. Am. See Chem. Soc., 135:5946-5949 (2013).

[0105] It is also conceivable that the tether length may change. For example, secondary alpha-helicopter When it is desirable to impose a relatively high degree of constraint on the hex structure, a short tether is used. It can be used, but in some cases it is restricted to secondary alpha-helix structures. The bundle should ideally be low, and therefore a long tether may be desired.

[0106] Furthermore, in order to give a tether mainly on one side of the alpha helix, amino acid i The total length of the tethers from i+3, i to i+4, and i to i+7 is the same. However, tethers can be synthesized to encompass any combination of the number of amino acids, and multiple It can also be used in combination with tethering.

[0107] In some cases, the hydrocarbon tethers (i.e., crosslinks) described herein are further It can be operated in one example, (for example, ring-closing metathesis using a ruthenium catalyst) Oxidizing the double bond of hydrocarbon alkenyl tethers (such as those synthesized using RCM), (For example, via epoxidation, aminohydroxylation, or dihydroxylation), the following We can provide one of the compounds. [ka]

[0108] Either the epoxide moiety or one of the free hydroxyl moieties is further regulated It can be activated. For example, epoxides can be used to bind to therapeutic agents. It can be treated with a nucleophile that gives additional functionality, or it can be treated with a nucleophile that gives additional functionality. Such derivatization is also achieved by synthetic operations of the amino or carboxyl terminus of polypeptides. This can be achieved via amino acid side chains. Other drugs, such as polypeptides, can enter cells. Drugs that promote penetration can be attached to functionalized tethers.

[0109] In some cases, alpha-disubstituted amino acids improve the stability of the alpha-helix secondary structure. It is used in polypeptides to improve them. However, alpha-disubstituted amino acids are essential. Instead, use mono-alpha substituents (for example, in tethered amino acids). This is also a possibility.

[0110] Staple polypeptides are drugs, toxins, and polyethylene glycol derivatives; secondly, poly Peptides; may contain carbohydrates, etc. Polymers or other agents may be stapled. When linked to peptides, it is sometimes desirable for the composition to be substantially homogeneous.

[0111] When polyethylene glycol (PEG) molecules are added, the pharmacokinetics of the polypeptide and Pharmacodynamic properties may be improved. For example, PEGylation can reduce renal clearance. This can lead to more stable plasma concentrations. PEG is a water-soluble polymer. And, Formula:XO--(CH2CH2O) n --CH2CH2-Y (In the formula, n is between 2 and 10,000, and X is H or terminal modification, e.g., C) 1~4 Al Kill; Y is the amine group of a polypeptide (but not limited to these, the lysine amine group) (It is an amide, carbamate, or urea linkage to a lonamine, or including the N-terminus.) It can be represented as being linked to the polypeptide. Y is also a thiol group ( This may also include maleimide linkage to a cysteine ​​thiol group (though not limited to these). Other methods for directly or indirectly linking PEG to polypeptides are available to those skilled in the art. If so, it is publicly known. PEG can be linear or branched. Various government Various forms of PEG containing active derivatives are commercially available.

[0112] PEGs having detachable links in their skeleton can be used. For example, PEG It can be prepared using ester links, which are the target of hydrolysis. Conjugates with PEG linkage capabilities are specified in WO99 / 34833 and WO99 / 1425. It is described in 9 and US6,348,558.

[0113] In certain embodiments, a polymer (e.g., PEG) is used via an intermediate linker. and is bound to the drugs described herein. In certain embodiments, a linker It is composed of 1 to 20 amino acids linked by peptide bonds (made (up of) The amino acids are selected from 20 naturally occurring amino acids. Some of the amino acids are glycosylated, as those skilled in the art will understand. In other embodiments, 1 to 20 amino acids may be glycine, alanine, proli A selection is made from phosphorus, asparagine, glutamine, and lysine. In other embodiments, phosphorus Car is composed of most sterically unhindered amino acids such as glycine and alanine. Non-peptide linkers are also possible. For example, alkyl linkers, e.g., -NH(CH 2) n C(O)- (wherein n=2~20) can be used. These alkyl groups The group is any sterically unhindered group, such as lower alkyl groups (e.g., C1-C6). Further substitutions with sil, halogens (e.g., Cl, Br), CN, NH2, phenyl, etc. It may be. U.S. Patent No. 5,446,090 specifies a bifunctional PEG linker, and each Its use in forming conjugates having peptides at the PEG linker terminus is described. It is being done.

[0114] Staple peptides are also, in some embodiments, further taken up intracellularly. It can be modified to promote or increase in vivo stability. For example, acylation or PEGylation of peptide-mimicking macrocyclic molecules enhances intracellular uptake. To promote, increase bioavailability, improve blood circulation, alter pharmacokinetics To reduce immunogenicity and / or the required frequency of administration.

[0115] In some embodiments, the staple peptides disclosed herein are (for example, It possesses an enhanced ability to penetrate the cell membrane (compared to non-staple peptides).

[0116] The method for synthesizing the stabilized peptides described herein is known in the art. Yes. Nevertheless, the following exemplary methods may be used. Various stats It is understood that the process may be carried out with an alternative sequence or order to obtain the desired compound. wax. Synthetic chemical transformations and useful in the synthesis of the compounds described herein. Methodologies for protecting functional groups (protection and deprotection) are publicly known in the art, for example R. Larock, Comprehensive Organic Transformations, VCH Publishers (198 9);TW Greene and PGM Wuts, Protective Groups in Organic Synthesis , 3d. Ed., John Wiley and Sons (1999);L. Fieser and M. Fieser, Fies er and Fieser's Reagents for Organic Synthesis, John Wiley and Sons ( 1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synth hesis, John Wiley and Sons (1995), and subsequent editions thereof This includes things that...

[0117] The peptides of the present invention can be prepared by chemical synthesis methods well known to those skilled in the art. For example, Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide See de, ed. Grant, WH Freeman & Co., New York, NY, 1992, p. 77. Please be illuminated. Therefore, peptides are, for example, in Applied Biosystems. For Peptide Synthesizer Model 430A or 431 And, using side-chain protected amino acids, either t-Boc or Fmoc chemistry Automated Merrifield technique for solid-phase synthesis using α-NH2 protected by It can be synthesized using the method.

[0118] One method for preparing the peptides described herein is solid-phase peptide synthesis (SPPS). The method involves using a linker molecule to break down the acid-unstable bond in the C-terminal amino acid. It is bonded to the cross-linked polystyrene resin via a solvent. This resin is not suitable for the solvent used in its synthesis. It is soluble, making it relatively simple and quick to wash away excess reagents and by-products. The terminals are protected by acid-stable Fmoc groups, but are removable by bases. Any side-chain functional group is protected by a group that is stable in a base and unstable in an acid.

[0119] Long peptides are synthesized using native chemical ligation to produce individual synthetic peptides. They may also be prepared by linking them together. Alternatively, long synthetic peptides can be prepared using well-known recombinant methods. It can be synthesized using DNA technology. Such technology includes detailed protocols. Provided as a well-known standard manual. The gene encoding the peptide of the present invention is constructed To build it, the amino acid sequence is back-translated, and preferably the gene is expressed in an organism that is optimal for that organism. Obtain a nucleic acid sequence that encodes an amino acid sequence containing a don. Next, the synthetic gene is typically... These are oligonucleotides that encode peptides and optional modifiers as needed. It is produced by synthesizing it. The synthetic gene is inserted into a suitable cloning vector. The peptide is then transfected into host cells. Subsequently, the peptide is expressed in a selected expression system and host cells. The peptides are primarily expressed under appropriate and suitable conditions. The peptides are purified and characterized by standard methods. Keru.

[0120] Peptides are available, for example, from Advanced Chemtech, and are high-through-protein. High-throughput setup using a multi-channel combinatorial synthesizer It can be prepared in combination form. Peptide bonds, for example, contribute to the physiological stability of peptides. To enhance this, retroinverso bond (C(O)-NH); reducing amide bond (NH-C H2); thiomethylene bond (S-CH2 or CH2-S); oxomethylene bond (O- CH2 or CH2-O); ethylene bond (CH2-CH2); thioamide bond (C(S) )-NH); trans-olefin bond (CH=CH); fluorosubstituted trans-olefin C(O)-CHR or CHR-C(O) )(wherein R is H or CH3); and fluoroketomethylene bond (C(O)- Replace with CFR or CFR-C(O) (where R is H, F, or CH3). It is possible.

[0121] Polypeptides can be acetylated, amidated, biotinylated, cinnamoylated, or farnesyl Formation, fluoresceination, formylation, myristoylation, palmitoylation, phosphorylation (Se By r, Tyr or Thr), stearoylation, succinylation and sulfurylation Further modifications are possible. As shown above, the peptide can be, for example, polyethylene. PEG (recall); alkyl groups (e.g., C1-C20 linear or branched alkyl groups) ); fatty acid radicals; and combinations thereof can be conjugated. Various α,α-disubstituted unnatural amino acids containing olefin side chains of a certain length can be synthesized by known methods. (Williams et al. J. Am. Chem. Soc., 113:9276, 1991; Schaf) meister et al., J. Am. Chem Soc., 122:5891, 2000; and Bird et al., Methods Enzymol., 446:369, 2008;Bird et al, Current Protocols in Chemi (Cal Biology, 2011). Regarding peptides, i units linked to the i+7th staple. When using the eye (helix 2 rotations are stabilized), a) one S5 amino acid and Use one R8 amino acid, or (b) use one S8 amino acid and one R5 amino acid. It is used. R8 is used except that the starting chiral additive yields an R-alkyl stereoisomer. It is synthesized using a similar pathway. Also, 8-iodoctene is used as a substitute for 5-iodopentene. It is used for this purpose. The inhibitor is synthesized using solid-phase peptide synthesis (SPPS) on MBHA resin. The material is synthesized on a solid support (see, for example, WO2010 / 148335).

[0122] Fmoc-protected α-amino acid (olefin amino acid Fmoc-S5-OH, Fm oc-R8-OH, Fmoc-R8-OH, Fmoc-S8-OH and Fmoc-R5 -Except for OH), 2-(6-chloro-1-H-benzotriazol-1-yl)-1,1, 3,3-tetramethylaminium hexafluorophosphate (HCTU), and Ri nk Amide MBHA is, for example, Novabiochem (San Diego It is commercially available from CA. Dimethylformamide (DMF), N-methyl-2-pyro Lydinone (NMP), N,N-diisopropylethylamine (DIEA), trifluoro Acetic acid (TFA), 1,2-dichloroethane (DCE), fluoroceine isothiocyanate (FITC) and piperidine are commercially available, for example, from Sigma-Aldrich. Olefin amino acid synthesis has been reported in the field of art (Williams). et al., Org. Synth., 80:31, 2003).

[0123] To obtain the stapled and purified peptides disclosed herein (e.g., Suitable methods for synthesis are also known in the art (e.g., Bird et al.) al., Methods in Enzymol., 446:369-386 (2008);Bird et al, Current Pr otocols in Chemical Biology, 2011; Walensky et al., Science, 305:1466-14 70 (2004);Schafmeister et al., J. Am. Chem. Soc., 122:5891-5892 (2000) ;U.S. Patent Application Publication No. 12 / 525,123, filed on March 18, 2010; and Please refer to U.S. Patent No. 7,723,468, registered on May 25, 2010. Each of these is incorporated herein by reference in its entirety.

[0124] In some embodiments, the peptide is substantially free of non-staple peptide contaminants. Alternatively, they are isolated. Methods for purifying peptides include, for example, solid-phase support. Peptide synthesis is performed on body. After cyclization, the solid support is isolated and dissolved in a solvent solution, such as DMS. O, may be suspended in a DMSO / dichloromethane mixture, or a DMSO / NMP mixture. . DMSO / dichloromethane or DMSO / NMP mixtures are approximately 30%, 40%, 5%. It may contain 0% or 60% DMSO. In certain embodiments, 50% / 50%. Use a DMSO / NMP solution. Incubate the solution for 1, 6, 12, or 24 hours. This may be done, and afterwards the resin may be washed with, for example, dichloromethane or NMP. In this embodiment, the resin is washed with NMP. Shaking and bubbling of inert gas into the solution. You may do so.

[0125] The properties of the staple (crosslinked) polypeptide of the present invention are, for example, described below. The assay can be performed using the method.

[0126] Assay for determining α-helix degree: The compound is placed in an aqueous solution (e.g., 5 ml at pH 7). Dissolve in potassium phosphate solution or distilled H2O to a concentration of 25-50 μM. Circularly polarized light. Dichroism (CD) spectra can be measured using a spectropolarimeter (e.g., Jasco J-710, Aviv). ) Standard measurement parameters (e.g., temperature, 20°C; wavelength, 190-260nm; st Top resolution, 0.5nm; speed, 20nm / sec; accumulation, 10; response, 1 sec; bandwidth, 1 nm; optical path length, 0.1 cm) obtained using each peptide. The α-helix content is determined by the average ellipticity of the residues relative to the decapeptide of the model helix. It is calculated by dividing by the reported value (Yang et al., Methods Enzymol. 130:2 08 (1986)).

[0127] Assay for determining the melting temperature (Tm): Crosslinked or unmodified template peptides are vaporized. Dissolve in H2O or other buffer or solvent (for example, at a final concentration of 50 μM), T m is the standard parameter for the change in ellipticity over a temperature range (e.g., 4 to 95°C). Turner (e.g., wavelength 222nm; step resolution, 0.5nm; speed, 20nm / sec; accu Ration: 10; Response: 1 second; Bandwidth: 1 nm; Temperature rise rate: 1°C / min; Optical path length, Measured using a spectropolarimeter with a 0.1 cm (e.g., Jasco J-710, Aviv) Determine by setting a standard.

[0128] In vitro protease resistance assay The amide bond in the peptide backbone is susceptible to hydrolysis by proteases. As a result, peptide compounds become vulnerable to rapid degradation in vivo. Typically, when a peptide helix is ​​formed, the amide skeleton is filled, and / or or twisted and / or shielded, as a result, the degradative cleavage of the protein is prevented. or substantially delayed. The peptide-mimicking macroring of the present invention is an in vitro enzyme protein It performs degradation of cereals (e.g., trypsin, chymotrypsin, pepsin) and controls the rate of degradation. Compared to comparable non-crosslinked or alternative staple polypeptides in terms of change of intent They may be evaluated as follows. For example, peptide-mimicking macrorings and comparable non-crosslinked polypeptides are The mixture was incubated with trypsin agarose and centrifuged at various time points. The reaction was quenched, then injected by HPLC, and the remaining residue was determined by UV absorption at 280 nm. The substrate is quantified. In short, the peptide mimetic macroring and peptide mimetic precursor (5 mc) are quantified. g) Trypsin agarose (Pierce) (S / E~125) and 0, 10, 20, 9 Incubate for 0 and 180 minutes. Quench the reaction rapidly by benchtop centrifugation. Then, the remaining substrate in the isolated supernatant was subjected to HPLC-based 280 nm pycnotomy. Quantification is performed by chromosome detection. The proteolytic reaction is a first-order reaction, and the rate constant k is time. This is determined from the plot of ln[S] for that value.

[0129] Peptide-mimicking macrocyclic and / or comparable non-crosslinked polypeptides are found in fresh mice, ra A smear and / or human serum (e.g., 1-2 mL) and, at 37°C, for example, 0, 1, 2 Macroring samples of different concentrations may be incubated for 4, 8, and 24 hours, respectively. Alternatively, it may be prepared by serial dilution using serum. The level of the compound remaining intact can be determined. To do this, the following procedure may be used: for example, 100 μL of serum in a 2 ml centrifuge tube. The sample is extracted by transferring the solution, followed by 10 μL of 50% formic acid and 50 μL of acetonitrile. Add 0 μL and centrifuge at 14,000 RPM at 4+ / -2°C for 10 minutes. Then, Transfer the clear liquid to a fresh 2 ml tube and steep in a Turbovap under N2 at less than 10 psi and 37°C. Evaporate. Reconstitute the sample in 100 μL of 50:50 acetonitrile:water and perform LC- Perform MS / MS analysis. (e.g., similar or equivalent procedures for testing vivo stability) This is a known method and may be used to determine the stability of macrorings in serum.

[0130] In vivo protease resistance assay: Stapling key peptides The point is that the in vitro resistance of the protease was clearly improved in vivo. This involves conversion to a pharmacokinetic state.

[0131] In vitro binding assay: A peptide mimetic macroring and peptide mimetic drug precursor To evaluate binding and affinity to the receptor protein, for example, fluorescence polarization FPA (Flat Panel Optics) can be used. FPA technology enables polarization and fluorescence trays. Molecular orientation and mobility are measured using SUR. When excited with polarized light, the apparent fraction It binds to molecules with a large molecular weight (for example, FITC-labeled peptides that bind to large proteins). The fluorescent tracer (e.g., FITC) is a small molecule (e.g., free in solution) The rotation speed is different compared to the fluorescent tracer bound to the FITC-labeled peptide. Because of its low molecular weight, it emits high levels of polarized fluorescence.

[0132] Stabilized NOXA peptide variant This disclosure also provides NOXA SAHB peptide variants. These variants The amino acid sequence described in Sequence ID No. 39 differs from that of one or more amino acids. In addition to amino acid substitutions, C-terminal and / or N-terminal deletions are also conceivable.

[0133] In some embodiments, the internally crosslinked peptide modifies the polypeptide of SEQ ID NO: 39. Such can be produced by (for example, by amino acid substitution). NOXA SAHB variant is compared to the peptide of SEQ ID NO. 39, or variant The stapled counterpart of the peptide (i.e., the peptide that does not have any variation from SEQ ID NO: 39). Compared to the stapled version, it has reduced hydrophobicity and / or overall positive charge. It may contain peptides. In some embodiments, the internal staples consist of two amino acid side chains and To replace, that is, each staple is replaced with, for example, 3, 4, or 6 amino acids. It is located between two separated amino acids. In some embodiments, the internal stitch is three The side chain of an amino acid is replaced, i.e., the stitch is, for example, 3 and 6 amino acid separations. It is a pair of crosslinks between three intervening amino acids. In some embodiments, internal Table and / or internal stitching includes at least two internal staples (four) It replaces the side chain of the amino acid, that is, each staple is, for example, 3-amino acid (Located between two amino acids that are separated by an acid). In some embodiments, internal staples A combination of an internal stitch and at least one internal staple is called an internal stitch. Includes. In some embodiments, the internal stitch contains a first amino acid as well as a second and second It replaces the side chain of the third amino acid, thereby replacing the first amino acid (the second amino acid and the third amino acid). The (between the 3rd amino acid) is crosslinked with the 2nd and 3rd amino acids by internal crosslinking. Here, the first and second amino acids are separated by 2, 3, or 6 amino acids, and the first And the third amino acid is separated by 2, 3, or 6 amino acids, and the second and third amino acids Mino acids are different amino acids. In some embodiments of the present disclosure, internally crosslinked polypeptides The side chains of the four amino acids in tide are replaced by two different internal staples. In some embodiments, the first of two different internal staples is 2, 3, and It bridges the first pair of 6-amino acid separated amino acids and at least two different internal stays The second of the pulls connects a second pair of amino acids separated by 2, 3, or 6 amino acids. Bridges. In some embodiments of the present disclosure, the internally crosslinked NOXA polypeptide variant is Prepared from the polypeptide of Sequence ID No. 39, for example, 1, 2, 3, 4, 5, 6, 7, It has 8 or 9 amino acid substitutions (for example, 1, 2, 3, 4, 5, 6, 7, 8, (or nine amino acids are substituted, either conservatively or nonconservatively). Example: NOXA Staple peptide variants are provided in Figures 6 and 10.

[0134] In some cases, the stabilized peptide is a helical of the stabilized NOXA peptide. The non-interacting surface of the NOXA BH3 helix (i.e., the NOXA BH3 helix that does not interact with BFL-1). (part of) 1 to 8 amino acid substitutions (for example, 1, 2, 3, 4, 5, 6, 7, or It may have 8. The “interaction surface” of the polypeptide described herein is BFL-1 Interacting with proteins (for example, specifically interacting with or specifically binding to them) ) Contains amino acid residues of an alpha helix. In certain cases, peptides are pepti On the interaction surface of the helix, there are 1-9, 1-8, 1-7, 1-6, and 1-5 elements. It has 1 to 4, 1 to 3, 1 to 2, or 1 amino substitution. In some cases, The amino acid at the interaction surface of any one of the peptides in row numbers 2-38 is changed to another amino acid. α-aminoisobutyric acid, for example, Ala or derivatives such as D-Ala, or α-aminoisobutyric acid It may be useful to replace it with. In certain embodiments, the amino acid substitution is conservative. This is an amino acid substitution. In some cases, the peptides described herein are one or more amino acids. Numbers (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-1 One of sequence numbers 2-38 having 1 or 1-12 conservative amino acid substitutions. It may include amino acids that are not on the interaction plane of the helix of the peptides described herein. Significant variability is acceptable. For example, these amino acids, if not all, are... Almost all of them may be substituted (for example, with conserved amino acids). "Substitution" refers to the process where one amino acid is replaced by another amino acid residue that has a similar side chain. This means replacement. A family of amino acid residues with similar side chains is considered a technical component. These are defined in the field. These families include amino acids with basic side chains. (For example, lysine, arginine, histidine), amino acids with acidic side chains (for example) , aspartic acid, glutamic acid), amino acids having non-charged side chains (e.g., glycan (Syn, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar Amino acids with a sexual side chain (e.g., alanine, valine, leucine, isoleucine, pro) (Phosphorus, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (For example, threonine, valine, isoleucine), and amino acids having aromatic side chains. (For example, tyrosine, phenylalanine, tryptophan, histidine) are some examples. .

[0135] In some cases, the peptides of this disclosure have slightly different amino acid sequences compared to SEQ ID NOs: 2-38. At least 14%, 15%, 20%, 27%, 34%, 40%, 47%, 50%, 53%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 97% It contains sequences with % identity. In certain cases, the variability in the amino acid sequence is It lies on the non-interacting surface of the helix. In some cases, variability in the amino acid sequence is due to the helix. It lies in the interaction surface of LIX. In certain cases, variability in the amino acid sequence is It exists on both the non-interacting and interacting surfaces of the Lix. In certain cases, A-1, T-2, Q-3, R-5, R-6, F-7, G-8, K-10, L-11, N-12, F -13, R-14, and Q-15 (numbering according to sequence number 39) Multiple substitutions are made. In some cases, the substitutions are L-alanine, D-alanine, and or α-aminoisobutyric acid. In some cases, C-terminus and / or N-terminus. One to five amino acids (for example, one, two, three, four, or five) are missing. A method for determining the percentage of identity between amino acid sequences is available in the art. It is publicly known. For example, sequences are aligned for the purpose of optimal comparison (for example, the most For proper alignment, the gap between the first and second amino acids or nucleic acid sequences (These may be introduced into one or both of these sequences, and non-homologous sequences may be ignored for comparison purposes.) In a preferred embodiment, the length of the reference array to be aligned for comparison is the length of the reference array. At least 30% of the length, preferably at least 40%, more preferably at least 50% %, more preferably at least 60%, even more preferably at least 70%, 80%, 90%, or 100%. Corresponding amino acid position or nucleotide position The amino acid residues or nucleotides in the first sequence are then compared. The position is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence. If this is the case, the molecules are identical at that position. Identity between two amino acid sequences. Percentage determination is achieved using the BLAST 2.0 program. Sequence comparison is , using gapless alignment, as well as the default parameters (Blo ssom 62 matrix, gap presence cost 11, gap cost per residue 1, This is done using a lambda ratio of 0.85. Used in the BLAST program. The mathematical algorithm described is by Altschul et al. (Nucleic Acids Res. 25:3389-3402) It is described in (1997).

[0136] Staple polypeptides are linked by internal intramolecular crosslinking (or "staples"). It contains at least two modified amino acids, where these at least two amino acids The acid is separated by 2, 3, or 6 amino acids. The stabilized peptides described herein. This includes staple peptides, which contain peptides having two staples, and / or s Contains tech peptide. At least two modified amino acids are non-natural alpha-A amino acids (including, but not limited to, α,α-disubstituted and N-alkylated amino acids) It may also be the case that there are many known non-natural amino acids, none of which are related to the present invention. It can be included in peptides. Some examples of unnatural amino acids are 4-hydroxypropyl Desmosine, gamma-aminobutyric acid, beta-cyanoalanine, norvaline, 4-(E )-Butenyl-4(R)-methyl-N-methyl-L-threonine, N-methyl-L-Ly Syn, 1-amino-cyclopropanecarboxylic acid, 1-amino-2-phenyl-cyclopropanecarboxylic acid Pancarboxylic acid, 1-amino-cyclobutanecarboxylic acid, 4-amino-cyclopentene carboxylic acid Rubonic acid, 3-amino-cyclohexanecarboxylic acid, 4-piperidylacetic acid, 4-amino- l-methylpyrrole-2-carboxylic acid, 2,4-diaminobutyric acid, 2,3-diaminopropyl Onic acid, 2,4-diaminobutyric acid, 2-aminoheptanediic acid, 4-(aminomethyl)benzoic acid Acids, 4-aminobenzoic acid, ortho, meta, and / or para substituted phenylalanine (for example) Substituted with -C(=O)C6H5, -CF3, -CN, -HALO, -NO2, CH3 (, disubstituted phenylalanine, substituted tyrosine (e.g., -Q=O)C6H5, -CF) 3, -CN, -halo, -NO2, CH3 (further substituted), and statins ru.

[0137] In a particular embodiment, the staple NOXA polypeptide variant is represented by Sequence ID No. 2 It contains or is derived from any one of the amino acid sequences listed in 2-34. (Without including the N-terminal nipecotinic acid).

[0138] In a particular embodiment, the staple NOXA polypeptide variant is SEQ ID NOXA 3 It contains or is derived from any one of the amino acid sequences listed in 5-38. (Without including the N-terminal nipecotinic acid).

[0139] In a particular embodiment, the staple NOXA polypeptide variant is sequence number 9 Structurally stabilized version of the amino acid sequence listed in any one of 4-109 It contains or consists of these. Non-limited examples of structural stabilization of these peptides include these By introducing unnatural amino acids at positions 2, 3, or 6 amino acid intervals in the sequence. This is achieved by hydrocarbon stapling.

[0140] In a particular embodiment, the staple polypeptide is [ka] It contains or consists of an amino acid sequence selected from the group comprising: Here, X1 and X2 are covalently bonded using a ring-closing metathesis (RCM) reaction. These are non-natural amino acids that can "staple together" to form cross-linked rings. be.

[0141] In some embodiments, the staple polypeptide variant is [ka] It contains or consists of an amino acid sequence selected from the group comprising: Here, "8" = R-octenylalanine and "X" = S-pentenylalanine. ru.

[0142] Stabilized NOXA peptide with warhead This disclosure includes a warhead, i.e., a reactive group such as an electrophilic unnatural amino acid. It is characterized by a stabilized NOXA peptide. Importantly, the NOXA peptide and B The interaction with FL-1 is between the electrophile and the cysteine ​​at position 55 of BFL-1. Because they are strengthened by covalent bonds in these peptides, Allows for significant variability in the amino acid sequence of the associated NOXA peptide (the warhead is (Higher than when it does not exist). Therefore, the BFL-1 interaction surface of the NOXA helix Even if substitutions and / or deletions exist in sequence number 39, including substitutions in the above, Peptides with these warheads are likely to be effective in binding to BFL-1. Furthermore, the presence of the warhead causes NOXA peptides (for example, among SEQ ID NOs. 39-59) to be affected. The size of any one of the following can be reduced in length (for example, from 15 to 14, 13, (To 12, 11, 10, 9, 8, 7, 6, or 5 amino acids).

[0143] Electrophiles can be introduced in relation to unnatural amino acids, as well as stabilized ( For example, as a chemical cap to the N-terminus or C-terminus of a cross-linked NOXA polypeptide. They can also be introduced. In some cases, electrophiles can be introduced into stabilized peptides. Stabilized peptides with such warheads interact with proteins It can form a covalent bond with at least some of the material. For example, NOXA peptides with a warhead can be covalently modified from BFL-1 (for example, (By covalently bonding with C55 of BFL-1), BFL-1 and BFL-1 are mutually bonded noncovalently. It may act upon and / or interact with MCL-1 in a non-covalent manner.

[0144] The warhead may be at the N-terminus, the C-terminus, or within the polypeptide sequence. It may also be present. In some cases, the warhead is an amino acid with a non-natural electrophile. In a particular embodiment, the warhead is a 3S-1-pyrrolidiamide at the end of the acrylamide. n-3-carboxylic acid, D-homoproline at the terminus of acrylamide, acrylamide L-homoproline at the terminal, isonipecotinic acid at the terminal of acrylamide, acrylic D-nipecotinic acid at the amide terminus, L-nipecotinic acid at the acrylamide terminus, D-proline at the terminal end of acrylamide, L-proline at the terminal end of acrylamide, Selected from the group consisting of trans-4-dimethylaminocrotonic acid and acrylic acid. .

[0145] In some embodiments, the electrophilic warhead is a cysteine-reactive D-nipecotinic acid moiety. In other embodiments, the electrophilic warhead is a cysteine-reactive portion.

[0146] In a particular embodiment, the warhead is (S)-1-acryloylpyrrolidine-3-cal Boxamide, 1-acrylopiperidine-4-carboxamide, (R)-1 acryloyl Piperidine-3-carboxamide, (S)-1-acryloylpiperidine-3-carboxamide Samide, (S)-1-acryloylpyrrolidine-2-carboxamide, (R)-1-Acryloylpyrrolidine-2-carboxamide Liloylpyrrolidine-2-carboxamide, (E)-4-(dimethylamino)buta-2- Non-natural electrophilic groups selected from the group consisting of enamide and acrylamide. It is an amino acid. In other embodiments, the warhead is not an amino acid. For example, an electrophile. The sex moiety and peptides are saturated nitrogen-containing heterocycles (aziridine, diaziridine, azetidine). Pyrrolidine, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, Thiazolidine, isothiazolidine, piperidine, piperazine, morpholine, thiomorphol (Azepane, or unsaturated nitrogen-containing heterocyclic compounds (azirine, diazirine, azeto, pyrrole) Imidazole, pyrazole, oxazole, isoxazole, thiazole, isothia They are linked via zole, pyridine, diazine, oxazine, thiazine, and azepine. Peptides and electrophiles also include substituted amino-functionalized rings (e.g., N-arylacrylics). Amides), for example, phenyl (aniline), or more complex bicyclic rings or These are polycyclic rings, such as naphthalene, anthracene, phenanthrene, indole, iso Indole, indoridine, quinolone, isoquinoline, quinoxaline, phthalidine, cina Zoline, purine, carbazole, indazole, benzimidazole, azaindole Therefore, they may be connected. In some embodiments, the electrophilic warhead is made of acrylic mesh. A carbonyl group, or more generally defined as an α,β-unsaturated carbonyl group, for example, α-Cyanocrylamide, propioamide, trans-4-dimethylamino-2-butyrate ¹namide, or trans-4-piperidinyl-2-buteneamide, or any other ion Acrylamide, or N-functionalized vinyl sulfonyl, alpha-fluoroacetyl, Alpha-chloroacetyl, alpha-bromoacetyl, and alpha-iodoacetyl or other electrophilic moieties. Electrophiles can be introduced in relation to unnatural amino acids. In addition, the N-terminus of the cross-linked (e.g., staple, stitch) polypeptide It can also be introduced as a chemical cap to the C-terminus.

[0147] In one embodiment, a NOXA peptide having a warhead that binds to BFL-1 is, SEQ ID NO: 94 It has an amino acid sequence described in any one of ~109. In another embodiment, BF NOXA peptides having a warhead that binds to L-1 are among the sequence numbers 94-109. Five or more of the sequences listed in any one of them (for example, 5, 6, 7, 8, 1) It has an amino acid sequence containing 0, 11, 12, 13, and 14 amino acids. In another embodiment, NOXA peptides with warheads that bind to BFL-1 are numbered SEQ ID NOs. 94-109. One to five deletions at the C-terminus or N-terminus of any one of the sequences described ( For example, it has an amino acid sequence containing 1, 2, 3, 4, or 5 amino acids. In some cases, electrophoresis The electrophilic warhead is located at the N-terminus of the peptide. In other cases, the electrophilic warhead is located within the peptide. It is located there.

[0148] In one embodiment, a NOXA peptide having a warhead that binds to BFL-1 is described below. It contains an array that is selected from the arrays listed. [ka]

[0149] In certain cases, the array having the above warhead is arbitrary and known in the art. The structure can be stabilized by the following method. For example, 2, 3, or 6 amino acid separation. Then, at least two amino acids (for example, 2, 3, 4, or 5) of the sequence are stapled. and / or can be replaced with non-natural amino acids that can form stitches. In some embodiments, the electrophilic warhead (J) is cysteine-reactive D-nipecotin This is the acid portion. In other embodiments, the electrophilic warhead (J) is a cysteine-reactive portion. ru.

[0150] In some embodiments, the staple polypeptide is [ka] It contains or consists of an amino acid sequence selected from the group comprising: Here, "X1" and "X2" are covalently bonded using a ring-closing metathesis (RCM) reaction. Non-natural axylomeres that can be joined together ("stapled together") to form a bridged ring. It is an amino acid, and "J" is either a non-natural electrophile-containing amino acid, or an amino acid. This is an electrophilic warhead presented in relation to the missing portion (the electrophile is a chemical cap). It can perform the function of). In certain embodiments, "J" is cysteine-reactive D-nipe This is the cotinic acid portion. In another specific embodiment, "J" is the cysteine-reactive portion. .

[0151] In some embodiments, staple polypeptides are listed above or in Sequence IDs 2-21. The amino acid sequence includes or consists of multiple residues on the non-interacting surface. For example, 1, 2, 3, 4, 5, 6) are Ala (alanine), D-Ala (D-alanine) N), Aib (α-aminoisobutyric acid), Sar (N-methylglycine), and Ser( A residue selected from the group consisting of serine, other substituted alanines, and glycine derivatives. They can be replaced. In addition, one or more of these residues (for example, 1, 2, 3, 4, 5, 6) can be added to the C-terminus of the peptide. In some cases, the above peptide 0 to 5 cydops (sequence number 60-75) or 0 to 5 cydops at the C-terminus of sequence number 2-21 The amino acids are Ala (alanine), D-Ala (D-alanine), and Aib (α-amino acid). Alanines substituted with other substituted alanines, including sobutyric acid, Sar (N-methylglycine), and Ser (serine). It is replaced by a residue selected from the group consisting of glycine derivatives, and glycine derivatives.

[0152] In some embodiments, the staple polypeptide is [ka] It contains or consists of an amino acid sequence selected from the group comprising: Here, "8" = R-octenylalanine and "X" = S-pentenylalanine. "J" = an amino acid containing a non-natural electrophile, or a part that is not an amino acid. The electrophilic warhead is presented in a series. In certain embodiments, "J" is a cysteine ​​antagonist. This is the responsive D-nipecotinic acid moiety. In another specific embodiment, "J" is the cysteine ​​reaction It is the sexual part.

[0153] In some embodiments, this disclosure is described in any one of SEQ ID NOs. 60 to 79. For the amino acid sequence, at least 14%, 15%, 20%, 27%, 34%, and 40%, 47%, 50%, 53%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, It features NOXA staple peptides with warheads that are 95% or 97% identical. Here, the modified peptide is covalently bound to BFL-1. Some embodiments Therefore, these modified peptides are also non-co-transferable to BFL-1 and / or MCL-1. They bind via bond. In some embodiments, these modified peptides exhibit amino acid variation. Reduced hydrophobicity and / or overall positive properties compared to the previous staple NOXA peptide. It has an electric charge. In some embodiments, hydrophobicity or positive charge independently determines the cell permeability. They are enhanced to optimize the sequence. In some embodiments, these modified peptides are sequence It has reduced hydrophobicity and / or an overall positive charge compared to peptide number 16.

[0154] In certain embodiments, this disclosure is described in any one of Sequence IDs 60-79. Compared to the amino acid sequence, 1 to 10 (for example, 1, 2, 3, 4, 5, 6, 7, 8) NOXA staple peptides having a bullet head with amino acid substitutions (9 or 10) It is characterized by a modified peptide, which is covalently bound to BFL-1. In some embodiments, these modified peptides also include BFL-1 and / or MC It binds to L-1 noncovalently. In some embodiments, these modified peptides are Reduced hydrophobicity and / or compared to the staple NOXA peptide before amino acid substitution. It has an overall positive charge. In some embodiments, these modified peptides have a sequence It has reduced hydrophobicity and / or an overall positive charge compared to peptide number 16. In some embodiments, hydrophobicity or positive charge is independently used to optimize cell permeability. It will be strengthened.

[0155] Stabilized N with exemplary stabilized NOXA peptide variant and warhead OXA peptide In a specific embodiment, the staple peptides number from 0 to 6 (i.e., 0, 1, 2, 3) Amino acid sequences AT having 4, 5, or 6 amino acid substitutions, insertions, and / or deletions. This includes 8LREFGDXLNFRQ (sequence number 46), where "8" = R-octenyl. It is alanine, and "X" = S-pentenylalanine. In certain embodiments, stay Plu peptides are described in the sections "NOXA Peptide" and "Stabilized Peptide" above. Further includes one or more of the modifications described. In another specific embodiment, the tape The peptide consists of the amino acid sequence AT8LREFGDXLNFRQ (SEQ ID NO: 46). Here, "8" = R-octenylalanine and "X" = S-pentenylalanine. Yes. In certain embodiments, sequences 0-6 of sequence number 46 are substituted with other amino acids. The number of amino acids (i.e., 0, 1, 2, 3, 4, 5, 6) is the helic of sequence number 46. It is located on the BFL-1 / A1 non-interaction surface of the s. In some embodiments, in SEQ ID NO: 46 0-3 amino acids are removed from the C-terminus, or removed, resulting in alanine, D-alanine. Nin, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine It is replaced by 1 to 6 amino acids from the group consisting of derivatives. In some embodiments, The 0-6 amino acids on the non-interacting surface of sequence number 46 are alanine, D-alanine, and α - Aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives It is substituted with an amino acid selected from the group consisting of the following. In some embodiments, SEQ ID NO: 4 The following amino acids A1, L4, R5, F7, G8, L11, N12, and Q15 of the 6th generation One or more of these are alpha-methylated or alpha-ethylated natural amines. It is substituted with an acid. In some embodiments, the following amino acids A1 and L4 of SEQ ID NO 46 One or more of R5, F7, G8, L11, N12, and Q15 are L-A Lanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine It is substituted with an amino acid selected from the group consisting of , and glycine derivatives. In this embodiment, among the following amino acids T2, R6, F13, and R14 of SEQ ID NO: One or more of these are alpha-methylated or alpha-ethylated natural amino acids. It is substituted with an acid. In certain embodiments, the following amino acids T2, R of SEQ ID NO: 46 6, F13, and R14, one or more of these are L-alanine, D-alanine, α - Aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives It is substituted with an amino acid selected from the group consisting of the following. In a particular embodiment, the sequence number The following amino acids in No. 46: A1, T2, R5, R6, F7, G8, L11, N12, F13 One or more of R14 and Q15 are modified with alpha-methyl or ethyl. It is modified, or L-alanine, D-alanine, α-aminoisobutyric acid, N-methyl A amino acid selected from the group consisting of glycine, serine, substituted alanine, and glycine derivatives. It is substituted with an amino acid. In certain cases, the sequence number is substituted with another amino acid. 46, 1-6 amino acids, are the BFL-1 / A1 interaction plane of the helix of SEQ ID NO: 46. It is located there. In other cases, the first to sixth amino acids of sequence number 46 are substituted by other amino acids. The ano acid is present on the BFL-1 / A1 non-interacting and interacting surfaces of the helix of SEQ ID NO: 46. Yes. In a particular embodiment, the first to sixth amino acids of sequence number 46 are L-alanine. D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and It is substituted with an amino acid selected from the group consisting of glycine derivatives.

[0156] In another specific embodiment, the staple peptides number from 0 to 6 (i.e., 0, 1, 2) Amino acid sequences having 3, 4, 5, or 6 amino acid substitutions, insertions, and / or deletions. Includes AT8LRRAGDXLNFRQ (sequence number 44), where "8" = R-octave It is nyalanine, and "X" = S-pentenylalanine. In certain embodiments, Tablet peptides are described in the sections "NOXA peptide" and "stabilized peptide" above. Further includes one or more of the modifications described in [the relevant section]. In another specific embodiment, [the relevant section] The single peptide is derived from the amino acid sequence AT8LRRAGDXLNFRQ (SEQ ID NO: 44). Here, "8" = R-octenylalanine and "X" = S-pentenylalanine In certain embodiments, the 0 of SEQ ID NO: 44 is substituted with another amino acid. ~6 amino acids (i.e., 0, 1, 2, 3, 4, 5, 6) are the helical molecule of sequence number 44. It is located on the BFL-1 / A1 non-interacting surface of the casing. In some embodiments, it is located at SEQ ID NO: 44. 0-3 amino acids are removed from the C-terminus, or removed, and alanine, D- Alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine It can be replaced by 1 to 6 amino acids from the group consisting of lysine derivatives. In some embodiments, The 0-6 amino acids on the non-interacting surface of sequence number 44 are alanine and D-alanine. α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives It is substituted with an amino acid selected from the group consisting of conductors. In some embodiments, the sequence number Of the following amino acids A1, L4, R5, G8, L11, N12, and Q15 of No. 44 One or more of these are alpha-methylated or alpha-ethylated natural amino acids. It is substituted with an acid. In some embodiments, the following amino acids A1 and L4 of SEQ ID NO: 44 One or more of R5, G8, L11, N12, and Q15 are L-alanine. D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and It is substituted with an amino acid selected from the group consisting of glycine derivatives. Some embodiments So, choose one of the following amino acids from sequence number 44: T2, R6, F13, and R14. The or multiple are substituted with alpha-methylated or alpha-ethylated natural amino acids. In a particular embodiment, the following amino acids T2, R6, F1 of SEQ ID NO: 44 3, and one or more of R14 are L-alanine, D-alanine, α-amino It consists of isobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives. It is substituted with an amino acid selected from the group. In a particular embodiment, SEQ ID NO: 44 The following amino acids: A1, T2, R5, R6, G8, L11, N12, F13, R14, and Is one or more of Q15 modified with alpha-methyl or ethyl? , or L-alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, celery Substituted with an amino acid selected from the group consisting of , substituted alanine, and glycine derivatives. In certain cases, 1 to 6 of sequence number 44 are substituted by other amino acids. The amino acid is located on the BFL-1 / A1 interaction plane of the helix of sequence number 44. In the example, amino acids 1-6 of sequence number 44, which are substituted by other amino acids, are sequence numbers. It is located on the BFL-1 / A1 non-interacting and interacting surfaces of helix 44. In this embodiment, the first to sixth amino acids of SEQ ID NO: 44 are L-alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives It is substituted with an amino acid selected from a group of molecules.

[0157] In another specific embodiment, the peptide is bound to BFL-1 as described herein. It is a NOXA peptide having a warhead. For example, the peptides of SEQ ID NOs. 46 and 44 mentioned above. Tides, or their variants, are unnatural electrophilic amino acids or amino acids. The electrophilic warhead presented in relation to the missing portion (the electrophile acts as a chemical cap) (Capable of performing the function) (For example, the cysteine-reactive moiety, cysteine-reactive D-nipecotin) It has been modified to include the acidic portion.

[0158] In a specific embodiment, the NOXA peptide having a warhead that binds to BFL-1 is 0 ~6 amino acid substitutions, insertions, and / or 6 amino acids (i.e., 0, 1, 2, 3, 4, 5, 6) or the sequence JATQLREFGDKLNFRQ (SEQ ID NO: 12) has a deletion (as described above). 8) comprising, where the peptide is structured by any method known in the art The stabilized electrophilic warhead (J) has a cysteine-reactive moiety (for example, D-2). This is the pecotic acid portion. In certain embodiments, the N has a warhead that binds to BFL-1. OXA peptides contain 0 to 6 amino acids (i.e., 0, 1, 2, 3, 4, 5, or 6 amino acids). Sequence JATX1LREFGDX2 having substitutions, insertions, and / or deletions (as described above) Includes LNFRQ (SEQ ID NO: 62), where "X1" and "X2" are ring-closed metaces. The rings are linked together by covalent bonds ("stapled together") using the RCM reaction, and the bridged rings are formed. It is a non-natural amino acid that can form a compound, and "J" is a non-natural electrophile-containing amino acid. Electrophilic warheads presented in relation to a portion that is either an acid or not an amino acid (electrophilic) The substantia can function as a chemical cap (for example, "J" is cis The theine-reactive moiety, for example, "J", is the cysteine-reactive D-nipecotinic acid moiety. (This is the case.) In another specific embodiment, a NOXA pepto having a warhead that binds to the BFL-1 Tide contains 0 to 6 amino acid substitutions and insertions (i.e., 0, 1, 2, 3, 4, 5, or 6). The sequence JAT8LREFGDXLNFRQ (distributed) has a deletion (as described above). This includes column number 78), where "8" = R-octenylalanine and "X" = S-pe It is lentenylalanine, and "J" = an unnatural electrophilic amino acid, or amine. This is an electrophilic warhead presented in relation to the non-acid portion (for example, "J" is cis). The theine-reactive moiety, for example, "J", is the cysteine-reactive D-nipecotinic acid moiety. In certain embodiments, a NOXA peptide having a warhead that binds to BFL-1 is present. (Sequence ID 62, 78, or 128) is the above-mentioned "NOXA peptide" and "stable Further includes one or more of the modifications described in the section on "modified peptides".

[0159] In a specific embodiment, the NOXA peptide having a warhead that binds to BFL-1 is 0 ~6 amino acid substitutions, insertions, and / or 6 amino acids (i.e., 0, 1, 2, 3, 4, 5, 6) or the sequence JATQLREFGDKLNFRQ (SEQ ID NO: 12) has a deletion (as described above). 8) consists of, where the peptide is composed by any method known in the art. The electrophilic warhead (J) is stabilized and has a cysteine-reactive portion (for example, D- This is the nipecotinic acid portion. In certain embodiments, it has a warhead that binds to BFL-1. NOXA peptide consists of 0 to 6 amino acids (i.e., 0, 1, 2, 3, 4, 5, or 6 amino acids). Sequence JATX1LREFGDX having substitutions, insertions, and / or deletions (as described above) It consists of 2LNFRQ (sequence number 62), where "X1" and "X2" are ring-closing meta Using the cesis (RCM) reaction, covalent bonds are formed ("stapled together") and then bridged. It is a non-natural amino acid that can form a ring, and "J" contains a non-natural electrophile. Electrophilic warheads presented in relation to amino acids or non-amino acid portions ( Electrophiles can function as chemical caps (for example, "J" is The cysteine-reactive moiety, for example, "J", is cysteine-reactive D-nipecotinic acid. (This is a part). In another specific embodiment, NOXA has a warhead coupled to the BFL-1. Peptides can have 0 to 6 amino acid substitutions or insertions (i.e., 0, 1, 2, 3, 4, 5, or 6 amino acids). Sequence JAT8LREFGDXLNFRQ having inclusions and / or deletions (as described above) (Sequence ID 78) consists of, where "8" = R-octenylalanine and "X" = Is it S-pentenylalanine, and is "J" an amino acid containing a non-natural electrophile? This is an electrophilic warhead presented in relation to a non-amino acid portion (for example, "J"). The cysteine-reactive moiety is, for example, "J" is cysteine-reactive D-nipecotin. (This is the acidic part.)

[0160] In another specific embodiment, the NOXA peptide having a warhead that binds to BFL-1 is , containing the sequence JATQLRRAGDKLNFRQ (SEQ ID NO: 126), where, peptide It is structurally stabilized by any method known in the art, and is an electrophilic remnant. The head (J) is the cysteine-reactive moiety (e.g., the D-nipecotinic acid moiety). In this embodiment, the NOXA peptide having a warhead that binds to BFL-1 is JATX1 Includes LRRAGDX2LNFRQ (Sequence ID 60), where "X1" and "X2" This is achieved by covalent bonding ("staple together") using a ring-closing metathesis (RCM) reaction. It is a non-natural amino acid that can form a cross-linked ring, and "J" is a non-natural amino acid. The electrophiles presented are those that are electrophilic amino acids, or are related to the non-amino acid portion. It is an electronic warhead (an electrophile can function as a chemical cap) (even For example, "J" is the cysteine-reactive part, and for example, "J" is the cysteine-reactive D -This is the nipecotinic acid portion. In another specific embodiment, the warhead that binds to BFL-1 is The NOXA peptide contained is sequence JAT8LRRAGDXLNFRQ (SEQ ID NO: 76) Including, here, "8" = R-octenylalanine and "X" = S-pentenylalanine "J" means that it is an amino acid containing a non-natural electrophile, or a part that is not an amino acid. The electrophilic warhead presented in relation to the minute (for example, "J" is cysteine ​​reactive) It is a part, for example, "J" is the cysteine-reactive D-nipecotinic acid moiety). In one embodiment, a NOXA peptide (SEQ ID NO: 6) having a warhead that binds to BFL-1 is used. 0 or 76 or 126) are the above "NOXA peptide" and "stabilized peptide". Further including one or more of the modifications described in the section on "Chido". Another specific implementation form In this state, the NOXA peptide having a warhead that binds to BFL-1 has the sequence JATQLRR It consists of AGDKLNFRQ (SEQ ID NO: 126), where the peptide is in the relevant technical field. The structure is stabilized by any known method, and the electrophilic warhead (J) is cis This is the thein-reactive moiety (for example, the D-nipecotinic acid moiety). In certain embodiments, B The NOXA peptide with a warhead that binds to FL-1 is JATX1LRRAGDX2L It consists of NFRQ (Sequence ID 60), where "X1" and "X2" are ring-closed metaces. The rings are linked together by covalent bonds ("stapled together") using the RCM reaction, and the bridged rings are formed. It is a non-natural amino acid that can form a compound, and "J" is a non-natural electrophile-containing amino acid. electrophilic warheads presented in relation to a portion that is either a amino acid or not an amino acid ( An electron body can function as a chemical cap. (For example, "J" is a s This is the cysteine-reactive part; for example, "J" is the cysteine-reactive D-nipecotinic acid part. (This is a minute.) In another specific embodiment, the NOXA has a warhead coupled to the BFL-1. Petit is composed of the sequence JAT8LRRAGDXLNFRQ (sequence number 76), where, "8" = R-octenylalanine, "X" = S-pentenylalanine, and "J " = Amino acids containing unnatural electrophiles, or related to a portion that is not an amino acid. The electrophilic warhead shown (for example, "J" is the cysteine-reactive part) For example, "J" is the cysteine-reactive D-nipecotinic acid moiety.

[0161] Treatment method This disclosure is intended to help prevent and / or treat cancer, autoimmune diseases, or inflammatory diseases. Stabilized peptides (or pharmaceutical compositions containing stabilized peptides) as described in the details. It is characterized by using one of the following methods: "to treat" or "to treat". When used herein, the term means to alleviate a disease or condition that the subject is suffering from. It refers to inhibiting or mitigating something.

[0162] The peptides described herein are used to treat human subjects with cancer expressing BFL-1. It may be useful for this purpose. The peptides described herein are also BFL-1 dependent. It may also be useful for treating human subjects having cancer. In certain embodiments, cancer is , solid tumor or humoral tumor. In certain embodiments, BFL-1-dependent cancer is It is melanoma, leukemia, lymphoma, or other hematopoietic malignancies or solid tumors. In certain embodiments, hematopoietic malignancies include acute myeloid leukemia, acute lymphoblastic leukemia, Chronic myeloid leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome, or multiple myeloma In certain embodiments, BFL-1-dependent cancers include breast cancer, autonomic ganglion cancer, and pancreatic cancer. Internal organ cancer, skin cancer, CNS cancer, hematopoietic or lymphatic system cancer, lung cancer, colorectal cancer, stomach cancer It is cancer, soft tissue sarcoma, or bone cancer. In certain cases, solid tumors are melanoma, breast cancer, etc. It is cancer, or lung cancer. In some embodiments, it expresses BFL-1 or has Dependent cells cause autoimmune diseases or other inflammatory conditions characteristic of cellular excess diseases. In certain cases, autoimmune diseases include autoimmune colitis, thyroiditis, arthritis, and kidney disease. It is inflammation, dermatitis, vasculitis, systemic lupus erythematosus, diabetes, or Sjögren's disease. In some cases, inflammatory diseases include asthma, psoriasis, inflammatory bowel disease, thyroiditis, arthritis, nephritis, It is dermatitis or vasculitis. In a specific embodiment, BFL-1-dependent cancer is acute Myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, bone It is myelodysplastic syndrome or multiple myeloma.

[0163] The peptides described herein are used to treat human subjects with cancer expressing MCL-1. It may be useful for that purpose. The peptides described herein are also MCL-1 dependent. It may also be useful for treating human subjects having MCL. In certain embodiments, -1-dependent cancers include melanoma, leukemia, lymphoma, or other hematopoietic malignancies or solid cancers. It is a type of tumor. In certain embodiments, hematopoietic malignancies include acute myeloid leukemia, acute leukemia, and acute leukemia. Lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome, and This is multiple myeloma. In certain cases, the solid tumors are melanoma, breast cancer, or lung cancer. In some embodiments, cells expressing or dependent on BFL-1 are It causes autoimmune diseases or other inflammatory conditions characteristic of cellular excess diseases. Examples of autoimmune diseases include autoimmune colitis, thyroiditis, arthritis, nephritis, dermatitis, and vascular diseases. It is inflammation, systemic lupus erythematosus, diabetes, or Sjögren's disease. In some cases, Inflammatory diseases include asthma, psoriasis, inflammatory bowel disease, thyroiditis, arthritis, nephritis, dermatitis, or pulse It is ductitis. In a specific embodiment, the MCL-1-dependent cancer is acute myeloid leukemia. ru.

[0164] The peptides described herein are used in cancers that co-express BFL-1 and MCL-1. It may be useful for treating human subjects. The peptides described herein are also B It may also be useful in treating human subjects with FL-1 and MCL-1-dependent cancers. In certain embodiments, BFL-1 and MCL-1-dependent cancers include melanoma and white leukoma. A hematopoietic disease, lymphoma, or other malignant tumor of the hematopoietic system or a solid tumor. A certain implementation In terms of conditions, hematopoietic malignancies include acute myeloid leukemia, acute lymphoblastic leukemia, and chronic myeloid leukemia. It is a blood disease, chronic lymphocytic leukemia, myelodysplastic syndrome, or multiple myeloma. In these cases, the solid tumor is melanoma, breast cancer, or lung cancer. In some embodiments, Cells expressing or dependent on BFL-1 exhibit autoimmunity characteristic of cellular hyperactivity diseases. It causes epidemic diseases or other inflammatory conditions. In certain cases, autoimmune diseases are caused by the autoimmune process itself. Immunogenic colitis, thyroiditis, arthritis, nephritis, dermatitis, vasculitis, systemic lupus erythematosus, glucose It is a urine disease or Sjögren's disease. In some cases, inflammatory diseases include asthma, psoriasis, and inflammation. These include venereal colitis, thyroiditis, arthritis, nephritis, dermatitis, or vasculitis. In specific embodiments... BFL-1 and MCL-1-dependent cancers are acute myeloid leukemias.

[0165] In a particular embodiment, for human subjects who require it, Sequence ID 16 and Sequence ID 16 are used. A peptide selected from the group consisting of 60 to 79 is administered. In a particular embodiment, The peptide of Sequence ID No. 62 is administered to human subjects who require it. In this scenario, the peptide of sequence number 60 is administered to human subjects who require it. In this embodiment, for human subjects who require it, Sequence IDs 16 and 60-79 are used. For an amino acid sequence selected from the group consisting of the following, at least 14%, 15%, 20%, and 2% are present. 7%, 34%, 40%, 47%, 50%, 53%, 60%, 65%, 70%, 75%, 8 Peptides that are 0%, 85%, 90%, 95%, or 97% identical are administered.

[0166] In a specific embodiment, the peptide AT8LREFGDX is administered to a human subject who requires it. LNFRQ (Sequence ID 46) is administered, and here, "8" = R-octenylalanine. Therefore, "X" = S-pentenylalanine. In specific embodiments, it is necessary In human subjects, the peptide AT8LRRAGDXLNFRQ (SEQ ID NO: 44) was administered, Here, "8" = R-octenylalanine and "X" = S-pentenylalanine. In specific embodiments, the BFL- described herein is used for human subjects who require it. A NOXA peptide having a warhead that binds to 1 is administered. In a specific embodiment, B The NOXA peptide with a warhead that binds to FL-1 has the sequence JATQLREFGDKL It has NFRQ (SEQ ID NO: 128), where the peptide is known in the art. The structure is stabilized by any method, and the electrophilic warhead (J) is cysteine ​​reactive. This is a portion (for example, the D-nipecotinic acid portion). In certain embodiments, it is linked to BFL-1. The NOXA peptide having a matching warhead has the sequence JATX1LREFGDX2LNFRQ (Sequence ID 62) is obtained, where "X1" and "X2" are ring-closed metathesis (RCM). ) The reaction is used to form a covalent bond ("staple together") to create a bridged ring. It is a non-natural amino acid that can do so, and "J" is a non-natural electrophile-containing amino acid. Or, an electrophilic warhead presented in relation to a non-amino acid portion (the electrophile is (It can function as a chemical cap) (for example, "J" is a cysteine ​​antagonist) This is the reactive part; for example, "J" is the cysteine-reactive D-nipecotinic acid moiety. In another specific embodiment, the NOXA peptide having a warhead that binds to BFL-1 is The sequence JAT8LREFGDXLNFRQ (sequence number 78) is present, where "8" = R- It is octenylalanine, "X" = S-pentenylalanine, and "J" = unnatural Electrophiles present in relation to electrophilic amino acids or non-amino acid portions. It is a cysteine-reactive warhead (for example, "J" is the cysteine-reactive part, for example, "J (This is the cysteine-reactive D-nipecotinic acid moiety). In another specific embodiment, B The NOXA peptide with a warhead that binds to FL-1 has the sequence JATQLRRAGDKL It has NFRQ (SEQ ID NO: 126), where the peptide is known in the art. The structure is stabilized by any method, and the electrophilic warhead (J) is cysteine ​​reactive. This is a portion (for example, the D-nipecotinic acid portion). In certain embodiments, it is linked to BFL-1. NOXA peptide having a warhead that combines with JATX1LRRAGDX2LNFRQ( It has column number 60), where "X1" and "X2" are ring-closed metathesis (RCM) reactions. Using a bond to form a covalent bond ("staple together") to create a bridged ring. It is a non-natural amino acid that can produce [something], and "J" is a non-natural electrophile-containing amino acid. , or electrophilic warheads presented in relation to non-amino acid portions (electrophiles are chemical (It can function as a target cap) (for example, "J" is cysteine ​​reactive It is a part; for example, "J" is the cysteine-reactive D-nipecotinic acid moiety. In a particular embodiment, the NOXA peptide having a warhead that binds to BFL-1 is sequence It has JAT8LRRAGDXLNFRQ (sequence number 76), where "8" = R-octave It is tenylalanine, "X" = S-pentenylalanine, and "J" = unnatural electrophile. Electrophilicity presented in relation to the part containing the amino acid or the part that is not an amino acid. The warhead (for example, "J" is the cysteine-reactive part, for example, "J" is (This is the cysteine-reactive D-nipecotinic acid moiety). Those skilled in the art will know the ring-closing metathesis reaction. Using a bond to form a covalent bond ("staple together") to create a bridged ring. For peptides containing two unnatural amino acids, the ring-closing metathesis reaction is performed. Before administering the ptide (i.e., stabilized peptide) to a subject requiring treatment, You will understand what is happening to peptides. In other words, two unnatural amino acids The crosslinked ring produced by the covalent bond is administered to the stabilized peptide. Before that, it would likely be present in the stabilized peptide.

[0167] In some cases, the NOXA stabilization disclosed herein is used for human subjects that require it. Peptides and inhibitors of DNA damage response pathway members are co-administered. Non-exclusive examples of members include ataxia vasodilator mutation (ATM) kinase and vasodilation. Sexual ataxia and rad3-related (ATR) kinase, checkpoint kinase 1 (CHK 1) Checkpoint kinase 2 (CHK2), and poly(ADP-ribose polymer) One example is the enzyme paralytic enzyme (PARP). In some cases, it is used in human subjects who require it. NOXA-stabilized peptides, ATM kinases, or ATR kinase inhibitors disclosed in this document However, they are administered as co-administrations. ATM and ATR kinase inhibitors are known in the art. (For example, Weber et al., Pharmacology & Therapeutics, 149:124-0138) See Awasthi et al., J Cell Sci 2015 128: 4255-4262 (2015). Non-exclusive examples of ATM inhibitors include KU-559403, KU-55933, KU- Examples include 60019, CP-466722, CGK733, and AZD0156. In some cases, the NOXA stabilized palladium disclosed herein is used for human subjects who require it. Butydore, checkpoint kinase 1 / checkpoint kinase 2 (CHK1 / 2) inhibitor The harmful agent is co-administered. CHK1 / 2 inhibitors are known in the art (for example) For example, Zabludoff et al., 2008; Anderson, VE et al. Cancer Res 71, 463-472, doi:10.1158 / 0008-5472.CAN-10-1252 (2011), Scagliotti, G. et al. I nvest New Drugs 34, 625-635, doi:10.1007 / s10637-016-0368-1 (2016), Hong, D. et al. J Clin Oncol 34, 1764-1771, doi:10.1200 / JCO.2015.64.5788 (201 6), Jobson, AG et al. Pharmacol 72, 876-884, doi:10.1124 / mol.107.0358 32 (2007), Smaill, JB et al. Eur J Med Chem 43, 1276-1296, doi:10 .1016 / j.ejmech.2007.07.016 (2008), Jackson, JR et al. Cancer Res 60, 566-572 (2000), Teng, M. et al. J Med Chem 50, 5253 (2007), Booth, See L. et al. 786-796 (2018). Non-limiting examples of CHK1 / 2 inhibitors and For example, AZD7762 (CHK1 / 2), CCT 241533 (CHK2), LY 2603618(CHK1), LY 2606368(CHK1 / 2), NSC 109 555 ditosylate (CHK2), PD 407824 (CHKE1), PF47736 (CHEK1), SB 218078(CHK1), TCS 2312(CHK1), o Examples include SRA737(CHK1). In some cases, the person who needs it Elephants are co-administered with the NOXA-stabilized peptide and PARP inhibitor disclosed herein. PARP inhibitors are known in the art (e.g., Menear et al., 2008, Penning, TD et al. J Med Chem 53, 3142-3153, doi:10.1021 / jm90 1775y (2010), Donawho, CK et al. Clin Cancer Res 13, 2728-2737, do i:10.1158 / 1078-0432.CCR-06-3039 (2007), Daniel, RA et al. Clin Cancer Res 15, 1241-1249, doi:10.1158 / 1078-0432.CCR-08-1095 (2009), Curtin, N. J. ea Clin Cancer Res 10, 881-889 (2004), Liu, X. et al. Clin Ca ncer Res 18, 510-523, doi:10.1158 / 1078-0432.CCR-11-1973 (2012), Brock, W. A. et al. Cancer Lett 205, 155-160, doi:10.1016 / j.canlet.2003.10.029 ( 2004), Bridges, BA et al. Oncotarget 5, 5076-5086 (2014), Laird, J. H. et al. Clin Cancer Res 24, 5143-5152, doi:10.1158 / 1078-0432.CCR-18- 0401 (2018), Oplustil O'Connor, L. et al. Cancer Res 76, 6084-6094, d oi:10.1158 / 0008-5472.CAN-15-3240 (2016), McGonigle, S. et al. Oncotarget 6 See 41307-41323 (2015). Non-exclusive examples of PARP inhibitors include Laparib, A-966492, Beriparib, Lucaparib, AG-14361, Iniparib INO-1001, Niraparib, Thalassoparib, AZD2461, 2X-121, BM Examples include N 673 (thalassoparib) and E7449.

[0168] In a particular embodiment, for human subjects that require it, sequence number 16, and sequence number 16, and sequence number 16. Peptides selected from the group consisting of numbers 60-79, 151, and 153, and D Inhibitors of NA damage response pathway members are administered. In certain embodiments, it is necessary For the target human subjects, Sequence ID 16, and Sequence IDs 60-79, 151, and 15 For amino acid sequences selected from the group consisting of 3, at least 14%, 15%, and 20% are present. 27%, 34%, 40%, 47%, 50%, 53%, 60%, 65%, 70%, 75%, Peptides that are 80%, 85%, 90%, 95%, or 97% identical, as well as DNA damage Inhibitors of the injury response pathway members are administered. In certain embodiments, this is required. For humans, sequence number 16, and sequence numbers 60-79, 151, and 153 A peptide selected from the group, as well as an ATM inhibitor or ATR kinase inhibitor, It is administered. In certain embodiments, it is administered to human subjects who require it, such as sequence number 16. Furthermore, amino acid combinations selected from the group consisting of SEQ ID NOs: 60-79, 151, and 153. For each column, at least 14%, 15%, 20%, 27%, 34%, 40%, 47%, 50% %, 53%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and These peptides are 97% identical, as are ATM kinase or ATR kinase inhibitors. It is administered. In certain embodiments, it is administered to human subjects who require it, such as sequence number 16. Furthermore, peptides selected from the group consisting of SEQ ID NOs. 60-79, 151, and 153, In addition, a CHK1 / 2 inhibitor or a PARP inhibitor is administered. In a particular embodiment So, for humans who need it, sequence number 16, and sequence numbers 60-79, 15 For amino acid sequences selected from the group consisting of 1 and 153, at least 14%, 1 5%, 20%, 27%, 34%, 40%, 47%, 50%, 53%, 60%, 65%, 7 Peptides that are 0%, 75%, 80%, 85%, 90%, 95%, or 97% identical, Then, a CHK1 / 2 inhibitor or PARP inhibitor is administered.

[0169] In certain embodiments, inhibitors of DNA damage response pathway members (e.g., ATM inhibitors) are used. Along with harmful agents (ATR kinase inhibitors, CHK1 / 2 inhibitors, or PARP inhibitors) The peptide co-administered to human subjects requiring this is AT8LREFGDXLNFRQ( This is sequence number 46), where "8" = R-octenylalanine and "X" = S- Pentenylalanine is used in specific embodiments to inhibit members of the DNA damage response pathway. Agents (e.g., ATM inhibitors, ATR kinase inhibitors, CHK1 / 2 inhibitors, or PA) The peptide co-administered to human subjects requiring it, along with the RP inhibitor, is AT8LR. RAGDXLNFRQ (sequence number 44), where "8" = R-octenylalani In a specific embodiment, DNA damage Inhibitors of response pathway members (e.g., ATM inhibitors, ATR kinase inhibitors, CHK1 It is co-administered to human subjects who require it, along with a 2 inhibitor or PARP inhibitor. The peptide is a NOXA peptide having a warhead that binds to BFL-1 as described herein. In a specific embodiment, a NOXA peptide having a warhead coupled to BFL-1 The sequence JATQLREFGDKLNFRQ (sequence number 128) is present, where pep The cydo is structurally stabilized by any method known in the art, and is electrophilic. The sexual warhead (J) is the cysteine-reactive portion (for example, the D-nipecotinic acid portion). In a particular embodiment, the NOXA peptide having a warhead that binds to BFL-1 is sequence J It has ATX1LREFGDX2LNFRQ (Sequence ID 62), where "X1" and "X2" is covalently bonded using ring-closing metathesis (RCM) reaction ("stay together"). "J" is a non-natural amino acid that can pull (and form a cross-linked ring). , presented in relation to non-natural electrophilic amino acids or non-amino acid portions. It is an electrophilic warhead (the electrophile can function as a chemical cap). (For example, "J" is the cysteine ​​reactive part, and for example, "J" is cysteine (This is the reactive D-nipecotinic acid moiety.) In another specific embodiment, it binds to BFL-1. The NOXA peptide with a warhead has the sequence JAT8LREFGDXLNFRQ (Sequence ID). 78) has, where "8" = R-octenylalanine and "X" = S-pentenyl It is rualanine, and "J" = an amino acid containing a non-natural electrophile, or an amino acid It is an electrophilic warhead presented in relation to the part that does not exist (for example, "J" is a system It is a cysteine-reactive part, for example, "J" is the cysteine-reactive D-nipecotinic acid part. (In another specific embodiment, a NOXA peptide having a warhead coupled to BFL-1 The sequence JATQLRRAGDKLNFRQ (sequence number 126) is present, where pep The cydo is structurally stabilized by any method known in the art, and is electrophilic. The sexual warhead (J) is the cysteine-reactive portion (for example, the D-nipecotinic acid portion). In certain embodiments, the NOXA peptide having a warhead that binds to BFL-1 is JAT X1LRRAGDX2LNFRQ (Sequence ID 60) has "X1" and "X 2) is a ring-closing metathesis (RCM) reaction in which the molecules are covalently bonded (stapled together). ") and can form a cross-linked ring, and "J" is a non-natural amino acid. Presented in relation to naturally occurring electrophilic amino acids or non-amino acid portions. It is an electrophilic warhead (the electrophile can function as a chemical cap) ( For example, "J" is the cysteine-reactive part, and for instance, "J" is the cysteine ​​reaction (This is the D-nipecotinic acid moiety). In another specific embodiment, a warhead that binds to BFL-1. NOXA peptide having the part is sequence JAT8LRRAGDXLNFRQ (SEQ ID NO: 76) ) has, where "8" = R-octenylalanine and "X" = S-pentenylalanine It is ranine, and "J" = an amino acid containing a non-natural electrophile, or not an amino acid. The electrophilic warhead presented in relation to the part (for example, "J" is cysteine ​​anti This is the reactive part; for example, "J" is the cysteine-reactive D-nipecotinic acid moiety. Those skilled in the art can use ring-closing metathesis reactions to bond covalently ("staple together"). A peptide containing two unnatural amino acids that can form a cross-linked ring. Regarding this, the ring-closing metathesis reaction processes the peptide (i.e., the stabilized peptide) Before administering it to the target population that requires it, it will be important to understand what is being done to the peptide. In other words, the cross-linked ring produced by the covalent bond between two unnatural amino acids Before administering the stabilized peptide to the target, the substances present in the stabilized peptide are It is likely.

[0170] In some cases, it is necessary for human subjects (e.g., BFL-1 and MCL-1) In human subjects who express both or have cancers dependent on them, the information disclosed herein applies to... NOXA-stabilized peptides and MCL-1 inhibitors are co-administered. This is publicly known in the relevant art (for example, clinical trial identification number NCT029793 66, Kotschy et al. Nature, 538(7626):477-482 (2016), Ayaz et al. ACS Chem Biol, 11(6):1710-1719 (2016), Clinical Trial Identification Number NCT02992483, Clinical Floor test identification number NCT03465540, Caenepeel et al., Proceedings: AACR Ann See ual Meeting 2017, DOI: 10.1158 / 1538-7445.AM2017-2027). MCL -1 Non-exclusive examples of inhibitors include S64315, S63845, and BAY 10003. Examples include the 94, MIK665, AMG397, and AMG176. In terms of form, for human subjects who require it, Sequence ID 16, and Sequence IDs 60-79, Peptides selected from the group consisting of 151 and 153, as well as MCL-1 inhibitors It is administered. In certain embodiments, it is administered to human subjects who require it, such as sequence number 16. Furthermore, amino acid combinations selected from the group consisting of SEQ ID NOs: 60-79, 151, and 153. For each column, at least 14%, 15%, 20%, 27%, 34%, 40%, 47%, 50% %, 53%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and The peptides, which are 97% identical, and the MCL-1 inhibitor are administered. Morphologically, it is a peptide co-administered with an MCL-1 inhibitor to human subjects who require it. This is AT8LREFGDXLNFRQ (sequence number 46), where "8" = R- It is octenylalanine, and "X" = S-pentenylalanine. Specific embodiment So, what peptides are co-administered with MCL-1 inhibitors to human subjects who require them? AT8LRRAGDXLNFRQ (sequence number 44), where "8" = R - octave It is tenylalanine, and "X" = S-pentenylalanine. In a specific embodiment, The peptide co-administered with the MCL-1 inhibitor to human subjects who require it is, This is a NOXA peptide having a warhead that binds to BFL-1 as described in the detailed document. In one embodiment, the NOXA peptide having a warhead that binds to BFL-1 has the sequence JAT The sequence QLREFGDKLNFRQ (Sequence ID 128) is present, where the peptide is the technology The structure is stabilized by any method known in the field, and the electrophilic warhead (J) is , which is the cysteine-reactive moiety (for example, the D-nipecotinic acid moiety). In certain embodiments The NOXA peptide having a warhead that binds to BFL-1 has the sequence JATX1LREF GDX2LNFRQ (Sequence ID 62) is present, where "X1" and "X2" are ring closing elements. Using the metathesis (RCM) reaction, they are joined by covalent bonds ("stapled together"), It is a non-natural amino acid that can form a bridged ring, and "J" is a non-natural electrophile. electrophilic receptacles presented in relation to body-containing amino acids or non-amino acid portions. The head (where the electrophile can function as a chemical cap) (for example, "J " is the cysteine-reactive moiety, for example, "J" is the cysteine-reactive D-nipeko (This is the tinic acid portion). In another specific embodiment, the warhead has a BFL-1 binding N The OXA peptide has the sequence JAT8LREFGDXLNFRQ (SEQ ID NO: 78), Here, "8" = R-octenylalanine and "X" = S-pentenylalanine. "J" = an amino acid containing a non-natural electrophile, or related to a non-amino acid portion. The electrophilic warhead presented is (for example, "J" is the cysteine-reactive part) For example, "J" is the cysteine-reactive D-nipecotinic acid moiety. In one embodiment, the NOXA peptide having a warhead that binds to BFL-1 has the sequence JAT The peptide has QLRRAGDKLNFRQ (SEQ ID NO: 126), where the peptide is the technology The structure is stabilized by any method known in the field, and the electrophilic warhead (J) is , which is the cysteine-reactive moiety (for example, the D-nipecotinic acid moiety). In certain embodiments The NOXA peptide having a warhead that binds to BFL-1 is JATX1LRRAGD X2LNFRQ (Sequence ID 60) is present, where "X1" and "X2" are ring-closing meta Using the cesis (RCM) reaction, covalent bonds are formed ("stapled together") and then bridged. It is a non-natural amino acid that can form a ring, and "J" contains a non-natural electrophile. electrophilic warheads presented in relation to amino acid-containing or non-amino acid portions (An electrophile can function as a chemical cap) (For example, "J" is This is the cysteine-reactive moiety; for example, "J" is the cysteine-reactive D-nipecotin (This is the acid portion). In another specific embodiment, NOX has a warhead that binds to BFL-1. Peptide A has the sequence JAT8LRRAGDXLNFRQ (SEQ ID NO: 76), and here "8" = R-octenylalanine, "X" = S-pentenylalanine, "J" = an amino acid containing a non-natural electrophile, or related to a non-amino acid portion. The electrophilic warhead presented is (for example, "J" is the cysteine-reactive part) (For example, "J" is the cysteine-reactive D-nipecotinic acid moiety). Then, using a ring-closing metathesis reaction, they are joined by covalent bonds ("stapled together") and cross-linked. For peptides containing two unnatural amino acids that can form a ring, ring closure Metathesis is a reaction that requires the processing of a peptide (i.e., a stabilized peptide). Before administering it to the target, you will understand what is happening to the peptide. In other words, 2 A cross-linked ring produced by the covalent bonding of two unnatural amino acids is stabilized. It is likely that the stabilized peptide will be present before the peptide is administered to the target.

[0171] In some cases, it is necessary for human subjects (e.g., BFL-1 and BCL-2) In human subjects who express both or have cancers dependent on them, the information disclosed herein applies to... NOXA-stabilized peptides and BCL-2 inhibitors are co-administered. This is publicly known in the art (for example, Roberts et al. N Engl J Med See 374(4):311-322 (2016). An example of a BCL-2 inhibitor is venetocrac. It is. In a particular embodiment, for a human subject that requires it, Sequence ID 16, Furthermore, peptides selected from the group consisting of SEQ ID NOs. 60-79, 151, and 153, Then a BCL-2 inhibitor is administered. In certain embodiments, humans who require it are given. The target group consists of sequence number 16, and sequence numbers 60-79, 151, and 153. For amino acid sequences selected from the above, at least 14%, 15%, 20%, 27%, and 34%. %, 40%, 47%, 50%, 53%, 60%, 65%, 70%, 75%, 80%, 85 Peptides that are 90%, 95%, or 97% identical, as well as BCL-2 inhibitors, are administered It is administered. In certain embodiments, it is administered along with a BCL-2 inhibitor to a human who requires it. The peptide co-administered to elephants is AT8LREFGDXLNFRQ (SEQ ID NO: 46). Here, "8" = R-octenylalanine and "X" = S-pentenylalanine In a specific embodiment, a BCL-2 inhibitor is administered to a human subject who requires it. The peptide to be co-administered is AT8LRRAGDXLNFRQ (SEQ ID NO: 44), Here, "8" = R-octenylalanine and "X" = S-pentenylalanine. In specific embodiments, it is administered together with a BCL-2 inhibitor to human subjects who require it. The peptide provided is NOX having a warhead that binds to BFL-1 as described herein. It is peptide A. In a specific embodiment, NOX has a warhead that binds to BFL-1. Peptide A has the sequence JATQLREFGDKLNFRQ (SEQ ID NO: 128), and here The peptide is then structurally stabilized by any method known in the art. The electrophilic warhead (J) is composed of a cysteine-reactive moiety (for example, the D-nipecotinic acid moiety). In a particular embodiment, the NOXA peptide having a warhead that binds to BFL-1 is , has the sequence JATX1LREFGDX2LNFRQ (sequence number 62), where "X1 " and "X2" are covalently bonded using a ring-closing metathesis (RCM) reaction ("together"). It is a non-natural amino acid that can be stapled to form a cross-linked ring. "J" is either a non-natural electrophilic amino acid or related to a non-amino acid portion. The electrophilic warhead presented (where the electrophile can function as a chemical cap) ) (for example, "J" is the cysteine ​​reactive part, for example, "J" is cysteine (This is the stain-reactive D-nipecotinic acid moiety.) In another specific embodiment, BFL-1 NOXA peptides having a binding warhead have the sequence JAT8LREFGDXLNFRQ( It has sequence number 78), where "8" = R-octenylalanine and "X" = S- It is pentenylalanine, and "J" = an unnatural electrophilic amino acid, or A This is an electrophilic warhead presented in relation to the non-mino acid portion (for example, "J" is, This is the cysteine-reactive moiety; for example, "J" is cysteine-reactive D-nipecotinic acid. (This is a part). In another specific embodiment, NOX has a warhead coupled to the BFL-1. Peptide A has the sequence JATQLRRAGDKLNFRQ (SEQ ID NO: 126), and here The peptide is then structurally stabilized by any method known in the art. The electrophilic warhead (J) is composed of a cysteine-reactive moiety (for example, the D-nipecotinic acid moiety). In a particular embodiment, the NOXA peptide having a warhead that binds to BFL-1 is , has JATX1LRRAGDX2LNFRQ (Sequence ID 60), where "X1" "X2" is covalently bonded using a ring-closing metathesis (RCM) reaction. It is a non-natural amino acid that can form a cross-linked ring (table), and is "J " is either a non-natural electrophile-containing amino acid, or related to a non-amino acid portion. In the presented electrophilic warhead (where the electrophile can function as a chemical cap): (For example, "J" is a cysteine ​​reactive part, and for example, "J" is cysteine (This is the in-reactive D-nipecotinic acid moiety.) In another specific embodiment, it binds to BFL-1. NOXA peptide having a warhead has the sequence JAT8LRRAGDXLNFRQ (sequence) Number 76) is such that "8" = R-octenylalanine and "X" = S-pentene It is tenylalanine, and "J" = an amino acid containing a non-natural electrophile, or ami This is an electrophilic warhead presented in relation to the non-acid portion (for example, "J" is cy This is the cysteine-reactive part; for example, "J" is the cysteine-reactive D-nipecotinic acid part. (It is a minute). Those skilled in the art can use ring-closing metathesis reactions to bond the bonds covalently (together). It contains two unnatural amino acids that can be stapled to form a cross-linked ring. Regarding peptides, the ring-closing metathesis reaction is performed on the peptide (i.e., stabilized peptide) Before administering the peptide to a subject requiring treatment, understand what is happening to the peptide. In other words, a frame produced by the covalent bond between two unnatural amino acids. The bridged ring is present in the stabilized peptide before administration to the target. They probably are.

[0172] In some cases, it is necessary for human subjects (e.g., BFL-1 and BCL-X) (In human subjects expressing both L and cancer dependent on them) as disclosed herein NOXA-stabilized peptides and BCL-XL inhibitors are co-administered. The inhibitor is known in the art (for example, Zhu et al. Aging Cell; See 15(3):428-35 (2016). An example of a BCL-XL inhibitor is Navitoclax. In a particular embodiment, for a human subject that requires it, sequence number 16, and If the peptide is selected from the group consisting of SEQ ID NOs. 60-79, 151, and 153, then In one particular embodiment, a BCL-XL inhibitor is administered to a person who requires it. The target group consists of sequence number 16, and sequence numbers 60-79, 151, and 153. For amino acid sequences selected from the above, at least 14%, 15%, 20%, 27%, and 34%. %, 40%, 47%, 50%, 53%, 60%, 65%, 70%, 75%, 80%, 85 Peptides that are 90%, 95%, or 97% identical, as well as BCL-XL inhibitors It is administered. In certain embodiments, it is needed along with a BCL-XL inhibitor. The peptide co-administered to the subjects is AT8LREFGDXLNFRQ (SEQ ID NO: 46). Here, "8" = R-octenylalanine and "X" = S-pentenylalanine. It is a human. In a specific embodiment, it is used in conjunction with a BCL-XL inhibitor to help humans who require it. The peptide co-administered to the subjects is AT8LRRAGDXLNFRQ (SEQ ID NO: 44). Yes, here, "8" = R-octenylalanine and "X" = S-pentenylalanine In a specific embodiment, a BCL-XL inhibitor is used along with a human who requires it. The peptide co-administered to the elephant has a warhead that binds to BFL-1 as described herein. It is a NOXA peptide. In a specific embodiment, it has a warhead that binds to BFL-1. The NOXA peptide has the sequence JATQLREFGDKLNFRQ (SEQ ID NO: 128). Here, the peptide is structurally stabilized by any method known in the art. The electrophilic warhead (J) is composed of a cysteine-reactive moiety (for example, D-nipecotin). (The acid portion) In certain embodiments, NOXA has a warhead that binds to BFL-1. Petit has the sequence JATX1LREFGDX2LNFRQ (sequence number 62), where "X1" and "X2" are covalently bonded using a ring-closing metathesis (RCM) reaction. Non-natural amino acids that can be stapled together to form a cross-linked ring. Therefore, "J" is either a non-natural electrophile-containing amino acid or a part that is not an amino acid. In relation to this, electrophilic warheads (where the electrophile functions as a chemical cap) are presented. (for example, "J" is the cysteine ​​reactive part, for example, "J (This is the cysteine-reactive D-nipecotinic acid moiety). In another specific embodiment, BF The NOXA peptide with a warhead that binds to L-1 has the sequence JAT8LREFGDXLN It has FRQ (Sequence ID 78), where "8" = R-octenylalanine and "X " = S-pentenylalanine, and "J" = a non-natural electrophilic amino acid, Or it is an electrophilic warhead presented in relation to a non-amino acid portion (for example, "J" is the cysteine-reactive moiety; for example, "J" is the cysteine-reactive D-nipe (This is the cotinic acid portion). In another specific embodiment, the warhead has a binding to BFL-1. The NOXA peptide has the sequence JATQLRRAGDKLNFRQ (SEQ ID NO: 126) Here, the peptide is structurally stabilized by any method known in the art. The electrophilic warhead (J) is composed of a cysteine-reactive moiety (for example, D-nipecotin). (The acid portion) In certain embodiments, NOXA has a warhead that binds to BFL-1. Petitdo has JATX1LRRAGDX2LNFRQ (Sequence ID 60), where, X1 and X2 are covalently bonded using a ring-closing metathesis (RCM) reaction. These are unnatural amino acids that can be stapled together to form a cross-linked ring. "J" is either a non-natural electrophilic amino acid, or a non-amino acid component. Related to this, electrophilic warheads (where the electrophile functions as a chemical cap) are presented. (to obtain) (for example, "J" is the cysteine ​​reactive part, for example, "J" is (This is the cysteine-reactive D-nipecotinic acid moiety). In another specific embodiment, BFL- The NOXA peptide having a warhead that binds to 1 has the sequence JAT8LRRAGDXLNFR Q (sequence number 76) is present, where "8" = R-octenylalanine and "X" = S-pentenylalanine is an amino acid containing a non-natural electrophile, or or is an electrophilic warhead presented in relation to a non-amino acid portion (for example, "J " is the cysteine-reactive moiety, for example, "J" is the cysteine-reactive D-nipeko (This is the tinic acid portion). Those skilled in the art can use a ring-closing metathesis reaction to bond them covalently. Two non-natural meshes that can "staple together" to form a bridged ring. For peptides containing ano acids, the ring-closing metathesis reaction is performed on the peptide (i.e., stabilized) Before administering the peptide to a subject requiring treatment, it is important to understand what has been done to the peptide. You will understand. In other words, it is produced by the covalent bond between two unnatural amino acids. The cross-linked ring is used to target the stabilized peptide before administration. It probably exists in Do.

[0173] In some cases, it is necessary for human subjects (e.g., BFL-1 and BCL). -Cancers that express or are dependent on XL, BCL-2, and / or MCL-1 In human subjects having the following characteristics, NOXA-stabilized peptides, BCL-XL, as disclosed herein, Inhibitors of BCL-2 and / or MCL-1 are co-administered. In some cases, Human subjects requiring this (e.g., BFL-1, BCL-XL, BCL-2, and M) In human subjects who express each of CL-1 or have cancers that are dependent on it, NOXA-stabilized peptides, BCL-XL, BCL-2, and MCL- disclosed in the document Inhibitor 1 is co-administered. In some cases, it is necessary for human subjects (for example, Expressing or depending on BFL-1, BCL-XL, and BCL-2 respectively. In human subjects with sexually transmitted cancer, the NOXA-stabilized peptide disclosed herein, BCL -XL and BCL-2 inhibitors are co-administered. In some cases, this is necessary. Human subjects (for example, those expressing BFL-1, BCL-XL, and MCL-1 respectively) In human subjects having cancer or a cancer dependent thereon, the NOXA benzodiazepines disclosed herein Statistical peptides, BCL-XL, and MCL-1 inhibitors are co-administered. In some cases... This is for human subjects that require it (e.g., BFL-1, BCL-2, and MCL-1) In human subjects who express or have cancer dependent on each of the following, the following are described herein: The NOXA-stabilized peptide, BCL-2, and MCL-1 inhibitors shown were co-administered. BCL-XL inhibitors are known in the art (for example, Zhu et al.). See Aging Cell; 15(3):428-35 (2016). Examples of BCL-XL inhibitors are: Navitoclax is a BCL-2 inhibitor. BCL-2 inhibitors are known in the art (for example) For example, see Roberts et al. N Engl J Med 374(4):311-322 (2016). An example of a BCL-2 inhibitor is venetoclax. MCL-1 inhibitors are also available. It is publicly known in the field (for example, clinical trial identification number NCT02979366, Kotschy). et al. Nature, 538(7626):477-482 (2016), Ayaz et al. ACS Chem Biol, 11(6):1710-1719 (2016), Clinical Trial Identification Number NCT02992483, Clinical Trial Identification Number No. NCT03465540, Caenepeel et al., Proceedings, AACR Annual Meeting See 2017, DOI: 10.1158 / 1538-7445.AM2017-2027. MCL-1 inhibitors Non-restrictive examples include S64315, S63845, BAY 1000394, MIK Examples include the 665, AMG397, and AMG176. In certain embodiments, For humans who require this, Sequence ID 16, and Sequence IDs 60-79, 151, and Peptides selected from a group consisting of 153, as well as BCL-XL inhibitors, BCL-2 Inhibitors and / or MCL-1 inhibitors are administered. In certain embodiments, For humans who require this, Sequence ID 16, and Sequence IDs 60-79, 151, and For amino acid sequences selected from the group consisting of 153, at least 14%, 15%, and 2% are present. 0%, 27%, 34%, 40%, 47%, 50%, 53%, 60%, 65%, 70%, 7 Peptides that are 5%, 80%, 85%, 90%, 95%, or 97% identical, as well as B CL-XL inhibitors, BCL-2 inhibitors, and / or MCL-1 inhibitors are administered. In certain embodiments, BCL-XL inhibitors, BCL-2 inhibitors, and / or MCs are used. The peptide co-administered with the L-1 inhibitor to human subjects in need is AT8LR. EFGDXLNFRQ (sequence number 46), where "8" = R-octenylara. It is nin, and "X" = S-pentenylalanine. In a specific embodiment, BCL- It is needed along with XL inhibitors, BCL-2 inhibitors, and / or MCL-1 inhibitors. The peptide co-administered to human subjects is AT8LRRAGDXLNFRQ (SEQ ID NO: 4). 4) where "8" = R-octenylalanine and "X" = S-pentenylalanine It is rualanine. In specific embodiments, it is a BCL-XL inhibitor, a BCL-2 inhibitor, Peptide co-administered to human subjects who require it, along with an MCL-1 inhibitor. The do is a NOXA peptide having a warhead that binds to BFL-1 as described herein. In a specific embodiment, the NOXA peptide having a warhead that binds to BFL-1 is It has the sequence JATQLREFGDKLNFRQ (SEQ ID NO: 128), where the peptide is The structure is stabilized by any method known in the art, and the electrophilic warhead The part (J) is a cysteine-reactive moiety (e.g., the D-nipecotinic acid moiety). In this embodiment, the NOXA peptide having a warhead that binds to BFL-1 has the sequence JATX It has 1LREFGDX2LNFRQ (Sequence ID 62), where "X1" and "X2 " is a ring-closing metathesis (RCM) reaction that uses covalent bonding ("staple together"). ) and can form a cross-linked ring; "J" is a non-natural amino acid. This is presented in relation to a naturally occurring electrophilic amino acid or a non-amino acid portion. It is an electrophilic warhead (the electrophile can function as a chemical cap) (for example For example, "J" is the cysteine-reactive part, and for example, "J" is cysteine-reactive (This is the D-nipecotinic acid portion.) In another specific embodiment, a warhead that binds to BFL-1. The NOXA peptide having the sequence JAT8LREFGDXLNFRQ (SEQ ID NO: 78) It has such that, where "8" = R-octenylalanine and "X" = S-pentenylalanine It is a nin, and "J" = a non-natural electrophilic amino acid, or not an amino acid. The electrophilic warhead presented in relation to the part (for example, "J" is the cysteine ​​reaction) This is the cysteine-reactive part; for example, "J" is the cysteine-reactive D-nipecotinic acid moiety. In another specific embodiment, a NOXA peptide having a warhead that binds to BFL-1 is, It has the sequence JATQLRRAGDKLNFRQ (SEQ ID NO: 126), where the peptide is The structure is stabilized by any method known in the art, and the electrophilic warhead The part (J) is a cysteine-reactive moiety (e.g., the D-nipecotinic acid moiety). In this embodiment, the NOXA peptide having a warhead that binds to BFL-1 is JATX1L It has RRAGDX2LNFRQ (Sequence ID 60), where "X1" and "X2" are , using ring-closing metathesis (RCM) reactions, they are bonded covalently ("stapled together"). It is a non-natural amino acid that can form a cross-linked ring, and "J" is a non-natural Electrophiles present in relation to electrophilic amino acids or non-amino acid portions. It is a ferronuclear warhead (an electrophile can function as a chemical cap) (for example) "J" is the cysteine-reactive moiety; for example, "J" is the cysteine-reactive D- (This is the nipecotinic acid portion). In another specific embodiment, the warhead has a binding to BFL-1. The NOXA peptide has the sequence JAT8LRRAGDXLNFRQ (SEQ ID NO: 76). Here, "8" = R-octenylalanine and "X" = S-pentenylalanine Therefore, "J" = an amino acid containing a non-natural electrophile, or a part that is not an amino acid. The electrophilic warhead presented in relation to (for example, "J" is the cysteine ​​reactive part) This is a fraction, for example, "J" is the cysteine-reactive D-nipecotinic acid moiety. If you are a person, you can use a ring-closing metathesis reaction to bond covalently ("staple together"). Regarding peptides containing two unnatural amino acids that can form a cross-linked ring... Therefore, the ring-closing metathesis reaction requires treatment of the peptide (i.e., the stabilized peptide). Before administering it to the target, you will understand what is happening to the peptide. In other words... Then, the cross-linked ring produced by the covalent bond between two unnatural amino acids becomes stable. Before administering the stabilized peptide to the target, the presence of the stabilized peptide is likely to be present. .

[0174] In some embodiments, human subjects are MCL-1 and / or BFL-1 expression / dependence Cancer (for example, melanoma, leukemia, lymphoma, or other hematopoietic malignancies or solid cancers) Having a tumor, or another cancer as described herein. In some cases, solid tumors are black It is chromoma, breast cancer, or lung cancer. In some embodiments, it expresses BFL-1 or Cells dependent on it are characteristic of autoimmune diseases or other inflammatory conditions associated with cellular hyperactivity. (This causes...) In certain cases, autoimmune diseases include autoimmune colitis, thyroiditis, Arthritis, nephritis, dermatitis, vasculitis, systemic lupus erythematosus, diabetes, or Sjögren's syndrome. It is a disease. In some cases, inflammatory diseases include asthma, psoriasis, inflammatory bowel disease, thyroiditis, and joint problems. This includes inflammation, nephritis, dermatitis, or vasculitis. In specific embodiments, human subjects are treated with AML. To possess.

[0175] Generally, the method involves selecting the target, as well as cancer, such as melanoma or lymphoma. For the prevention or treatment of tumors, an effective amount of one of the peptides specified herein is administered to the target. This means administering multiple substances, for example, in or as a pharmaceutical composition. This includes repeating the administration as needed, whether orally, intravenously, or topically. It may be administered. For example, if the subject has cancer that expresses BFL-1. Determination, determination that the subject has cancer expressing MCL-1, or determination that the subject expresses BFL-1 Treatment may be selected based on the determination that the patient has cancer expressing both MCL-1 and MCL-1. The peptides of this disclosure are used to determine whether a target cancer expresses BFL-1, or It can be used to determine whether the target cancer is BFL-1 dependent.

[0176] Specific medication dosages and treatment regimens for any particular patient are influenced by various factors. The factors involved include the activity of the specific compound used, age, body weight, and General health status, sex, diet, administration time, excretion rate, drug combinations, disease, condition or symptom The severity and course of the condition, the patient's tendencies toward the disease, condition or symptoms, and the doctor treating the patient. The teacher's judgment is at play.

[0177] The effective dose may be administered as one or more doses, applications, or medications. Therapeutic effectiveness The amount of therapeutic compound (i.e., the effective dosage) depends on the selected therapeutic compound. It can be administered once or multiple times per day, or once or multiple times per week. This includes once every other day. A person skilled in the art would know that this does not limit the severity of the disease or disability. This includes the degree, medical history, the subject's overall health and / or age, and any other pre-existing medical conditions. Certain factors influence the essential dosage and timing of medication for effectively treating the target. It will be understood that this may sometimes be the case. Furthermore, the therapeutic effects described herein Treatment of the target using a certain amount of therapeutic compound may include a single treatment or a series of treatments. For example, an effective dose may be administered at least once.

[0178] Pharmaceutical composition One or more of the stabilized peptides described herein (for example) For example, sequence numbers 2-38, and one of 60-79, 151, and 153. (plural) means to be formulated for use as a pharmaceutical composition or in a pharmaceutical composition. This is possible. The pharmaceutical composition can be used in the treatment method described herein (see above). It can be used in this way. In a particular embodiment, the pharmaceutical composition is 1 to 10, 1 to 9 pieces, 1-8 pieces, 1-7 pieces, 1-6 pieces, 1-5 pieces, 1-4 pieces, 1-3 pieces, 1-2 pieces, and Except for one amino acid substitution, insertion, or deletion, SEQ ID NOs. 2-38 and 60-79 It includes amino acid sequences that are identical to the amino acid sequences described. The changes in these peptides affect the BFL-1 non-interacting alpha-helix plane and / or This can be done on the BFL-1 interaction alpha-helix plane. Such compositions are Any route, for example, Food and Drug Administration ( Formulated for administration to subjects via any route approved by the FDA or It may be compliant. An example method is the FDA's CDER Data Standards M annual, version number 004 (fda.give / cder / dsm / DRG / d It is described in (available at rg00301.htm). For example, the composition is Administration by inhalation (e.g., oral and / or intranasal inhalation (e.g., nebulizer) as well) Administered by means of spray, injection (e.g., intravenous, intra-arterial, subdermal, or intraperitoneal). Intramuscular, intramuscular, and / or subcutaneous administration, as well as oral administration, transmucosal administration, / or topical administration (including topical (e.g., intranasal) sprays and / or solutions) It can be formulated or adapted for this purpose.

[0179] In some cases, the pharmaceutical composition contains an effective amount of one or more stabilized peptides. The terms “effective amount” and “effective for treatment” may be used herein. , its administration to produce the intended effect or physiological outcome (e.g., infection The duration of effectiveness within the context of the treatment (including acute or chronic administration and periodic or continuous administration) One or more compounds or pharmaceutical compositions described herein that are used in ) It refers to quantity or concentration.

[0180] The pharmaceutical composition of the present invention comprises one or more peptides and any pharmaceutically acceptable carrier. and / or may include a vehicle. In some cases, the pharmacopoeia may include one or more additional The therapeutic agent, in an amount effective to achieve modulation of the disease or disease symptoms, and further May include. For example, a pharmaceutical composition may contain ATM or ATR inhibitors. ATMKina Non-specific examples of enzyme inhibitors include KU-559403, KU-55933, and KU-6 Examples include 0019, CP-466722, CGK733, and AZD0156. Non-exclusive examples of TR kinase inhibitors include cisandrin B, NU6027, and NVP- BEZ235, VE-821, VE-822 / VX-970, AZ20, and AZD6 738 were listed.

[0181] The term "pharmaceutically acceptable carrier or adjuvant" is used in conjunction with the compound of the present invention. It may be administered to the patient without destroying its pharmacological activity and in order to deliver a therapeutic dose of the compound. This refers to a carrier or adjuvant that is non-toxic when administered in sufficient doses.

[0182] A pharmaceutically acceptable carrier, adjuvant, etc. may be used in the pharmaceutical composition of the present invention. While not limited to these, the vehicles include ion exchangers, alumina, Aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS), e.g., d- α-Tocopherol polyethylene glycol 1000 succinate, in the form of pharmaceutical medication. Surfactants used in conjunction with this, such as Tween® or other similar polymer delivery molecules, Trix, serum proteins, e.g., human serum albumin, buffering substances, e.g., phosphate Glycine, sorbic acid, potassium sorbate, partially glyceridized saturated vegetable fatty acids Compounds, water, salts, or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, or aqueous phosphate. Potassium chloride, sodium chloride, zinc salt, colloidal silica, magnesium trisilicate, polypropylene Nylpyrrolidone, cellulose-based substances, polyethylene glycol, carboxymethylcellulose -Sodium, polyacrylate, wax, polyethylene-polyoxypropylene- These include block polymers, polyethylene glycol, and lanolin. Cyclodextrin For example, α-, β-, and γ-cyclodextrins are also included in the formulations described herein. It may be advantageously used to enhance the delivery of the compound.

[0183] The pharmaceutical composition of the present invention is any conventional non-toxic, pharmaceutically acceptable carrier, adjuvant Alternatively, it may contain a vehicle. In some cases, the pH of the formulation is determined by the formulation compound or It uses pharmaceutically acceptable acids, bases, or buffers to enhance the stability of its delivery form. It may be adjusted accordingly. The term parenteral, as used herein, refers to subcutaneous, intradermal, and sterilized. Intravascular, intramuscular, intra-articular, intra-arterial, intra-synovial, intrasternal, intrathecal, intra-lesional, and intracranial injections. This includes injection techniques.

[0184] The pharmaceutical composition may be in the form of a liquid or powder for inhalation and / or nasal administration. Good. Such compositions may contain suitable dispersants or wetting agents (e.g., Tween (registered trademark)). )80) and a suspending agent are used to formulate according to techniques known in the art. Injectable sterile preparations may also be in non-toxic parenterally acceptable diluents or solvents. For example, it can be used as a sterile solution or suspension for injection in the form of a solution in 1,3-butanediol. One of the permissible vehicles and solvents that may be used is mannitol, water, and ri. These are a gel solution and an isotonic sodium chloride solution. Furthermore, a sterilizing and fixing oil is added as a solvent or suspension. It is conventionally used as a turbidifier. For this purpose, it contains synthetic mono or diglycerides. A sterile fixative oil of your choice may be used. Fatty acids, such as oleic acid and its glyceride derivatives. It is useful in injectable preparations and is found in naturally occurring, pharmaceutically acceptable oils, such as The same applies to polyoxyethylated olive oil or castor oil. These oily liquids Alternatively, the suspension may contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose. Formulations in which the drug is administered or in pharmaceutically acceptable forms, such as emulsions and / or This may also include similar dispersants commonly used in suspensions. Other commonly used interfaces Activators, e.g., Tween® or Span and / or pharmaceutically acceptable Other similar emulsifiers commonly used in the manufacture of solid, liquid, or other forms of medication. Alternatively, bioavailability enhancers may also be used for formulation purposes.

[0185] The pharmaceutical composition can be administered orally in any orally permissible dosage form, and The forms of medication are not limited to these, but include capsules, tablets, emulsions, and There are aqueous suspensions, dispersants, and liquids. For tablets for oral use, the commonly used dispersants are... In terms of composition, it includes lactose and corn starch. Lubricants include, for example, magnesium stearate. Nesium is also usually added. For oral administration in capsule form, it is used as a useful diluent. These include lactose and dried corn starch. Aqueous suspensions and / or emulsions When the drug is administered orally, the active ingredient is combined with an emulsifier and / or suspending agent. It may be suspended or dissolved in an oily phase. If necessary, a certain sweetener and Flavorings and / or colorants may be added.

[0186] Alternatively, the pharmaceutical composition may be administered by nasal aerosol or inhalation. i. Such compositions are prepared according to well-known techniques in the field of pharmaceutical formulations, Benzyl alcohol or other suitable preservative, to enhance bioavailability Absorption enhancers, fluorocarbons, and / or other solubilizers known in the art Alternatively, it may be prepared as a solution in physiological saline using a dispersant.

[0187] In some cases, one or more peptides disclosed herein are, for example, Such conjugate compositions can be conjugated to carrier proteins. It may be monovalent or polyvalent. For example, the conjugate composition may be a carrier protein. It may contain one peptide disclosed herein that is conjugated. Alternatively, the conjugate composition may consist of two or more conjugated on a carrier. This specification may include peptides exceeding those disclosed herein.

[0188] As used herein, when two entities are “conjugated” to one another, These are connected by direct or indirect shared or non-shared interactions. In one embodiment, the meeting is communal. In another embodiment, the meeting is non-common. Interacting interactions include hydrogen bonding, van der Waals interactions, hydrophobic interactions, and magnetic phases. Examples include interactions and electrostatic interactions. Indirect co-existence interactions involve two entities being jointly connected. If present, a linker group is used as needed.

[0189] Carrier proteins are any proteins that increase or enhance immunogenicity in a target. It may contain a protein. Exemplary carrier proteins are those described in the Art. (For example, Fattom et al., Infect. Immun., 58:2309-2312, 1990; Dev et a l., Proc. Natl. Acad. Sci. USA 88:7175-7179, 1991;Li et al., Infect. Immun. 57:3823-3827, 1989;Szu et al., Infect. Immun. 59:4555-4561,1991 ;Szu et al., J. Exp. Med. 166:1510-1524, 1987; and Szu et al., Infe See ct. Immun. 62:4440-4444, 1994. The polymer carrier is one or more Natural or The material may be a synthetic material. The carrier may be water-soluble. [Examples]

[0190] The following examples are provided to further illustrate the claimed invention and to limit the scope of the invention. It should not be judged as such. To the extent that specific materials are mentioned, it is an example. This is merely an objective and not intended to limit the present invention. Those skilled in the art will be able to invent. Without using the invention and without departing from the scope of the invention, an equivalent means or reaction product may be generated. It can be expanded.

[0191] (Example 1) Potent selective cell permeability cysteine-reactive BFL-1 staple NOXA BH3 inhibitor Development of harmful substances The inventors have previously developed a high-affinity non-covalent bond of a natural BH3 domain helix. The interaction is combined with irreversible blockade obtained by the covalent bonding reaction of the electrophilic warhead. Based on the BIM BH3 sequence, selective covalent staple peptide of BFL-1 We developed an inhibitor that selectively neutralizes the anti-apoptotic activity of BFL-1 in vitro. Furthermore, BFL-1 effectively triggers apoptosis in melanoma. The design and characterization of coupled BIM SAHB was successful (Huhn et al., Cell Ch em Biol., 23(9):1123-1134 (2016). This cysteine-reactive BIM SAHB is It targets only BFL-1 via covalent bonding, but BCL-X L , MCL-1, and possible Non-covalent interactions with a broad range of BCL-2 family proteins, including BAX, in terms of sex. It retains the ability to form. NOXA, a BH3-only protein, however However, the spectrum of BCL-2 protein binding activity is narrower, and MCL-1 and BF It exhibits L-1 biselectivity (Stewart et al., Nature Chemical Biology, 6, 5 95-601 (2010)). In this specification, such staple peptides are ultimately used in therapeutic applications. The NOXA BH3 template is made strong enough or cell-permeable for use. Using the SAHB with a warhead, covalent and non-covalent bonding to the BFL-1 The selective properties were fine-tuned. These new constructs target only BFL-1. (by covalent and / or non-covalent means), or BFL-1 (covalent (by bonded and / or non-covalent means) and MCL-1 (non-covalent means) It is unique in that it either exhibits exclusive dual targeting (by means of) .

[0192] Specific binding selectivity, cell penetration properties, and inv enable the development of drugs for clinical use. To produce constructs with stability and biological activity in IVO, first, Using the pull-scanning method, a library of cysteine-reactive NOXA SAHB was created. We generated a compound to optimize its binding efficacy, selectivity, and cell permeability. Importantly, the structural design Based on the data, it was not thought that the classical method would make important contact with the protein target. A novel, shortened template lacking the N-terminal and C-terminal residues of the typical BH3 domain sequence. The NOXA sequence was designed (Figure 1). Over the length of the peptide template, (i, i+4) and (i, i+7) staples are arranged in a continuous pattern, while N-terminal D-nipecotinic acid (D-NA) is arranged on the other side. Staple scanning was achieved by keeping the reactive groups constant (Figure 2). Each peptide was incubated with BFL-1ΔC, and then reduced and denatured gels were incubated. By detecting high molecular weight conjugates using pneumatophoresis and staining them with Coomassy, The reactivity of the NOXA library was measured (Figure 3). Covalent bonding reaction with BFL-1 was possible. The effective peptide was then used in cell-based studies to investigate peptide uptake. HA-BFL-1ΔC 293T cells transfected with C4S / C19S were treated with a peptide (20 μM, 6 hours later). The sample is treated in between, then collected and the lysate is removed by anti-HA Western blotting. The analysis was performed (Figure 4). The presence of the BFL-1 doublet indicates that the peptide with the warhead is located inside the cell. The goal is to both gain access and covalently bond to BFL-1. It demonstrated what is possible. The NOXA peptide, which has (i, i+7) staples in 15th place, BIM SAHB A Compared to -3, it showed a dramatic increase in covalent targeting efficiency. However, in LDH release tests in A375P cells, this peptide showed unexpected results. This was found to cause membrane disruption (Figure 5).

[0193] To compensate for this drawback, NOXA 15 constructs can reduce dissolution behavior. To identify changes in composition, the cells were subjected to alanine scanning mutagenesis (Figure 6). In particular, the sparse cells were subjected to mutagenesis. As one design strategy to eliminate solubility by reducing aqueous contents, The aqueous residues were converted to alanine. In fact, a sequence of alanine over the length of the NOXA sequence Due to a specific mutation, NOXA S has a warhead with little to no lytic activity. Three AHBs were obtained (Figure 7). Notably, each of the mutations that reduced lysis was This reduced the overall hydrophobicity of the peptide. Surprisingly, the sequence of NOXA was modified. Most of the differences did not have a negative effect on covalent BFL-1 binding activity. However, "L The two residues of the minimal consensus BH3 domain binding motif, XXXGDE, are Leu Excluding 29 and Asp34 (Figure 8) (Day et al., J. Mol. Biol., 380(5):958- 971 (2008). These data indicate the electrophilic projectiles for targeting C55 with BFL-1. Staple peptides with a head have BFL-1 interaction with the NOXA helix. Even when present on the surface, it is extremely sensitive to modifications of specific amino acid residues. This is shown to be the case for cysteine-reactive NOXA-15 F32A and NOXA-15 L All 36A SAHBs are cell-permeable and covalently target BFL-1. (Figure 9).

[0194] Alternative repeating method to neutralize the solubility activity of cysteine-reactive NOXA-15 peptide In this approach, the goal is to reduce the overall positive charge of the peptide, using two methods. Different arginine molecules were mutated into glutamic acid residues within the NOXA sequence (Figure 10). In this process, the overall charge of each peptide was reduced from +2 to 0, and the co-charge against BFL-1 was reduced. It retains binding activity (Figure 12) and shows little to no cell lysis (Figure 1 1) We achieved improved cellular uptake (Figure 13).

[0195] Therefore, we will use two different approaches to biochemical characterization and cell research. To advance this process, we inventively produced a non-soluble, cell-permeable, cysteine-reactive NOXA SAHB. I did it.

[0196] (Example 2) The biochemical characterization of the selectivity of cell-permeable cysteine-reactive staple NOXA peptide is , along with maintaining covalent selectivity for BFL-1, and non-covalent selectivity for MCL-1 Shows a decrease in target affinity. The native NOXA BH3 sequence, along with the peptide length, staple arrangement, and mutations, are modified. After making significant changes, including dysentence induction, we then used two lead cell permeable peptides, systo These for the selectivity of in-reactive D-NA-NOXA-15 F32A and R31E We attempted to determine the effect of the modification. First, in combination with the addition of a cysteine-reactive electrophile... These changes affect the non-covalent and covalent nature of the anti-apoptotic protein mixture. We investigated how it affected the balance of binding SAHB interactions. (Biotin) Formula D-NA-NOXA-15 F32A SAHB or the corresponding acetylated construct, Recombinant BFL-1, BCL-X L , and in an equimolar mixture with MCL-1 The solution was baited and then subjected to streptavidin pull-down. Acetylated (Ac-)NO XA-15 F32A SAHB is a new technology featuring a significantly shortened native BH3 sequence. In line with the design, BFL-1, BCL-X L It did not show binding to MCL-1 and was not detected. It showed possible but minimal direct interaction (Figure 14). However, D-NA -NOXA-15 F32A SAHB exhibits a shift in interaction tendency toward BFL-1. As a result of covalent BFL-1ΔC conjugation, many notable BFLs -1 bond was present (Figure 14). The contrast between the same covalent and non-covalent interactions is Under the same experimental conditions, Ac-NOXA-15 R31E and D-NA-NOXA-15 R Observed in comparison with 31E (Figure 15). In addition, preferential interactions of these peptides. This was evaluated using endogenous proteins in lysates derived from the A375P melanoma cell line. It was valuable. When subjected to streptavidin pulldown, biotinylated Ac- and D-NA -NOXA-15 F32A or R31E SAHB is approximately equivalent to MCL-1 Although the coupling was shown, the structure with the warhead also showed remarkable coupling of BFL-1. It showed an increase (Figures 16 and 17).

[0197] Direct action of lead D-NA-NOXA-15 SAHB on anti-apoptotic proteins In addition to studying binding, their ability to competitively disrupt the inhibitory BFL-1 complex. The force was also evaluated. Regarding clinically useful biological activity, it was evaluated for lowering the apoptosis threshold. To achieve this, inhibit the unoccupied anti-apoptotic protein, and apot To induce systole, pro-apoptotic proteins compete with anti-apoptotic complexes. The ability to displace integrally is one of two different desirable functionalities. For competitive experiments Transient transfect tBID with HA-BFL-1ΔC C4S / C19S. The mixture was added to a lysate derived from 293T cells, and biotinylated Ac or D- Incubate with NA-NOXA-15 F32A SAHB and perform anti-HA immunoprecipitation. The following procedure was performed, and blotting was performed for HA and tBID. Acetylated NOXA-15 F32A was unable to compete with tBID for binding to HA-BFL-1. However, on the other hand, the D-NA-NOXA-15 F32A structure with a warhead is protein Examples include complete conversion of high molecular weight species and almost complete inhibition of tBID co-immunoprecipitation. As demonstrated, HA-BFL-1 was captured by covalent bonding (Figure 18, left). Notably, Using lysates derived from 293T cells that transiently express FLAG-MCL-1 When the experiment was repeated, all NOXA-15 F32A peptides showed tBID / F LAG-MCL-1 coimmunoprecipitation can be disrupted non-covalently (Figure 18, right), N A clear shift in the binding selectivity of OXA-15 F32A SAHB to BFL-1 was demonstrated. This result shows that Ac-NOXA-15 R31E and D-NA-NOXA-15 R31 When compared with E, they are identical (Figure 19), and for this structure, the same functional connection Prioritization has been established.

[0198] Regarding binding to anti-apoptotic proteins, it competes with the FITC-BID BH3 peptide. The combined ability of acetylated NOXA-15 R31E SAHB and cysteine ​​reactivity of NO Fluorescence polarization experiments comparing the capabilities of XA-15 R31E SAHB showed similar effects. As shown (Figures 20-23). ​​D-NA-NOXA-15 R31E SA with warhead. Only HB is related to binding to BFL-1ΔC C4S / C19S according to FITC-BID B. It can compete with H3 (Figure 20), and this action is BFL-1ΔC C4S / C19S / This activity was not observed in C55S, therefore, Cys5 in the BH3 binding pocket. It was dependent on the presence of 5 (Figure 21). The Ac-NOXA-15 R31E peptide was also D- NA-NOXA-15 R31E peptide is also MCL-1 of FITC-BID BH3. The interaction with either ΔNΔC (Figure 22) or BCL-XLΔC (Figure 23) was significant. It could not be dissociated. These biochemical experiments conferred cellular uptake, Various modifications made to the NOXA BH3 sequence to reduce solubility led to competitive binding. Related to this, strong covalent selectivity for BFL-1 and reduced selectivity for MCL-1 It was shown that this resulted in a staple peptide with non-covalent affinity.

[0199] (Example 3) Cysteine-reactive NOXA SAHB is effective in BFL-1-dependent AML cell lines, and in AT It has a synergistic effect with M inhibition. Selectivity of cysteine-reactive NOXA-15 R31E SAHB for BFL-1 Considering this, the ideal environment for testing its cellular activity is one in which survival is particularly important for BFL-1 expression. It was hypothesized that this would be the case in the cancer cell lines to which it is dependent. To map the inheritance dependency, an attempt at large-scale whole-genome RNAi screening is being made. This is being done (Tsherniak, Cell, 170(3):564-576 (2017)). Next-generation C cells of cancer cell lines. RISPR-Cas9 screening datasets, particularly those focused on AML. We then queried and tested whether the cells showed a dependence on BFL-1 (Figure 24). Cell lines with a positive Z-score value exhibit almost no dependence on BFL-1. Or it does not show at all (for example, MV4-11, upper right), on the other hand, it is more negative The dependency Z-score value represents the increase in dependency on BFL-1 (for example, U937, (Bottom left). Most cancer cell lines express a range of anti-apoptotic proteins. Therefore, a combination of cysteine-reactive NOXA-15 R31E SAHB and a second dependency was used. In particular, BFL-1 dependence is in the moderate range (Z-score value is -4~ (In the case of -2), it was further hypothesized that this could potentially enhance cytotoxicity. To carry out this attempt, we evaluated genes that showed a significant codependency with BFL-1 dependence. ATM, a serine / threonine kinase that is activated by DNA double-strand breaks, It was the most highly correlated codependent gene in AML (Figure 25). BFL-1 The AML cell line U937 showed the highest dependence on cysteine-responsive NOX. Co-treatment with A-15 R31E SAHB and the small molecule ATM inhibitor KU-55933 When analyzed by CalcuSyn software, highly synergistic cytotoxicity was observed. A strong synergistic effect was observed (CI < 0.3, Figure 26). In addition, cysteine-reactive N When treated with OXA-15 R31E and KU-55933 alone or in combination, U9 37 cells showed a significant increase in caspars over time with combination therapy compared to monotherapy. It exhibits Ze-3 / 7 activation (Figure 27), and the cause of its cytotoxic effect is apoptosis induction. A synergistic effect was suggested. In response to this, the least dependent on BFL-1 was observed. The AML cell line MV4-11, over the same dose range applied above, contains cysteine. Reactive NOXA-15 R31E SAHB alone is essentially insensitive, and ATM inhibition is used. When combined with harmful agents, it showed little to no cytotoxic synergistic effect (Figure 28). ), and regarding combination therapy, compared to monotherapy, it did not show an increase in caspase-3 / 7 activation. (Figure 29)

[0200] (Example 4) Anti-apoptotic BFL-1 and its complex with covalent staple peptide inhibitors Crystal structure The structure of the anti-apoptotic target and its mimetic in complex with the BH3 helix is ​​as follows: The structure of BFL-1 alone can provide a roadmap for molecular purification and therapeutic drug development. And, for the first time, in combination with covalent staple peptide inhibitors, we have elucidated the following: We attempted to do this. The structure of apo BFL-1 is a ligand-unbound anti-apoptotic protein. It provides an opportunity to compare qualitative structures, and also uses cysteine-reactive staple peptide inhibitors. This study aims to elucidate the structural importance and molecular characteristics of the covalent targeting of BFL-1.

[0201] Structure of Apo BFL-1 The structure of ligand-unbound BFL-1 was determined by X-ray crystallography at a resolution of 1.69 Å. Decoded using (Figure 30, PDB identifier: 5WHI), typically a glycerin cutting helix It consists of a series of α-helices connected by loops containing ¹ or proline. The globular protein was identified (Figure 31A-B). BFL-1 is α-helix 1 A multi-domain BC containing core α5-α6 hairpins surrounded by ~4 and 7~8. L-2 family proteins share all the essential elements of their conserved structure. . α9 shortening is performed to promote the expression and crystallization of anti-apoptotic proteins. The region has canonical surface grooves (grooves) where residues derived from α-helix 2-4 are lined up. ) are particularly the anti-apoptotic proteins MCL-1 and BCL-X L The unparalleled other Regarding the APO structure, the relative position, accessibility, and three-dimensional structure of the grooves show differences. The overlays of all three structures are alpha-helix 1, 5, 6, 7, and 8. It shows almost complete overlap, but the distal portion of α3 and the adjacent α2 and the proximal portion of α4 The orientation of the positional portion has significantly poor conservation (Figure 31C). The latter is a canonical with a variable gradient. BCL-X L The one that is displaced the most downwards, followed by Then, in MCL-1 and BFL-1, they progressively shift horizontally. These uncoupled units In the structure of the state, the distance between conserved residues in the roof and floor of the groove is measured. When evaluated by doing so, the width of the joint pockets also differs. Empty BH3 joint The width of the joint pocket is essentially the same in MCL-1 and BFL-1 across multiple measurements. However, BCL-X L The groove is particularly located between the midpoints of α-helix 3 and 4. It is narrower (Figure 31D). The latter is BCL-X L In this case, they are arranged in opposite parallels to each other, MC In L-1 and BFL-1, these α-helicopters are arranged in a progressively more V-shape. This arises from significant differences in the relative arrangement of the components. Based on the structure of anti-apoptotic inhibitors. The design is largely derived from protein complexes bound to BH3, but a Such differences in three-dimensional structure between PO structures lead to alternative topography for molecular targeting. It becomes clear.

[0202] The most interesting structural differences between ligand-unbound anti-apoptotic proteins One of them is a cysta exposed to solvent within the BH3 bond groove of apo BFL-1. The presence of (Figure 31B). apo MCL-1 also has BCL-X L Furthermore, Such cysteine ​​is not found in the structure of other BCL-2 family proteins. (Figure 31E-F). This cysteine ​​is actually used in vitro and in cells. The covalent targeting of BFL-1 based on staple BIM and NOXA BH3 is beneficial. It can be used. A cysteine-reactive warhead can be used with a BCL-2 or MCL-1 inhibitor. By incorporating it into a mimicked small molecule, it is possible to switch to target specificity that is advantageous for BFL-1 inhibition. This can be possible. To provide structural plans for such small molecule development, and also for B in covalent bonding To analyze the steric importance of binding with H3, the cis in the complex with BFL-1 Next, we studied the crystal structure of the thein-reactive staple BH3 peptide.

[0203] Covalent targeting of BFL-1 C55 by D-NA-NOXA SAHB Following the BH3 domain of human NOXA BH3, the sequence composition, staple type, and A series of D-nipecotinic acid (D-NA) derivatized BCLs were modeled by modifying their positions. -2 domain stabilized alpha helix (SAHB) is generated, and the construct is crystallized. The product was subjected to leaning. BFL-1ΔC was inserted at amino acid positions 26-40 and 28, 35. After being compounded with NOXA SAHB containing i, i+7 staples, promising regeneration A suitable crystal was formed (hereinafter referred to as D-NA-NOXA SAHB in this specification). (Figure 32A). Site-selective derivative of BFL-1 by D-NA-NOXA SAHB. To verify the effects of the modification, a series of BFL-1 constructs (WT, C55S, C4S / C19) were used. S, C4S / C19S / C55S) are acetyl or D-NA cap NOXA SA Incubate with either HB and crosslink BFL-1 by electrophoresis and kumming. Monitoring was performed by C-staining. Peptides and t pesticides that resulted in individual increases in molecular weight. The protein combination is a BFL-1 construct containing C55, i.e., wild type and C4S The D-NA-NOXA SAHB was with / C19S (Figure 32A). In fact, the inter By mass spectrometry, BFL-1 C4S / C19 was detected by peptide incubation. Complete conversion from S to appropriately sized D-NA-NOXA SAHB adduct Confirmed (Figure 32B). Targeting of BFL-1 by biotinylated D-NA-NOXA SAHB. The capabilities are all due to recombinant MCL-1 and BCL-X, which contain cysteine. L , and BFL -1 Using a protein mixture, in a competitive streptavidin pull-down assay... We then tested both Ac-NOXA SAHB and D-NA-NOXA SAHB. Although a similar small amount of MCL-1 was pulled down, only D-NA-NOXA SAHB showed results. , robust BFL-1 derivatization as found in the input lane, and BF This resulted in both a substantial pulldown of L-1 (Figure 32C). Finally, the complex protein To verify this activity with respect to lysates, Ac- and D-NA derivatized NOX A SAHB is derived from a lysate of A375P melanoma cells expressing BFL-1. When incubated, the NOXA SAHB with a warhead also has a native A significant increase in the pulldown of the BFL-1 protein was observed (Figure 32D).

[0204] Crystal structure of the / BFL-1 / D-NA-NOXA SAHB complex After documenting the potent BFL-1 targeting capability of D-NA-NOXA SAHB, The crystal structure of the complex with FL-1ΔC(C4S / C19S) was deciphered at a resolution of 2.38 Å. (Figures 33A-B, Figure 30, PDB identifier: 5WHH). All ligand atoms are superior. This results in a high electron density, and staple peptides bonded to grooves defined by α2~α4 The calculated interface area per molecule is 771 Å. 2 As shown above, the parent of the amphipathic α-helix Aqueous residues R30, D34, and N37 are E80 / D81 on the BFL-1 surface, respectively. R88 and K147 interact with each other through complementary electrostatic and hydrogen bonding (Figure) 33A). Residues L29, F32, and L3 of the hydrophobic surface of D-NA-NOXA SAHB 6 is the canonical hydrophobic groove, with residues F95, V44 / V48, and V40 / V It is directly coupled with 90 (Figure 33C). The i, i+7 staples are observed in the cis configuration. This is mainly exposed to the solvent (Figure 33A-C). In addition, C55 and D-NA The covalent bonds between the acrylamide moiety and the acrylamide moiety are easily visualized (Figure 33D). The crillamide portion has the same composition as NOXA BH3 C25 for BFL-1 C55. The direction is maintained (Figure 33E~F), but the piperidine group of D-NA is, coincidentally, in the BFL-1 table. Complementary hydrophobicity is formed by residues L52, L56, V74, and F95 on the surface. It binds to the cellular pocket (Figures 33F-3G). Therefore, the introduced acrylamide is present. The chemical structure of the part itself promotes further BFL-1 interactions in the hydrophobic groove. Provide opportunities.

[0205] By analyzing the structures of both the apo and SAHB derivatization of BFL-1, Riga It became possible to compare the three-dimensional structures of the ligand-unbound and ligand-bound forms. Through its use, D-NA-NOXA SAHB induces the three-dimensional structural "opening" of the groove, and Therefore, for example, between the α-carbons of residues 55 and 70 in the apo form and the BH3-bound form When comparing distances, α2 / α3 and α4 are displaced by 2.8 Å from each other (Figure 34). A~B). These individual structural changes are compared with BFL-1 / D-NA-NOXA SAHB and ap This is quantitatively shown by a distance difference matrix compared with BFL-1 (Figure 34C~D). The RMSD value of 0.897 for comparisons between all conserved α-carbons in the BFL-1 structure is, The importance of three-dimensional structural changes is emphasized.

[0206] Observed SAHB-induced remodeling of the BFL-1 canonical groove is BF The formation of a new surface pocket adjacent to L-1 C55 should be noted (Figure 34E). This surface change also involves the interaction of BFL-1 with the non-staple NOXA BH3 peptide. This was also evident at that time, and this discovery relates to the hydrocarbon staple or acrylamide portion. The general correlation beyond structural adaptation is emphasized (Figure 35A). Importantly, NOX This change in groove topography during BH3 interaction is due to the ligand-unbound and N A structural comparison of MCL-1 with the OXA BH3-bound form demonstrates such remodeling. In that, the distance measurements between residues in the upper and lower parts of the groove do not change significantly. This is specific to BFL-1 (Figures 34B, 35B). In fact, BH3 binding accessibility The similarity of the MCL-1 groove between the unbonded and bonded structures reflects the diverse BH3 domains. This may explain the relatively high affinity of MCL-1 for the nehelix. This difference in stereochemistry between -1 and -1 is similar to the Ac-derivative form of NOXA SAHB. Both the D-NA derivatized form and the D-NA derivatized form can bind to MCL-1 non-covalently. The shortened Ac-NOXA SAHB will not be BFL unless it is equipped with a cysteine-reactive warhead. This may explain why there is little or no interaction with -1. Pocket opening and Considering the associated potential energy requirements, the surface-accessible C55 of BFL-1 The irreversible nature of covalent reactions with residues can provide a substantial advantage in binding. Specifically, it mimics NOXA BH3 in the C25 region, inducing the formation of this characteristic pocket. The deriving compounds (Figures 33F-G, 34E) are illustrated by D-NA-NOXA SAHB. Therefore, a novel drug that has preferential selectivity for BFL-1 over MCL-1. This can result in (Figure 32C).

[0207] conclusion By analyzing the structure of apo BFL-1, the BH3-ligand binding of BFL-1 was determined. This provides the first opportunity to compare the type morphology and the ligand-unbound morphology, and the structure in the surface grooves. Differences in structure became clear. Importantly, the characteristic topography of ligand-unbound grooves - can be used for small molecule discovery by in silico screening, BF The structure of the proposed hit in the complex with L-1 is obtained by immersing the target molecule in an apo crystal. Obtained by the same method. Unoccupied BFL-1 grooves are similarly cysteine-reactive stays. Selective covalent targeting by pluteptides and potentially their small molecule mimetic forms. This demonstrates the surface accessibility of uniquely located cysteine ​​residues that can be utilized. In fact, D-nipecotinic acid derivatized NO, reinforced with i, i+7 staples. The structure of BFL-1 in complex with XA BH3 α-helix indicates that the ligand Disulfide bonds suitable for induced surface pocket opening and auxiliary hydrophobic interactions Both the formation of pores tangent to the surface and the formation of pores tangent to the surface become apparent.

[0208] This first structure of the covalent complex between BFL-1 and staple BH3 peptide In this configuration, the introduced i and i+7 staples are localized on the non-interacting surface of the helix. Furthermore, it preserves the native interaction between NOXA BH3 and BFL-1, and the ligand- It was shown that the groove topography can be remodeled to maximize target binding. BFL-1 contains acrylamide that irreversibly reacts with C55 located in the canonical groove. By introducing the part that does this, this shortened i, i+7 staple NOXA BH3 structure The non-covalent bonding preference of the building to MCL-1 is a strong covalent inhibitor of BFL-1. It was converted into a harmful agent. Combined, these structural and biochemical results are for clinical use. We are developing highly selective BFL-1 targeting agents and BFL-1 / MCL-1 dual inhibitors. It brings new opportunities.

[0209] (Example 5) Cysteine ​​in reactivating cell death in BFL-1-dependent AML cell lines The selective synergistic effect of reactive NOXA SAHB and targeted inhibition of the DNA damage response pathway Further verification NOXA-15 R31E SAHB has the ability to synergistically interact with members of the DNA damage response pathway. To investigate the effects, U937 cells or MV4;11 cells were subjected to D-NA-NOXA S Treatment with the AHB and / or ATM inhibitor AZD0156 to assess cell viability and caspar levels. Ze-3 / 7 activation was measured. NOXA-15 R31E SAHB is AZD0156 When combined with this, in U937 cells (which are BFL-1 dependent), selection It showed selective cytotoxicity and synergistic effects, but in MV4;11 cells (this is BFL-1 dependent). (Not related to sex) was not shown (Figures 40-43).

[0210] AZD01 is present in the BFL-1-dependent U937 cell line but not in the MV4;11 cell line. The selective cytotoxicity of NOXA-15 R31E SAHB when combined with 56 To further verify the synergistic effect (Figures 40-43), the inventors conducted a series of confirmation studies. The inventors have identified another cancer cell line that exhibits dependence on BFL-1, for example, O CI-AML-3 AML and Jurkat T-cell leukemia cells (Kim et al., 200 5. Meyers et al., 2017) conducted the above-mentioned cytotoxic study and in U937 cells Similar pharmacological synergistic effects were observed (Figures 44-45). Next, the inventors They added two additional ATM inhibitors, Ku-60019 (Golding et al., 2009) and Ku-55933 (Hickson et al., 2004) was tested and observed in AZD0156. The same synergistic pattern was observed in U937 cells (Figures 46-47). ), was not observed in MV4;11 cells (Figures 48-49). The inventors of CHK1 / 2 inhibitor AZD7762 (Zabludoff et al., 2008) and PARP inhibitor Olapa Other inhibitors of the DNA damage response (DDR) pathway, including Rib (Menear et al., 2008), A synergistic analysis comparing U937 (Figures 50-51) and MV4;11 (Figures 52-53) We found that it behaved similarly to ATM inhibitors. As a further measure of specificity... The compound was tested in KG-1 AML cells, but these cells showed pro-apoptotic activity. It has been shown to be relatively unresponsive to the addition of ATM inhibitors in response to stimulation. (Boehrer et al., 2009). The inventors found that, consistent with BFL-1 dependence, D-NA -Dose-responsive cytotoxicity in response to monotherapy with NOXA SAHB-15 R31E. Although the properties were observed, no synergistic effect of AZD0156 was observed when applied in combination. (Figure 54). Finally, the inventors have shown that the MCL-1 selective inhibitor S63845 is M In CL-1-dependent MV4;11 cells, monotherapy activity and combination therapy with ATM inhibition were observed. It shows both synergistic effects (Kotschy et al. 2016), but it is BFL-1 dependent. In U937 cells, which are responsive to D-NA-NOXA SAHB-15 R31E, We found that it did not show activity (Figure 55). In fact, MV4;11 cells and U937 S63845 and D-NA-NOXA SAHB-15 R3 in comparison with cells The contrasting activities of 1E are those of the two anti-apoptotic inhibitors, MCL-1 and B, respectively. This emphasizes the strong functional specificity for FL-1.

[0211] (Example 6) Intracellular and mechanical activity of BFL-1 and DNA damage response pathway inhibitors in treated cells All of the inventors' lead constructs showed that the canonical BH3 binding pocket in cancer cells Regarding interactions in the t, there are potential competitors with D-NA-NOXA SAHB. The full-length BFL-1 protein, which has a C-terminal helix, can be covalently crosslinked. Confirmed. The inventors have confirmed that recombinant full-length BFL-1 and expression in 293T cells We generated HA-tagged full-length BFL-1 cells, and then used them in vitro and in treated cells. Each sample underwent crosslinking analysis. The inventors consistently identified D-NA-NOXA S Both F32A and R31E analogs of AHB-15 were recombinant and expressed. Covalent crosslinking to full-length BFL-1 was observed, but this was compared with treatment of the corresponding C-terminal shortened species. Therefore, efficiency was somewhat reduced (Figures 56A-B). Thus, in AML Confirm effective targeting of native BFL-1 and a planned series of cytotoxic components To identify the best compounds for analysis, we showed the strongest BFL-1 dependency score. Lysate derived from the AML cell line U937 was biotinylated with NOXA SAHB. -15 Treatment with F32A and R31E peptides followed by streptavidin pulldown. And BFL-1 Western analysis was performed. The results of the crosslinking shown above (for example, implemented Consistent with Examples 1-3), the acrylamide portion was incorporated into the NOXA SAHB-15 peptide. By incorporating this, BFL-1 targeting is achieved, in this case, by using a gene derived from U937 AML cells. Active targeting of the BFL-1 protein was significantly enhanced (Figure 56C). Therefore, D-NA-NOXA SAHB-15 R31E is native between two structures. It was further identified as a more effective binder for the active BFL-1 protein, and therefore Therefore, it was selected for a series of cancer cell tests.

[0212] From a mechanistic perspective, the inventors of this invention have found that U937 cells can be treated with D-NA-NOXA SAHB-1 5. Cytotoxic and synergistic effects of treatment with a combination of R31E and AZD0156 , and the observed caspase 3 / 7 activation, when measured by TMRE staining, Similarly, we further confirmed that it resulted in mitochondrial membrane depolarization (Figure 57). AZD To demonstrate that 0156 effectively targets ATMs at the applicable dose, this invention The researchers treated U937 cells with an ATM inhibitor for 2 hours and compared them to ATM autologous cells in S1981. Phosphorylation and downstream CHK2 phosphorylation at S33 / 35, Western Blotti Monitoring was performed using a monitoring system. In fact, in U937 cells, the constitutive level is normally elevated. Dose-responsive inhibition of S1981 autophosphorylation, which should be occurring, and the lowest treatment dose Even in this case, strong blockade of CHK2 phosphorylation was observed (Figure 58). When evaluating the potential mechanistic principles of the action, the inventors responded to the AZD0156 treatment. In response, a time-dependent increase in the protein level of pro-apoptotic BAX was observed (Figure 59). ), BAX-mediated apoptosis-related anti-apoptotic inhibitors in U937 cells Upregulation of BAX, combined with pharmacological blockade of BFL-1, enhances the finer points. This suggests that it may explain cell damage. Finally, the inventors developed a monotherapy for U937 cells. D-NA-NOXA SAHB-15 R31E used in treatment and combination treatments When treated with serial dilutions, perform an LDH release assay at 30 minutes and 4 hours. When measured, no nonspecific cell membrane lysis activity was observed (Figure 60). Target protein binding (e.g., BFL-1), cell permeability without membrane disruption, and Regarding the cytotoxic activity by the mechanism, it is necessary to optimize staple peptides. The series of experiments described above are for the development of staple peptides, such as cysta, for therapeutic drug development. This highlights the non-trivial nature of the design of the reactive NOXA SAHB. As a further example, D-Nipex A series of constructs containing cotinic acid warheads were treated with 20 μM peptide for 2 hours in HeLa. In cells, various trends in nonspecific membrane lysis were observed, ranging from 0% to approximately 60% (Figure 61). Overall, BFL-1 gene-dependent and codependent analysis (Figure 62) showed that cysteine Selective BFL-1 targeting activity of reactive NOXA SAHB, and DNA damage response pathways. To effectively apply and verify the synergistic effects of these when combined with inhibitors, The selection of cancer cell subtypes was presented.

[0213] Materials and methods used in the examples Synthesis of staple peptides N-terminal derivatization with acetyl, FITC-βAla, or electrophilic warhead, B Hydrocarbon staple peptide corresponding to the BH3 domain of CL-2 family proteins The present inventors synthesized, purified, and quantified the substance using methods previously reported by the inventors (Bird et al., Methods Enzymol., 446:369-86 (2008), Bird et al., Curr. Proto c. Chem. Biol., 3(3):99-117 (2011). A peptide containing acrylamide, Coupling lylic acid or trans-crotonic acid to the N-terminus of the peptide, or Using standard Fmoc coupling and deprotection methods, Fmoc-protected cyclic amino acids (Chem-Impex International) is coupled first, The synthesized product was obtained by either deprotecting Fmoc and acyling it with acrylic acid. (Huhn et al., Cell Chem Biol., 23(9):1123-1134 (2016)).

[0214] Recombinant protein expression and purification Recombinant anti-apoptotic BFL-1ΔC (amino acids 1-151) and its cysteine The serine variant is PET17b (Novagen, N-terminal hexahistidine tag). It was cloned into Escherichia coli LOBSTR BL21(DE 3) Expressed in (Kerafast), continuous Ni affinity and size exclusion cross Purified by matrix as described (Pitter et al., Methods Enz Ymol., 446:387-408 (2008). Recombinant anti-apoptotic BCL-XLΔC (amino acid 1). ~212) and MCL-1ΔNΔC (amino acids 172~329), PGEX-4T- Cloned into an expression vector 1 (GE Healthcare, N-terminal GST tag), BL21(DE3) Escherichia coli(Sigma Aldrich ) was expressed in and purified as previously described (Huhn et al., 2016 (above ), Pitter et al., 2008 (above). When purifying BCL-XLΔC, For easy identification of proteins by silver staining in the putavidin pull-down experiment, G The ST tag was cut using thrombin (12-15 units) and BCL-XLΔC and G This resulted in a size difference compared to ST-MCL-1ΔNΔC.

[0215] Recombinant full-length BFL-1 (175 amino acids) containing an N-terminal hexahistidine tag, PT Cloned into the YB1 vector (New England Biolabs), as described above. The product was purified in the manner described above, however, the chitin affinity chromatography and DTT elution steps were not performed. Excluding the addition of the mouse monoclonal anti-His6 tag. Protein purity and identity, mouse monoclonal anti-His6 tag Using the antibody (Abcam), Coomassi staining and Western blotting analysis are performed. Therefore, I have confirmed it.

[0216] In vitro covalent conjugation assay His-BFL-1ΔC C4S / C19S protein (5 μM) is mixed with 50 mM Tr In 10 mM DTT and 100 mM NaCl in IS (pH 8.0), at room temperature... Pre-treat for 30 minutes (final volume 9.5 μL), then incubate at room temperature for another 1 hour. For incubation, mix with cysteine-reactive NOXA SAHB in a molar ratio of 10:1. The sample was then boiled in a 3x loading buffer containing DTT, and 12% After electrophoresis on Bis-Tris gels, the samples were subjected to Coomassie staining.

[0217] Cellular uptake The cells contain 10% fetal bovine serum (FBS) and penicillin-streptomycin. The cultures were grown using standard culture media (293T, A375P:DMEM;U937). (MV4-11; RMP1). For the uptake of cysteine-reactive peptides, 293T Apply lipofectamine LTX Plus (Thermo Scientific) to the cells. Use 2 μg of pCMV plasmid containing HA-BFL-1ΔC C4S / C19S. The cells were transfected. 24 hours after transfection, the cells were treated with 5% FBS. Cells were treated with 20 μM of the indicated SAHB in DMEM containing [the specified substance] for 8 hours. 1% CHAPS lysis buffer (150mM NaCl, 50mM Tris pH 7.4 (dissolved by incubation with 100 mM DTT). Soluble fraction Follow the manufacturer's instructions (Thermo Scient) to determine the protein concentration in minutes for the BCA kit. The measurements were taken using the method specified in the ific. The sample was then boiled in LDS buffer and subjected to antimicrobial action. HA antibody (Sigma Aldrich, no. 12CA5) and anti-actin antibody (Si Using a 1:1000 dilution of GMA Aldrich (No. A1978), Wester It was subjected to analysis.

[0218] Streptoavidin pulldown Wild-type His-BFL-1ΔC, BCL-XLΔC (untagged), and GST-MC Combine L-1ΔNΔC (1 μM each) with 3 mM DTT in PBS at room temperature. The mixture was incubated for 30 minutes. The mixture was then heated with the vehicle or the indicated C-terminal biotin. Treat with NOXA SAHB (1 μM) for 4 hours, then wash with 30 μL of PBS. High-performance streptavidin (SA) agarose beads (Thermo Fisher The beads were added to Pierce and incubated at room temperature for 2 hours while rotating. Next, NP-40 lysis buffer (1% NP-40, 50mM Tris pH8, 1 The samples were washed three times with 00 mM NaCl and 2.5 mM MgCl2, and then washed three times with PBS. To elute the bound protein, the beads contain 10 mg / mL biotin. The sample was boiled in 0% SDS for 10 minutes. After elution, the sample was treated with 3x LDS and 2M DTT. Boiled for 20 minutes. 12% Bis-T added to the input (2%) and eluent (5 μL). Gel electrophoresis was performed using a RIS gel, and the gel was stained with a Pierce silver staining kit (The It was deployed using RMO Fisher.

[0219] BFL-1 targeting in cancer cell lysates Cultured A375P cells and U937 cells were either treated with trypsin or harvested, respectively. Then, wash with PBS and 1% CHAPS lysis buffer (1% CHAPS, 150 mM N). aCl, 50mM Tris (pH 7.4, 100μM DTT) were incubated together. The protein was dissolved by using a BCA kit. The protein concentration of the soluble fraction was measured using a BCA kit. Measurements were taken using the product according to the manufacturer's instructions (Thermo Scientific). The lysate sample (1 mg) was incubated overnight at 4°C in 1% CHAPS lysis buffer. Alternatively, it was incubated with C-terminal biotinylated NOXA SAHB (30 μM). Otin capture is mixed with high-performance SA agarose (Thermo Scientific). The material was incubated at 4°C for 2 hours, then centrifuged to dissolve the pelletized beads. This was achieved by washing three times with desorption buffer (1 mL). The protein bound to the beads... The quality is determined by boiling for 10 minutes in a 10% SDS containing 10 mg / mL biotin. It is eluted by and then BFL-1 antibody (Abcam, no. 125259) and M Using CL-1 antibody (Rockland, number 600-401-394S), electrophoresis It was subjected to dynamic and Western blotting.

[0220] Destruction of the BFL-1 / BH3 complex Regarding interactions with BFL-1 and MCL-1, biotinylated S competes with tBID. To evaluate the capacity of AHB, as described above, p3XFLAG-CM was introduced into 293T cells. In the V-10 vector (Sigma), HA-BFL-1ΔC C4S / C19S Either FLAG-MCL-1 or FLAG-MCL-1 was transfected. After 24 hours, the cells were transfected. Trypsin treatment, wash with PBS, dissolve in 1% CHAPS buffer, and BCA kit The supernatant was collected to determine the protein concentration. Lysate sample (0.5 mg) This was then mixed with 0.25 μM recombinant tBID (R&D Systems) and 5 μM biotinylated N. Incubated with OXA SAHB at room temperature for 6 hours. This mixture was also treated with HA. After being subjected to FLAG immunoprecipitation, HA antibody (Sigma Aldrich, no. 12CA) was used. 5) FLAG antibody (Sigma-Aldrich, F7425), and BID antibody ( Using a 1:1000 dilution of Santa Cruz (sc-11423), West An analysis was conducted.

[0221] Competitive fluorescence polarization coupled assay The fluorescence polarization (FP) assay was performed as previously described (Pitter et al., 2008 (above). Briefly speaking, using serial dilutions of anti-apoptotic proteins First, generate a direct coupling curve using FITC-BID BH3 (25nM), then 5 minutes At that time, the SpectraMax M5 microplate reader (Molecular FP was measured in the devices. For competitive assays, acetylation or Serial dilutions of NOXA SAHB with a warhead were determined by a direct binding assay. The recombinant protein was then added at approximately EC75 concentrations. FITC-BID BH3 (25 nM) was then added, and the fluorescence polarization was measured at equilibrium. PRISM Using software (GraphPad), nonlinear regression analysis of competitive couple curves is performed. The IC50 value was calculated.

[0222] LDH release assay. A375P and U937 cancer cells were treated with 10% fetal bovine serum (FBS) and The cells were cultured in DMEM and RPMI containing penicillin-streptomycin. Seeds were sown in a 96-well plate (5 x 10 per well). 3 (Individual cell). A375P cell For U937 cells, after overnight incubation, or immediately after seeding. The cells were then placed in DMEM or RPMI supplemented with 5% FBS, and the indicated concentrations of D-NA- The plates were treated with NOXA SAHB for the indicated durations (30 minutes, 4 hours). The plates were then cooled to 4°C. After centrifuging at 1,500 rpm for 5 minutes (Thermo Scientific Sorvall Four-Place Swinging Bucket Rotor [75006445], 478 × g), 100 μL of cell culture medium in a transparent plate (C Transfer to (orning) and shake for 30 minutes with 100 μL of LDH reagent (Roche). Incubate while allowing it to do so, then use a microplate reader (Spectrama Absorbance was measured at 490 nm in x M5 (Molecular Devices). By doing so, LDH release was quantified.

[0223] Cell viability and caspase-3 / 7 activation assay. Culture in RPMI containing 10% FBS and penicillin-streptomycin U937, MV4;11, OCI-AML-3, Jurkat, and KG-1 cells were used. Seeds were sown in a 96-well plate (5 x 10 per well). 3 (Individual cells), overnight incubation In RPMI supplemented with 5% FBS after baiting, the indicated concentration of D-NA-NOXA SAHB and / or AZD0156, Ku-60019, Ku-55933, AZD 7762, Olaparib, or S63845 (all Selleck Chemica) (Obtained from ls) The cells were treated for the period indicated. Cell viability and caspase-3 / 7 activity The chemicals are CellTiter-Glo and Caspase-Glo, respectively. 3 / 7 Chemical Development The luminescence was measured using a photoreagent (Promega) and measured with a Spectramax M5 microcontroller. The synergistic effects of drug interactions were detected using a cross-plate reader. The calculations were performed using Biosoft software.

[0224] Preparation of BFL-1 conjugated with SAHB BFL-1ΔC (30 μM) was treated with DTT (20 mM) at 4°C for 30 minutes, followed by Then, with peptide:protein molar ratios of 1.2x, 1.2x, and 1x, D-NA-NOX Incubate each of the SAHB peptides continuously at 4°C for 1 hour. BFL-1ΔC / D-NA-NOXA SAHB conjugate was prepared using the following method. The resulting conjugate was then subjected to size exclusion chromatography. The condyloma was purified and then analyzed by SDS-PAGE gel electrophoresis and intact mass spectrometry. We evaluated the efficiency of the communication process.

[0225] X-ray crystal structure analysis Apo His-BFL-1ΔC C4S / C19S was expressed and purified as described above. 20 mM HEPES pH 7.5, 300 mM NaCl, 5% glycerol, The buffer was replaced with 50 mM arginine and 1 mM DTT. (Equivolute: 100 nL) ) 6.3 mg / mL (340 μM) apo BFL-1ΔC is added to the reservoir solution (35 Mix % PEG1500 and 0.1M sodium cacodylate (pH 6) and crystallize. The solution was prepared by dropping it into a liquid at 20°C. The crystals were then rapidly converted to a solution containing 25% glycerol. After transferring to a crystallization buffer, it was flash-frozen in liquid nitrogen. apo BFL-1ΔC crystal? The diffraction data obtained was processed by Advanced Photon Source (Argon NE-CAT beamline at NE National Laboratory Collected with 24ID-C, integrated the dataset, and scaled using the XIA2 package. (Evans, Acta Crystallogr D Biol Crystallogr 62, 72-82 (2006), K absch, Acta Crystallogr D Biol Crystallogr 66, 133-144 (2010), Winter, J Appl Crystallogr 43, 186-190 (2010). Structure, program Phaser(Mc Coy et al., J Appl Crystallogr 40, 658-674 (2007)) and search model PD The sequence was deciphered by molecular substitution using entry B 1NB8. Phenix(Adams et al., Acta Crystallogr D Biol Crystallogr 66, 213-221 (2010)) and Coot(Emsley and Cowtan, Acta Crystallogr D Biol Crystallogr 60, Through iterative manual model construction and refinement using 2126-2132, (2004), the most A model with excellent statistics, including a large diffraction of 1.69 Å, was obtained (PDB:5WHI). .

[0226] His-BFL-1ΔC C4S / in complex with D-NA-NOXA SAHB C19S was prepared as described above, and mixed with 20mM HEPES pH7 and 100mM NaC 1 liter of (200 nL) of (400 μM) buffer was exchanged during 1 mM DTT. The His-BFL-1ΔC / D-NA-NOXA SAHB complex was added to the reservoir solution (20 Mix with % PEG3350 and 0.2M sodium malonate, and let the crystals rise to 20°C. Crystals were formed by suspension in a liquid. The crystals were rapidly converted in a crystallization buffer containing 25% glycerol. After transferring, it was flash-frozen in liquid nitrogen. His-BFL-1ΔC / D-NA-NOX Diffraction data obtained from A SAHB crystals, Advanced Photon Soul NE-C at RCE (Argonne National Laboratory) The samples were collected at AT beamline 24ID-C. After the initial molecular substitution, the ligand was added to B Placement was performed using uster and Rhofit (Smart et al., 2012), and preliminaryly The crystallographic data model (PDB: 5WHH, maximum diffraction 2.38 Å) was refined. It was generated as described above.

[0227] Plotting of the distance difference matrix To generate a heatmap, the alpha carbon coordinates stored between structures are used in the PDB. Extracted from a file. Distance difference matrix plot, Center for Structure ral Biology at Yale University(New Haven) It was generated using the DDMP program (CT).

[0228] Quantification and statistical analysis For structural analysis, the length and distance difference matrix plots of the bonds were quantified using pymol and The DDMP program was used. The refinement statistics of the X-ray structure were obtained using Phenix software. Generated by a toware package. Biochemical and cellular data were generated using a technical triplicate. The experiment was conducted on an object, repeated at least twice, and yielded the same result, with the mean ± standard deviation of the experiment. Therefore, p-values ​​lower than 0.05 can be statistically analyzed using an independent two-tailed Student t-test. We consider these to be academically significant and indicate them with an asterisk. The data was collected using Prism software 7. Analysis was performed using 0 (GraphPad) and CalcuSyn (Biosoft). .

[0229] Data and software availability X-ray crystal structure analysis data, accession numbers 5WHI(apo BFL-1) and 5WHH It was deposited with the PDB under the name (BFL-1 / D-NA NOXA SAHB).

[0230] Mitochondrial membrane potential assay U937 cells were seeded in a 6-well plate (2 x 10⁶ cells per well). 6 (individual cells), Treatment with the indicated concentrations of D-NA-NOXA SAHB and / or AZD0156 After 24 hours, the cells were placed with TMRE (1 μM) as instructed by the manufacturer (TMRE- Incubate for 30 minutes according to the mitochondrial membrane potential assay kit (Abcam). The cells were then resuspended in 0.2% BSA in PBS, 2× per well. 10 5 Individual cells were seeded in a black 96-well plate (Corning). TMRE - Mitochondrial membrane potential can be measured using a Spectramax M5 microplate reader. The reading was obtained by measuring fluorescence (excitation / emission = 549 / 575 nm).

[0231] Phosphotransduction analysis. Culture in RPMI containing 10% FBS and penicillin-streptomycin The U937 cells were seeded into a 6-well plate (2 x 10⁶ cells per well). 6 (Individual cells) The cells were then treated with AZD0156 at the indicated concentration for 2 hours. Next, the cells were washed with PBS. Dissolve in 1% CHAPS buffer and use a BCA kit to determine the protein concentration. The supernatant was collected. The sample was analyzed using phospho-ATM(S1981, Cell Signalin g, number 4526), ​​ATM (Abcam, number ab78), phospho-CHK2 (Ser 33 / 35, Cell Signaling, number 2665), CHK2 (Cell S Ignaling (No. 2662), and actin (Sigma, No. A1978) anti Electrophoresis and Western blotting were performed using a 1:1000 dilution of the body.

[0232] Dynamic evaluation of BAX protein levels U937 cells were seeded in a 6-well plate (2 x 10⁶ cells per well). 6 ), AZD0 Cells were treated with 156 (5 μM), harvested at the indicated time, and lysed. Next, BAX antibody (Santa Cruz sc-493) and actin antibody Using the body (Sigma, number A1978), electrophoresis and Western blotting were performed. It was used for that purpose.

[0233] Other Embodiments Although the present invention has been described in detail, the above description is intended to be illustrative. This does not limit the scope of the invention as defined by the attached claims. Other embodiments, advantages, and improvements are within the scope of the following claims. In certain embodiments, for example, the following items are provided: (Item 1) (i) an electrophilic warhead, (ii) A modified amino acid sequence of the sequence described in Sequence ID No. 39, which is at least It is also 5 amino acid long and contains at least one modification that stabilizes the peptide structure, and the sequence number Modified 39 amino acids in which one or more amino acids are substituted by other amino acids. Amino acid sequence and A peptide containing, (a) Binds to BFL-1 / A1, or (b) A peptide that binds to BFL-1 / A1 and MCL-1. (Item 2) The modified amino acid sequence is 5 to 35 amino acids long, as described in item 1. . (Item 3) The modified amino acid sequence is 8 to 18 amino acids long, as described in item 1. . (Item 4) The modification that stabilizes the peptide structure is stapling and / or stitching. Peptides as described in item 1, 2, or 3. (Item 5) The staples are i-position and i+3-position, i-position and i+4-position, or i-position and i+ One or more of the peptides listed in item 4 that are ranked 7th. (Item 6) The modification that stabilizes the peptide structure involves at least two amino acids of SEQ ID NO: 39, Any one of items 1 to 5, including substitution with a non-natural amino acid having an olefin side chain. The peptide described above. (Item 7) The aforementioned unnatural amino acids having olefin side chains include S-pentenylalanine and R-octane. Tenylalanine; R-propenylalanine, S-pentenylalanine; R-pentenylalanine Lanine, S-pentenylalanine; bis-pentenylglycine, S-pentenylalanine , R-octenylalanine; and bis-pentenylglycine, S-octenylalanine A peptide selected from the group consisting of R-octenylalanine, as described in item 6. (Item 8) Modifications that stabilize the peptide structure include hydrocarbon staples / stitches and lactamstems. Staples / stitching, UV-cyclized staples / stitching, oxime staples / stitching Thioether staples / stitches, double-click staples / stitches, screws Lactam staples / stitches, bis-arylated staples / stitches, or these Any two or more combinations of the items described in any one of items 1 through 5. peptide. (Item 9) The one or more amino acids of SEQ ID NO: 39 that are substituted with other amino acids are One of items 1 through 8 located on the BFL-1 / A1 non-interaction surface of the helix with column number 39 Peptides as described in item 1. (Item 10) In SEQ ID NO: 39, 0 to 5 amino acids have been removed from the C-terminus, or Removed, alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, Replaced with 1 to 6 amino acids from the group consisting of substituted alanine and glycine derivatives. A peptide as described in any one of items 1 to 9. (Item 11) The 0 to 6 amino acids on the non-interacting surface of Sequence ID No. 39 are alanine, D-alanine. N, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine Substituted with an amino acid selected from the group consisting of derivatives, one of items 1 to 10 The peptide described in item 1. (Item 12) The following amino acids of SEQ ID NO: 39, A1, L4, R5, F7, G8, L11, N12, and One or more of Q15 are alpha-methylated or alpha-ethylated. A peptide as described in any one of items 1 through 11, which is substituted with a naturally occurring amino acid. (Item 13) The following amino acids of SEQ ID NO: 39, A1, L4, R5, F7, G8, L11, N12, and One or more of Q15 are L-alanine, D-alanine, and α-aminoisobutyric acid Selected from the group consisting of N-methylglycine, serine, substituted alanine, and glycine derivatives. A peptide described in any one of items 1 through 11, which is substituted with a selected amino acid. (Item 14) The amino acids T2, Q3, R6, K10, F13, and R14 of SEQ ID NO: 39 One or more of these are alpha-methylated or alpha-ethylated natural amines. A peptide described in any one of items 1 through 12, which is substituted with an anoic acid. (Item 15) The amino acids T2, Q3, R6, K10, F13, and R14 of SEQ ID NO: 39 One or more of these are L-alanine, D-alanine, α-aminoisobutyric acid, N-methyl A amino acid selected from the group consisting of glycine, serine, substituted alanine, and glycine derivatives. A peptide described in any one of items 1 through 12, which is substituted with an anoic acid. (Item 16) The one or more amino acids in Sequence ID No. 39 are L-alanine, D-alanine, α- Aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives One of items 1 to 9 is substituted with an amino acid selected from the following group. The peptide described above. (Item 17) (i) The overall hydrophobicity of the peptide is compared to the peptide of SEQ ID NO: 39 or 16. It is reduced, (ii) The overall positive charge of the peptide is compared to the peptide of SEQ ID NO: 39 or 16. It is reduced, or (iii) A combination of (i) and (ii), described in any one of items 1 through 16. Peptides. (Item 18) The electrophilic warhead is a non-natural amino acid having an electrophilic group, items 1 to 17. A peptide as described in any one of the items. (Item 19) The aforementioned unnatural amino acid having an electrophilic group is linked to the polypeptide skeleton. The peptide according to item 18, having acrylamide or a substituted acrylamide. (Item 20) The aforementioned unnatural amino acid having an electrophilic group is (S)-1-acryloylpyrrolidine- 3-Carboxamide, 1-Acrylopiperidine-4-Carboxamide, (R)-1 Acrylopiperidine Liloylpiperidine-3-carboxamide, (S)-1-acryloylpiperidine-3- Carboxamide, (S)-1-Acryloylpyrrolidine-2-carboxamide, (R)- 1-Acryloylpyrrolidine-2-carboxamide, (E)-4-(dimethylamino)br Ta-2-enamide, acrylamide, aziridine, diaziridine, azetidine, pyrrolidine N, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidin N, isothiazolidine, piperidine, piperazine, morpholine, thiomorpholine, azepa Neazirin, diazirin, azeto, pyrrole, imidazole, pyrazole, oxazole , isoxazole, thiazole, isothiazole, pyridine, diazine, oxazine, Thiazine, azepinephenyl (aniline), naphthalene, anthracene, phenanthrene Indole, isoindole, indoridine, quinolone, isoquinoline, quinoxaline Phthalidine, quinazoline, purine, carbazole, indazole, benzimidazole Azaindole, α-cyanoacrylamide, propioamide, trans-4-dimethicone Luamino-2-butenamide, trans-4-piperidinyl-2-butenamide, substituted activator Selected from the group consisting of lylamides and vinyl sulfonamides, as described in item 19. Peptides. (Item 21) The electrophilic warhead is a cysteine-reactive D-nipecotinic acid moiety, items 1 to 1 A peptide as described in any one of item 7. (Item 22) The electrophilic warhead is a cysteine-reactive portion, according to any one of items 1 to 17. The peptide described above. (Item 23) The electrophilic warhead is located at the N-terminus of the peptide, one of items 1 to 22. The peptide described above. (Item 24) The electrophilic warhead is not at the N-terminus of the peptide, any one of items 1 to 22. The peptide described above. (Item 25) (i) an electrophilic warhead, (ii) A0B0C0D0E0A1B1C1D1E1A2B2C2D2E2 (Sequence number) 93) Five or more amino acids (in the sequence, (a) A0 is not present, A, D-alanine, α-aminoisobutyric acid, or staple / Stitch position, (b) B0 does not exist, T, A, D-alanine, α-aminoisobutyric acid, I, M, B( Norleucine), or staple / stitch position, (c) C0 is not present, Q, A, D-alanine, α-aminoisobutyric acid, or stay This is the pull / stitch position. (d) D0 is not present, L, A, D-alanine, α-aminoisobutyric acid, I, or F And, (e) E0 is R, A, D-alanine, α-aminoisobutyric acid, K, T, or staple / Stitch position, (f) A1 is R, A, D-alanine, α-aminoisobutyric acid, E, or staple / s This is the touch position, (g)B1 is F, A, D-alanine, α-aminoisobutyric acid, I, L, V, or ste This is the pull / stitch position. (h)C1 is G, A, D-alanine, α-aminoisobutyric acid, or staple / step It is the position, (i) D1 is D, (j)E1 is absent, K, A, D-alanine, α-aminoisobutyric acid, or stay This is the pull / stitch position. (k)A2 is absent, L, A, D-alanine, α-aminoisobutyric acid, W, V, also This is the staple / stitch position. (l) B2 is absent, N, A, D-alanine, α-aminoisobutyric acid, S, D, also This is the staple / stitch position. (m)C2 is not present, F, A, L, D-alanine, α-aminoisobutyric acid, or S Table / stitch position, (n)D2 is absent, R, A, D-alanine, α-aminoisobutyric acid, E, or S Table / stitch position, (o)E2 is not present, Q, A, D-alanine, α-aminoisobutyric acid, L, or S (This is the table / stitch position) A peptide containing, (a) Binds to BFL-1 / A1, or (b) A peptide that binds to BFL-1 / A1 and MCL-1. (Item 26) A peptide described in item 25, with a length of 5 to 35 amino acids. (Item 27) A peptide described in item 25, with a length of 8 to 18 amino acids. (Item 28) (i) The overall hydrophobicity of the peptide is compared to the peptide of SEQ ID NO: 39 or 16. It is reduced, (ii) The overall positive charge of the peptide is compared to the peptide of SEQ ID NO: 39 or 16. It is reduced, or (iii) A combination of (i) and (ii), as described in item 25, 26, or 27. peptide. (Item 29) Claims 25 to 2, wherein the electrophilic warhead is a non-natural amino acid having an electrophilic group. A peptide as described in any one of item 8. (Item 30) The aforementioned unnatural amino acid having an electrophilic group is linked to the polypeptide skeleton. The peptide according to item 29, having an acrylamide or a substituted acrylamide. (Item 31) The aforementioned unnatural amino acid having an electrophilic group is (S)-1-acryloylpyrrolidine- 3-Carboxamide, 1-Acrylopiperidine-4-Carboxamide, (R)-1 Acrylopiperidine Liloylpiperidine-3-carboxamide, (S)-1-acryloylpiperidine-3- Carboxamide, (S)-1-Acryloylpyrrolidine-2-carboxamide, (R)- 1-Acryloylpyrrolidine-2-carboxamide, (E)-4-(dimethylamino)br Ta-2-enamide, acrylamide, aziridine, diaziridine, azetidine, pyrrolidine N, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidin N, isothiazolidine, piperidine, piperazine, morpholine, thiomorpholine, azepa Neazirin, diazirin, azeto, pyrrole, imidazole, pyrazole, oxazole , isoxazole, thiazole, isothiazole, pyridine, diazine, oxazine, Thiazine, azepinephenyl (aniline), naphthalene, anthracene, phenanthrene Indole, isoindole, indoridine, quinolone, isoquinoline, quinoxaline Phthalidine, quinazoline, purine, carbazole, indazole, benzimidazole Azaindole, α-cyanoacrylamide, propioamide, trans-4-dimethicone Luamino-2-butenamide, trans-4-piperidinyl-2-butenamide, substituted activator Selected from the group consisting of lylamides and vinyl sulfonamides, as described in item 30. Peptides. (Item 32) The electrophilic warhead is the cysteine-reactive D-nipecotinic acid portion, as stated in item 25. A peptide as described in any one of item 29. (Item 33) The electrophilic warhead is a cysteine-reactive portion, one of items 25 to 29. The peptides listed in the section. (Item 34) The electrophilic warhead is located at the N-terminus of the peptide, one of items 25 to 33 The peptides listed in the section. (Item 35) The electrophilic warhead is not at the N-terminus of the peptide, one of items 25 to 33 The peptides listed in the section. (Item 36) If the aforementioned staples are present, then position i and position i+3, position i and position i+4, or The amino acid between position i and position i+7, as described in any one of items 25 to 33. Petit Do. (Item 37) At least two amino acids of sequence number 93, having olefin side chains, are unnatural amino acids. Peptides as described in any one of items 25 to 36, including substitution with acid. (Item 38) The aforementioned unnatural amino acids having olefin side chains include S-pentenylalanine and R-octane. Tenylalanine; R-propenylalanine, S-pentenylalanine; R-pentenylalanine Lanine, S-pentenylalanine; bis-pentenylglycine, S-pentenylalanine , R-octenylalanine; and bis-pentenylglycine, S-octenylalanine A peptide selected from the group consisting of R-octenylalanine, as described in item 37. (Item 39) Hydrocarbon staples / stitches, lactam staples / stitches, UV-cyclization addition staples Staples / stitching, oxime staples / stitching, thioether staples / stitching Double-click staples / stitches, screws - lactum staples / stitches, screws - Arylated staples / stitches, or any combination of two or more of these. Peptides, including those listed in any one of items 25 to 36. (Item 40) A peptide that covalently binds to BFL-1 / A1, comprising an electrophilic warhead and the following: It includes a sequence that is at least 14% identical to the amino acid sequence described, [ka] A peptide that selectively binds to BFL-1 / A1 rather than MCL-1. (Item 41) A peptide described in item 40, with a length of 8 to 18 amino acids. (Item 42) The electrophilic warhead is located at the N-terminus of the peptide, as described in item 40 or 41. Petit Do. (Item 43) The electrophilic warhead is not at the N-terminus of the peptide as described in item 40 or 41. Petit Do. (Item 44) The electrophilic warhead is a non-natural amino acid having an electrophilic group, items 40 to 4 The peptide described in any one of item 3. (Item 45) The aforementioned unnatural amino acid having an electrophilic group is linked to the polypeptide skeleton. The peptide according to item 44, having an acrylamide or a substituted acrylamide. (Item 46) The aforementioned unnatural amino acid having an electrophilic group is (S)-1-acryloylpyrrolidine- 3-Carboxamide, 1-Acrylopiperidine-4-Carboxamide, (R)-1 Acrylopiperidine Liloylpiperidine-3-carboxamide, (S)-1-acryloylpiperidine-3- Carboxamide, (S)-1-Acryloylpyrrolidine-2-carboxamide, (R)- 1-Acryloylpyrrolidine-2-carboxamide, (E)-4-(dimethylamino)br Ta-2-enamide, acrylamide, aziridine, diaziridine, azetidine, pyrrolidine N, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidin N, isothiazolidine, piperidine, piperazine, morpholine, thiomorpholine, azepa Neazirin, diazirin, azeto, pyrrole, imidazole, pyrazole, oxazole , isoxazole, thiazole, isothiazole, pyridine, diazine, oxazine, Thiazine, azepinephenyl (aniline), naphthalene, anthracene, phenanthrene Indole, isoindole, indoridine, quinolone, isoquinoline, quinoxaline Phthalidine, quinazoline, purine, carbazole, indazole, benzimidazole Azaindole, α-cyanoacrylamide, propioamide, trans-4-dimethicone Luamino-2-butenamide, trans-4-piperidinyl-2-butenamide, substituted activator Selected from the group consisting of lylamides and vinyl sulfonamides, as described in item 45. Peptides. (Item 47) The electrophilic warhead is the cysteine-reactive D-nipecotinic acid portion, as stated in item 40. A peptide as described in any one of item 44. (Item 48) The electrophilic warhead is a cysteine-reactive portion, one of items 40 to 44 The peptides listed in the section. (Item 49) Hydrocarbon staples / stitches, lactam staples / stitches, UV-cyclization addition staples Staples / stitching, oxime staples / stitching, thioether staples / stitching Double-click staples / stitches, screws - lactum staples / stitches, screws - Arylated staples / stitches, or any combination of two or more of these. Peptides, including those listed in any one of items 40 to 48. (Item 50) At least 15%, 20%, 27%, 34%, 40%, and 47% of the aforementioned amino acid sequence. %, 50%, 53%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95 Any of items 40 through 49, which include sequences that are %, 97%, or 100% identical. Peptides as described in item 1. (Item 51) 86% variation from the aforementioned amino acid sequence indicates that the BFL-1 non-interaction of the aforementioned amino acid sequence Peptides located on the ulfa helix plane, as described in item 40 or 41. (Item 52) A peptide that covalently binds to BFL-1 / A1, and the amino acids listed below Includes sequences that are at least 14% identical to the sequence, [ka] The peptide selectively binds to BFL-1 / A1 rather than MCL-1, and the peptide is B It is covalently bonded to FL-1 / A1 and aloofly bonded to MCL-1. A peptide in which J is the electrophilic warhead and X1 and X2 are unnatural amino acids. (Item 53) A peptide described in item 52, with a length of 8 to 18 amino acids. (Item 54) The electrophilic warhead is a non-natural amino acid having an electrophilic group, item 52 or The peptide described in 53. (Item 55) At least 15%, 20%, 27%, 34%, 40%, and 47% of the aforementioned amino acid sequence. %, 50%, 53%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95 Items 52, 53, or 54 include sequences that are %, 97%, or 100% identical. Peptides. (Item 56) 86% variation from the aforementioned amino acid sequence indicates that the BFL-1 non-interaction of the aforementioned amino acid sequence Peptides located on the ulfa helix plane, as described in item 52 or 53. (Item 57) The 2nd, 3rd, 6th, 7th, 8th, 9th, 12th, 13th, 14th, and 15th positions of the above sequence , or one or more amino acids at position 16 (position 1 is "J") The peptides described in any one of items 52 to 56 may be substituted with other amino acids. Do. (Item 58) The 2nd, 3rd, 6th, 7th, 8th, 9th, 12th, 13th, 14th, and 15th positions of the above sequence , or one or more amino acids at position 16 (position 1 is "J") Substituted with alanine, D-alanine, or α-aminoisobutyric acid, items 52 to 5 A peptide as described in any one of item 6. (Item 59) The amino acid at position 7 and / or 15 is substituted with glutamic acid. The amino acid at position 13 is substituted with aspartic acid, and / or the amino acid at position 16 is substituted with leucine, and / or position 2 , 3rd, 6th, 8th, 9th, 12th, 14th, and 15th place (one or more of these) The amino acid in is substituted with alanine, D-alanine, or α-aminoisobutyric acid. The peptides listed in item 52 or 53 (the number one is "J"). (Item 60) The amino acid at position 8 and / or 12 is alanine, D-alanine, or α- The peptide described in item 59, which is substituted with aminoisobutyric acid. (Item 61) The peptide contains the sequence JATX1LREFGDX2LNFRQ (SEQ ID NO: 62) or derived from, The peptide selectively binds to BFL-1 / A1 rather than MCL-1, and the peptide It binds to BFL-1 / A1 by covalent bonds and to MCL-1 by non-covalent bonds, J is an electrophilic warhead, and X1 and X2 are unnatural amino acids, item 52. The peptide described in 53 or 54. (Item 62) The peptide contains the sequence JATX1LRRAGDX2LNFRQ (SEQ ID NO: 60) or derived from, The peptide selectively binds to BFL-1 / A1 rather than MCL-1, and the peptide It binds to BFL-1 / A1 by covalent bonds and to MCL-1 by non-covalent bonds, J is an electrophilic warhead, and X1 and X2 are unnatural amino acids, item 52. The peptide described in 53 or 54. (Item 63) The electrophilic warhead has an electrophilic portion linked to a polypeptide skeleton, and a linker However, nitrogen-containing heterocyclic amino acids, or amino-functionalized benzene rings, carbon A peptide, as described in any one of items 52 to 62, which is cyclic, polycyclic, or heterocyclic. (Item 64) The electrophilic moiety is an electrophilic acrylamide, a substituted acrylamide, or a vinyl sulfonamide. A peptide as described in item 63, which is an amide or α,β unsaturated amide. (Item 65) The electrophilic warhead is a non-natural amino acid having an electrophilic group, items 52 to 6 The peptide described in any one of item 2. (Item 66) The aforementioned unnatural amino acid having an electrophilic group is (S)-1-acryloylpyrrolidine- 3-Carboxamide, 1-Acrylopiperidine-4-Carboxamide, (R)-1 Acrylopiperidine Liloylpiperidine-3-carboxamide, (S)-1-acryloylpiperidine-3- Carboxamide, (S)-1-Acryloylpyrrolidine-2-carboxamide, (R)- 1-Acryloylpyrrolidine-2-carboxamide, (E)-4-(dimethylamino)br Ta-2-enamide, acrylamide, aziridine, diaziridine, azetidine, pyrrolidine N, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidin N, isothiazolidine, piperidine, piperazine, morpholine, thiomorpholine, azepa Neazirin, diazirin, azeto, pyrrole, imidazole, pyrazole, oxazole , isoxazole, thiazole, isothiazole, pyridine, diazine, oxazine, Thiazine, azepinephenyl (aniline), naphthalene, anthracene, phenanthrene Indole, isoindole, indoridine, quinolone, isoquinoline, quinoxaline Phthalidine, quinazoline, purine, carbazole, indazole, benzimidazole Azaindole, α-cyanoacrylamide, propioamide, trans-4-dimethicone Luamino-2-butenamide, trans-4-piperidinyl-2-butenamide, substituted activator Selected from the group consisting of lylamides and vinyl sulfonamides, as described in item 65. Peptides. (Item 67) The electrophilic warhead is the cysteine-reactive D-nipecotinic acid portion, as stated in item 52. A peptide as described in any one of item 62. (Item 68...

Claims

[Claim 1] The invention described in the specification.

Images