Selective targeting of ubiquitin- and ubiquitin-like e1-activating enzymes by structurally-stabilized peptides

Abstract

To provide selective targeting of ubiquitin- and ubiquitin-like E1-activating enzymes by structurally-stabilized peptides.SOLUTION: This disclosure features structurally stabilized and / or warhead-bearing structurally stabilized peptide inhibitors for targeting ubiquitin activating enzymes (E1). Methods of using such structurally stabilized peptides and warhead-bearing structurally stabilized peptides in the treatment of E1-expressing cancers or diseases or E1-dependent cancers or diseases are also disclosed. Combination therapies comprising such structurally stabilized and / or warhead-bearing structurally stabilized peptides for the treatment of E1-expressing diseases or E1-dependent diseases are also provided.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 835,721, filed on April 18, 2019, the content of which is hereby incorporated by reference in its entirety. Description of Research Funded by the Federal Government

[0002] This invention was made with government support under grant numbers R35 CA197583 and F30 CA221087 awarded by the National Institutes of Health. The government has certain rights in this invention. Reference to the Sequence Listing

[0003] This application contains a sequence listing submitted electronically in ASCII format, which is hereby incorporated by reference in its entirety. The ASCII copy was created on April 17, 2020, has the name 00530 - 0348WO1_SL.txt, and is 720,896 bytes in size. Technical Field

[0004] The present disclosure relates to structurally stabilized peptides that target ubiquitin and ubiquitin - like E1 activating enzymes, and to methods for using such peptides in the treatment of cancer and other diseases related to the survival of diseased cells.

Background Art

[0005] The ubiquitin-proteasome system (UPS) is a highly regulated enzymatic network responsible for intracellular proteolysis. The ubiquitin-activating enzyme (E1) catalyzes the transfer of ubiquitin (Ub) to the ubiquitin-conjugating enzyme (E2). E1 first catalyzes the adenylation of the C-terminus of Ub and then activates Ub by forming a high-energy thioester bond between the C-terminus of Ub and the catalytic cysteine of E1. E1 then binds to E2 and transfers Ub from the catalytic cysteine of E1 to the catalytic cysteine of E2, thereby effecting the transfer to E2. E2 then forms a complex with the ubiquitin ligase (E3) and the substrate protein and transfers the carboxyl group at the C-terminus of Ub to the substrate protein by a covalent bond. The diverse configurations of single and multiple Ub bindings to the target protein represent a complex code that modulates protein function and targets the protein for proteasomal degradation. Several drugs targeting the UPS, including immunomodulatory imide drugs (IMiDs) that redirect E3 activity and proteasome inhibitors (e.g., bortezomib for multiple myeloma), have been clinically successful in multiple myeloma and mantle cell lymphoma.

[0006] The human genome contains over 600 ubiquitin E3 enzymes, approximately 40 ubiquitin E2 enzymes, and only two ubiquitin E1 enzymes, namely UBA1 and UBA6. UBA6 is an E1 for only one ubiquitin E2 (USE1), whereas UBA1 is an E1 for all other ubiquitin E2s and correspondingly activates more than 99% of cellular ubiquitin (Jin et al., Nature, 447: 1135-1138 (2007)). Since the entire UPS system depends almost entirely on UBA1, inhibition of UBA1 This results in the stabilization of substrates that are normally degraded, endoplasmic reticulum stress, and cell cycle arrest (Hyer et al., Nature Medicine, 24(2):186-193 (2018)). The clinical-grade UBA1 inhibitor TAK243 / MLN7243 has been developed and shown to be highly effective in preclinical mouse models of solid tumors and multiple myeloma (Hyer et al., Nature Medicine, 24(2):186-193 (2018)), and recently completed a phase 1 clinical trial for advanced solid tumors (NCT02045095). However, very recent studies have identified a resistance mechanism based on point mutations to the mechanism of action of the drug (Barghout et al., Leukemia, 2018 Jun 8. doi: 10.1038 / s41375-018-0167-0), highlighting the need for and novelty of potential alternative modes related to inhibition of targeting of the E1 enzyme. The need and novelty of potential alternative modes related to inhibition of targeting of the E1 enzyme are emphasized.

[0007] In parallel with the ubiquitin system, several other ubiquitin-like proteins (UBLs) covalently attach to substrate proteins and serve as signals for various fates. These UBLs have their own activating and conjugating enzymes. UBLs that show the highest homology to the ubiquitin conjugation system include NEDD8 and SUMO (Hochstrasser, Nature, 458: 422-429 (2009)). The E1s for NEDD8 and SUMO They are UBA3-NAE1 and UBA2-SAE1, respectively, which are heterodimeric complexes. Inhibitors of UBA3-NAE1 (MLN4924 / pevonedistat) and UBA2-SAE1 (ML-792) with mechanisms similar to MLN7243 have been developed (Soucy et al., Nature, 458(7239):732-736 (2009); He et al., Nat Chem Biol., 13(11):1164-1171 (2017)). NEDD8 controls the activity of cullin-RING ubiquitin E3 ligase, and correspondingly, MLN4924 exerts its therapeutic effect by inhibiting cullin-RING-mediated protein turnover. SUMO has multiple cellular roles including the regulation of mitosis, and indeed, ML-792 causes mitotic perturbations. Both molecules show potent anti-cancer activity in vitro, and MLN4924 is currently in multiple Phase 2 and Phase 3 trials for solid tumors and hematological malignancies (NCT03268954, NCT02610777, NCT03228186, NCT03238248, NCT03330821, NCT03319537). MLN7243, MLN4924, and ML-792 are adenosyl sulfamates that bind to the ATP-binding sites of UBA1, UBA3, and UBA2, respectively. Cancer cell resistance to ATP-competitive inhibitors often occurs due to mutations at or around the target enzyme's ATP-binding site (Krishnamurty and Maly, ACS Chem Biol., 5(1):121-138 (2010)); indeed, due to the selective pressure in cancer cells, UBA3 cells, UBA3 A171T , UBA3 A171D , UBA3 I310N and UBA3 Y352HThrough positive selection of a clone population expressing a mutant version of UBA3 that contains, over time, results in reduced sensitivity to MLN4924 (Milhollen et al., Cancer Cell, 21(3):388-401 (2012); Toth et al., Cell Rep., 1(4):309-316 (2012); Xu et al., PLoS One, 9(4):e93530 (2014)). UBA1 A580T UBA1 A580S and UBA1 Y583C are also resistant to MLN7243 (Misra et al., Structure, 25(7): 1120-1129 (2017), Barghout et al., Leukemia, 2018 Jun 8. doi: 10.1038 / s41375-018-0167-0), and UBA2 S 95N,M97T is resistant to ML-792 (He et al., Nat Chem Biol., 13(11):1164-1171 (2017)). Thus, despite the initial success of these inhibitors, the clear threat of resistance due to active-site mutations requires the development of alternative modes of inhibition for these enzymes for the treatment of hematological malignancies and solid tumors. To date, no other drug lead compounds have been developed that exhibit alternative modes of ubiquitin E1 activating enzyme inhibition.

Prior Art Documents

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

[0009] Therefore, there is a need for new inhibitors of these ubiquitin and ubiquitin-like activating enzymes.

[0010] E1 first catalyzes the adenylation of the C-terminus of Ub and then activates Ub by forming a high-energy thioester bond between the C-terminus of Ub and the catalytic cysteine of E1. Then, E1 binds to E2 and transfers Ub from the catalytic cysteine of E1 to the catalytic cysteine of E2 for delivery to E2. E2 then forms a complex with ubiquitin ligase (E3) and the substrate protein and transfers the carboxyl group at the C-terminus of Ub to the substrate protein by covalent bond. The interaction between E1 and E2 buries 3000 Å of the protein surface area, of which 1000 Å 2 is filled, and 1000 Å 2is buried at the interface between helix A of E2 (E2 hA) and the E1 ubiquitin fold domain (E1 UFD). The interaction between E1 and E2 hA is important in the formation of the E1-E2 encounter complex. The present disclosure can act as a direct inhibitor of E1, for example, by competitive inhibition of the E1-E2 interaction (see, e.g., E1 and E2 in Tables 1-5), a structurally stabilized (e.g., stapled) alpha-helical peptide that mimics E2 hA (e.g., SEQ ID NO Provide a stapled version of 1-37, 39, 55-63, or 792-806. In certain embodiments, a structurally stabilized (e.g., stapled) E2 hA peptide binds to an E1 UFD (e.g., amino acids 950-1058 of SEQ ID NO: 845; amino acids 444-552 of SEQ ID NO: 846; amino acids 445-561 of SEQ ID NO: 847; amino acids 950-1052 of SEQ ID NO: 848; or amino acids 911-1012 of SEQ ID NO: 849). In some cases, a structurally stabilized (e.g., stapled) E2 hA peptide has 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions in any one of SEQ ID NOs: 1-37, 39, 55-63, or 792-806. In certain examples, when methionine is present in the peptide or the structurally stabilized peptide, methionine is replaced with norleucine. In some cases, a structurally stabilized (e.g., stapled) peptide has 1-5 deletions at the N-terminus or C-terminus. These structurally stabilized variant E2 hA peptides inhibit the E1-E2 interaction. In some cases, these peptides inhibit E1-mediated thioester transfer to E2 in a dose-dependent manner. The present disclosure also provides versions having a warhead of the stabilized (e.g., stapled) E2 hA peptides described herein that can covalently bind to cognate E1. The present disclosure features methods for using such stabilized peptides (or versions having their warheads) alone or in combination with other therapeutic agents in the treatment of E1-dependent cancers and / or cancers expressing E1 (e.g., hematological malignancies, solid tumors, or other specific cancers described herein below) and other diseases involved in the survival of diseased cells (e.g., antibody-mediated transplant rejection, autoimmune disorders, or inflammatory disorders).

[0011] Provide structural stabilization (e.g., internal cross-linking, e.g., stapling) to the E2 described herein Modifications for introduction into the hA peptide can be located on (i) the face of E2 hA that is not involved in direct interaction with its cognate E1 enzyme, (ii) the interface between the polar and non-polar faces of E2 hA, and / or (iii) the face of E2 hA that directly interacts with its cognate E1 enzyme. In certain examples, the structurally stabilized (e.g., internally cross-linked, e.g., stapled) E2 hA peptides described herein may also contain one or more (e.g., 1, 2, 3, 4, 5) additional amino acid substitutions (compared to the wild-type E2 hA peptide sequence), e.g., one or more (e.g., 1, 2, 3, 4, 5) conservative and / or non-conservative amino acid substitutions (i.e., in addition to the amino acid substitutions made to confer structural stability to E2 hA, one or more amino acid substitutions). In certain examples, where methionine is present within the peptide or the structurally stabilized peptide, methionine is replaced with norleucine. In certain examples, these additional substitutions are amino acid substitutions that directly interact with the cognate E1 enzyme of E2 hA. In certain examples, these additional substitutions are amino acid substitutions that are not involved in direct interaction with the cognate E1 enzyme of E2 hA. In certain examples, these additional substitutions are substitutions of both amino acids that directly interact with the cognate E1 enzyme of E2 hA and amino acids that are not involved in direct interaction with the cognate E1 enzyme of E2 hA. In certain examples, the structurally stabilized (e.g., internally cross-linked, e.g., stapled) E2 hA peptides described herein may contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions from the N-terminus and / or C-terminus of E2 hA. For example, the structurally stabilized (e.g., internally cross-linked, e.g., stapled) E2 hA peptide can be 5 or more (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) amino acids in length. In certain examples, the structurally stabilized (e.g., internally cross-linked, e.g., stapled) E2 hA peptide is 5-11 amino acids in length. In certain examples, the structurally stabilized (e.g., internally cross-linked, e.g., stapled) E2 hA peptide is 5-16 amino acids in length.In certain instances, the structurally stabilized (e.g., internally cross-linked, e.g., stapled) E2 hA peptide is 11 to 16 amino acids in length.

[0012] Accordingly, provided herein are peptides comprising the amino acid sequence of at least 8 contiguous amino acids of the sequence of SEQ ID NO: 132 having from 0 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions. In certain embodiments, the substitutions, when present, are not at positions 5 and 12 of SEQ ID NO: 132. In certain embodiments, the peptide inhibits the human E1-human E2 interaction. In certain embodiments, the peptide inhibits human E1-mediated thioester transfer to human E2. In certain embodiments, position 5 is R-octenylalanine and position 12 is S-pentenylalanine. Also provided herein are salts of the peptides. In some examples, the salt is an acetate, sulfate, or chloride.

[0013] In certain embodiments, the peptide has from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions, and the substitutions are not at positions 7 and 9 of SEQ ID NO: 132, or positions 7 or 9. In certain embodiments, the peptide has from 1 to 10 amino acid substitutions, and the substitutions are not at one or more (e.g., 1, 2, 3, 4, 5) of positions 7, 9, 10, 13, and 16 of SEQ ID NO: 132. In certain embodiments, the peptide has from 1 to 10 amino acid substitutions, and the substitutions are at one or more of positions 7, 9, 10, 13, and 16 of SEQ ID NO: 132, and the substitutions are substitutions to alanine at one or more of these positions. In certain embodiments, the peptide has from 1 to 10 amino acid substitutions, and the substitutions are at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12) of positions 1, 2, 3, 4, 6, 8, 11, 13, 14, 15, 16, or 17 of SEQ ID NO: 132. In some cases, these positions are substituted with alanine. In some cases, positions 6 and 8 of SEQ ID NO: 132, or positions 6 or 8, are substituted with glutamic acid. In certain embodiments, the peptide has from 1 to 10 amino acid substitutions, and the substitutions include one or more of positions 6 or 8 of SEQ ID NO: 132. In some cases, these positions are substituted with glutamic acid. In certain embodiments, the peptide further comprises a reactive group capable of forming a covalent bond with a cysteine residue in human E1, and optionally, the reactive group is a non-natural amino acid having an electrophilic group or electrophilic chemical cap at the N-terminus. The reactive group may be present in or from N to C of SEQ ID NO: 132 (i.e., the amino acids between the N-terminus and the C-terminus may be substituted with a non-natural amino acid having an electrophilic group or electrophilic chemical cap at the N-terminus).

[0014] In certain embodiments, the peptide is 8 to 50, 8 to 40, 8 to 30, 8 to 25, 8 to 20, 8 to 17, 10 to 50, 10 to 40, 10 to 30, 10 to 25, 10 to 20, 10 to 17, 15 to 50, 15 to 40, 15 to 30, 15 to 25, 15 to 20, 15 to 17, 17 to 50, 17 to 40, 17 to 30, 17 to 25, 17 to 20, or 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In certain embodiments, the peptide or a salt thereof is internally cross-linked. In some cases, the side chains of non-natural amino acids containing olefin side chains are linked.

[0015] A peptide or a salt thereof, wherein the peptide comprises, from the N-terminus to the C-terminus, the following residues: Xaa1 = B, A (where B is norleucine or absent); Xaa2 = S, A, or absent; Xaa3 = T, A, or absent; Xaa4 = P, A, or absent; Xaa5 = stapling amino acid Xaa6 = R, E, or A; Xaa7 = R or A; Xaa8 = R, E, or A; Xaa9 = L, A, or a reactive group capable of forming a covalent bond with a cysteine residue in human E1 (optionally, the reactive group is a non-natural amino acid having an electrophilic group or an electrophilic chemical cap at the N-terminus); Xaa10 = B or A (where B is norleucine); Xaa11 = R or A; Xaa12 = stapling amino acid; Xaa13 = F, A, a reactive group capable of forming a covalent bond with a cysteine residue in human E1 (optionally, the reactive group is a non-natural amino acid having an electrophilic group or an electrophilic chemical cap at the N-terminus), or absent; Xaa14 = K, R, A, or absent; Xaa15 = R, A, or absent Xaa16 = L, A, or absent; and Xaa17 = Q, A, or absent comprising at least 8 contiguous amino acids of the sequence having A peptide or a salt thereof that inhibits the interaction between human E1 and human E2 is also disclosed herein. In some cases, the peptide inhibits human E1-mediated thioester transfer to human E2.

[0016] In certain embodiments, the peptide is 8 - 50, 8 - 40, 8 - 30, 8 - 25, 8 - 20, 8 - 17, 10 - 50, 10 - 40, 10 - 30, 10 - 25, 10 - 20, 10 - 17, 15 - 50, 15 - 40, 15 - 30, 15 - 25, 15 - 20, 15 - 17, 17 - 50, 17 - 40, 17 - 30, 17 - 25, 17 - 20, or 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In certain embodiments, the peptide or a salt thereof is crosslinked internally. In some cases, the side chains of non-natural amino acids containing olefin side chains are linked.

[0017] In another aspect, the disclosure provides a peptide comprising an amino acid sequence of any one of SEQ ID NOs: 680, 727, 752, 841, 842, or 844 having 1 - 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions, wherein the stapled positions of these peptides (i.e., non-natural The peptide is characterized in that the position having the natural amino acid) is not substituted. In some cases, the residues predicted to interact directly with E1 are either not substituted or are substituted with alanine or a conservative amino acid. All other residues may be substituted. In certain examples, if methionine is present in the peptide or the structurally stabilized peptide, the methionine is substituted with norleucine. These peptides inhibit the E1-E2 interaction. In some examples, they inhibit E1-mediated thioester transfer to E2 in a dose-dependent manner.

[0018] Methods for making structurally stabilized peptides are also provided herein. In some examples, the method includes obtaining a peptide as described herein and crosslinking the peptide. In some examples, the crosslinking is by an RCM reaction. In some examples, the method further includes formulating the crosslinked peptide as a pharmaceutical composition.

[0019] Pharmaceutical compositions comprising any of the peptides or salts thereof disclosed herein and a pharmaceutically acceptable carrier are also provided herein.

[0020] Also provided herein is a method of treating a disease that expresses E1 or a disease that depends on E1 in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of any of the peptides, salts thereof, or pharmaceutical compositions disclosed herein. In some examples, the disease that expresses E1 or the disease that depends on E1 is cancer. In some examples, the disease that expresses E1 or the disease that depends on E1 is a hematologic malignancy, a solid tumor, an antibody-mediated transplant rejection, an autoimmune disorder, or an inflammatory disorder. In some examples, the human subject has been non-responsive or has developed resistance to treatment for treating a disease that expresses E1 or a disease that depends on E1.

[0021] In another aspect, a peptide comprising a modified amino acid sequence shown by any one of amino acids A1 to A11 with SEQ ID NOs: 1 to 33, wherein one or more of the A1 to A11 amino acids are replaced by another amino acid, and the modified amino acid sequence comprises a modification that stabilizes at least one peptide structure, and the peptide binds to ubiquitin-activating enzyme 1 (UBA1) and inhibits it, the peptide is provided herein.

[0022] In certain embodiments, the modification that stabilizes the peptide structure comprises substitution of at least two amino acids of the sequence shown by any one of amino acids A1 to A 11 with a non-natural amino acid, and the non-natural amino acid comprises an olefin side chain.

[0023] In certain embodiments, the modification that stabilizes the peptide structure is a hydrocarbon staple / stitch, a lactam staple / stitch; a UV cycloaddition staple / stitch; an oxime staple / stitch; a thioether staple / stitch; a double click staple / stitch; a bis-lactam staple / stitch; a bis-arylation staple / stitch; or any combination of two or more thereof. In certain embodiments, the modification that stabilizes the peptide structure is a stitch.

[0024] In certain embodiments, the modification that stabilizes the peptide structure is a staple. In certain embodiments, the staple is present at one or more of two positions in the amino acid sequence, and the two positions are i and i + 3, i and i + 4, i and i + 6, or i and i + 7. In certain embodiments, the staple is present at one or more of two positions in the amino acid sequence, and the two positions are A2 and A9, A5 and A9, A8 and A 12 、A9 and A 13 、A1 and A8, A4 and A 11 、as well as A5 and A 12Selected from the group consisting of. In certain embodiments, the staples are present at two positions, the two positions being A2 and A9. In certain embodiments, the staples each contain an amino acid substitution at each of the two positions, each of the amino acid substitutions being a substitution with a non-natural amino acid, the non-natural amino acid containing an olefin side chain(s).

[0025] In certain embodiments where the peptide of the first aspect contains a non-natural amino acid containing an olefin side chain, the non-natural amino acid containing an olefin side chain is selected from the group consisting of S-pentenylalanine, R-octenylalanine; R-propenylalanine, S-pentenylalanine; R-pentenylalanine, S-pentenylalanine; Bis-pentenylglycine, S-pentenylalanine, R-octenylalanine; and Bis-pentenylglycine, S-octenylalanine, and R-octenylalanine.

[0026] In certain embodiments, (a) the modified amino acid sequence contains a modified amino acid sequence of the sequence shown for amino acids A1 - A of SEQ ID NO: 4, and the modified amino acid sequence contains 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 4; or (b) the modified amino acid sequence contains a modified amino acid sequence of the sequence shown for amino acids A1 - A of SEQ ID NO: 6, and the modified amino acid sequence contains 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 6; or (c) the modified amino acid sequence contains a modified amino acid sequence of the sequence shown for amino acids A1 - A of SEQ ID NO: 1, and the modified amino acid sequence contains 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 1. 11 including a modified amino acid sequence of the sequence shown, and the modified amino acid sequence contains 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 4; 11 or (b) the modified amino acid sequence includes a modified amino acid sequence of the sequence shown for amino acids A1 - A of SEQ ID NO: 6, and the modified amino acid sequence contains 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 6; 11 or (c) the modified amino acid sequence includes a modified amino acid sequence of the sequence shown for amino acids A1 - A of SEQ ID NO: 1, and the modified amino acid sequence contains 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 1. 11 or (c) the modified amino acid sequence contains a modified amino acid sequence of the sequence shown for amino acids A1 - A of SEQ ID NO: 1, and the modified amino acid sequence contains 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 1. 11 including a modified amino acid sequence of the sequence shown, and the modified amino acid sequence contains 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 1; 11comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of; (d) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 2, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 2 11 and comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 2, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 2 11 comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of; (e) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 3, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 3 11 and comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 3, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 3 11 comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of; (f) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 5, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 5 11 and comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 5, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 5 11 comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of; (g) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 7, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 7 11 and comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 7, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 7 11 comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of; (h) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 8, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 8 11 and comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 8, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 8 11 comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of; (i) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 9, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 9 11 and comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 9, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 911 comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of; (j) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 10, and the modified amino acid sequence is A1 to A of SEQ ID NO: 10 11 comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 10, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 10 11 comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of; (k) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 11, and the modified amino acid sequence is A1 to A of SEQ ID NO: 11 11 comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 11, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 11 11 comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of; (l) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 12, and the modified amino acid sequence is A1 to A of SEQ ID NO: 12 11 comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 12, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 12 11 comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of; (m) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 13, and the modified amino acid sequence is A1 to A of SEQ ID NO: 13 11 comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 13, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 13 11 comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of; (n) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 14, and the modified amino acid sequence is A1 to A of SEQ ID NO: 14 11 comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 14, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 14 11 comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of; (o) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 15, and the modified amino acid sequence is A1 to A of SEQ ID NO: 15 11comprising a modified amino acid sequence of the sequence shown in 11 and the modified amino acid sequence contains 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of 11 ; (p) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in 11 and the modified amino acid sequence contains 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of 11 ; (q) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in 11 and the modified amino acid sequence contains 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of 11 ; (r) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in 11 and the modified amino acid sequence contains 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of 11 ; (s) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in 11 and the modified amino acid sequence contains 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of 11 ; (t) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in 11at the amino acid positions selected from the group consisting of 0, 1, 2, 3, 4, or 5 amino acid substitutions; (u) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 21, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 21 11 comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 2, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A 11 at the amino acid positions selected from the group consisting of 0, 1, 2, 3, 4, or 5 amino acid substitutions; (v) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 22, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 22 11 comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 3, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A 11 at the amino acid positions selected from the group consisting of 0, 1, 2, 3, 4, or 5 amino acid substitutions; (w) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 23, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 23 11 comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 4, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A 11 at the amino acid positions selected from the group consisting of 0, 1, 2, 3, 4, or 5 amino acid substitutions; (x) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 24, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 24 11 comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 5, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A 11 at the amino acid positions selected from the group consisting of 0, 1, 2, 3, 4, or 5 amino acid substitutions; (y) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 25, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 25 11 comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 6, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A 11 at the amino acid positions selected from the group consisting of 0, 1, 2, 3, 4, or 5 amino acid substitutions; (z) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 26, and the modified amino acid sequence is A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 26 11comprising a modified amino acid sequence of the sequence shown therein, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 26; or (aa) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 27, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 27; or (bb) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 28, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 28; or (cc) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 29, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 29; or (dd) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 30, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 30; or (ee) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 31, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 31; 11 amino acid substitutions; or (aa) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 27, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 27; or (bb) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 28, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 28; or (cc) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 29, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 29; or (dd) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 30, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 30; or (ee) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 31, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 31; to A of SEQ ID NO: 27; or (bb) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 28, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 28; or (cc) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 29, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 29; or (dd) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 30, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 30; or (ee) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 31, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 31; 11 comprising a modified amino acid sequence of the sequence shown therein, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 27; or (bb) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 28, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 28; or (cc) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 29, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 29; or (dd) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 30, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 30; or (ee) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 31, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 31; 11 amino acid substitutions; or (bb) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 28, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 28; or (cc) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 29, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 29; or (dd) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 30, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 30; or (ee) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 31, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 31; 11 comprising a modified amino acid sequence of the sequence shown therein, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 28; or (cc) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 29, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 29; or (dd) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 30, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 30; or (ee) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 31, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 31; 11 amino acid substitutions; or (cc) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 29, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 29; or (dd) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 30, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 30; or (ee) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 31, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 31; 11 comprising a modified amino acid sequence of the sequence shown therein, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 29; or (dd) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 30, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 30; or (ee) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 31, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 31; 11 amino acid substitutions; or (dd) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 30, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 30; or (ee) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 31, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 31; 11 comprising a modified amino acid sequence of the sequence shown therein, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 30; or (ee) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 31, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 31; 11 amino acid substitutions; or (ee) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 31, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 31; 11 comprising a modified amino acid sequence of the sequence shown therein, the modified amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 31; 11comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at an amino acid position selected from the group consisting of; (ff) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 32, and the modified amino acid sequence has 0, 1, 2, 3, 4, or 5 amino acid substitutions at an amino acid position selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 32; or (gg) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 33, and the modified amino acid sequence has 0, 1, 2, 3, 4, or 5 amino acid substitutions at an amino acid position selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 33. 11 comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 32, and the modified amino acid sequence has 0, 1, 2, 3, 4, or 5 amino acid substitutions at an amino acid position selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 32; or (gg) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 33, and the modified amino acid sequence has 0, 1, 2, 3, 4, or 5 amino acid substitutions at an amino acid position selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 33. 11 comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at an amino acid position selected from the group consisting of; or (gg) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 33, and the modified amino acid sequence has 0, 1, 2, 3, 4, or 5 amino acid substitutions at an amino acid position selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 33. 11 comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 33, and the modified amino acid sequence has 0, 1, 2, 3, 4, or 5 amino acid substitutions at an amino acid position selected from the group consisting of A1, A2, A3, A5, A8, A9, and A of SEQ ID NO: 33. 11 comprises an amino acid sequence having 0, 1, 2, 3, 4, or 5 amino acid substitutions at an amino acid position selected from the group consisting of.

[0027] In certain embodiments, (a) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 4, and the modified amino acid sequence has 0, 1, or 2 conservative amino acid substitutions at an amino acid position selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 4; (b) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 6, and the modified amino acid sequence has 0, 1, or 2 conservative amino acid substitutions at an amino acid position selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 6; (c) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 1, and the modified amino acid sequence has 0, 1, or 2 conservative amino acid substitutions at an amino acid position selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 1; (d) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 2, and the modified amino acid sequence has 0, 1, or 2 conservative amino acid substitutions at an amino acid position selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 2. 11 comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 4, and the modified amino acid sequence has 0, 1, or 2 conservative amino acid substitutions at an amino acid position selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 4; (b) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 6, and the modified amino acid sequence has 0, 1, or 2 conservative amino acid substitutions at an amino acid position selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 6; (c) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 1, and the modified amino acid sequence has 0, 1, or 2 conservative amino acid substitutions at an amino acid position selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 1; (d) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 2, and the modified amino acid sequence has 0, 1, or 2 conservative amino acid substitutions at an amino acid position selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 2. 10 comprises an amino acid sequence having 0, 1, or 2 conservative amino acid substitutions at an amino acid position selected from the group consisting of; (b) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 6, and the modified amino acid sequence has 0, 1, or 2 conservative amino acid substitutions at an amino acid position selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 6; (c) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 1, and the modified amino acid sequence has 0, 1, or 2 conservative amino acid substitutions at an amino acid position selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 1; (d) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 2, and the modified amino acid sequence has 0, 1, or 2 conservative amino acid substitutions at an amino acid position selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 2. 11 comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 6, and the modified amino acid sequence has 0, 1, or 2 conservative amino acid substitutions at an amino acid position selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 6; (c) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 1, and the modified amino acid sequence has 0, 1, or 2 conservative amino acid substitutions at an amino acid position selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 1; (d) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 2, and the modified amino acid sequence has 0, 1, or 2 conservative amino acid substitutions at an amino acid position selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 2. 10 comprises an amino acid sequence having 0, 1, or 2 conservative amino acid substitutions at an amino acid position selected from the group consisting of; (c) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 1, and the modified amino acid sequence has 0, 1, or 2 conservative amino acid substitutions at an amino acid position selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 1; (d) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 2, and the modified amino acid sequence has 0, 1, or 2 conservative amino acid substitutions at an amino acid position selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 2. 11 comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 1, and the modified amino acid sequence has 0, 1, or 2 conservative amino acid substitutions at an amino acid position selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 1; (d) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 2, and the modified amino acid sequence has 0, 1, or 2 conservative amino acid substitutions at an amino acid position selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 2. 10 comprises an amino acid sequence having 0, 1, or 2 conservative amino acid substitutions at an amino acid position selected from the group consisting of; (d) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 2, and the modified amino acid sequence has 0, 1, or 2 conservative amino acid substitutions at an amino acid position selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 2. 11comprising a modified amino acid sequence of the sequence shown in, the modified amino acid sequence having 0, 1, or 2 conservative amino acid substitutions at amino acid positions selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 2; (e) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in, the modified amino acid sequence having 0, 1, or 2 conservative amino acid substitutions at amino acid positions selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 3; (f) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in, the modified amino acid sequence having 0, 1, or 2 conservative amino acid substitutions at amino acid positions selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 5; (g) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in, the modified amino acid sequence having 0, 1, or 2 conservative amino acid substitutions at amino acid positions selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 7; (h) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in, the modified amino acid sequence having 0, 1, or 2 conservative amino acid substitutions at amino acid positions selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 8; (i) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in, the modified amino acid sequence having 0, 1, or 2 conservative amino acid substitutions at amino acid positions selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 9; (j) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in, the modified amino acid sequence having 0, 1, or 2 conservative amino acid substitutions at amino acid positions selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 10; 10 from the group consisting of; (e) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in, the modified amino acid sequence having 0, 1, or 2 conservative amino acid substitutions at amino acid positions selected from the group consisting of A1 to A of SEQ ID NO: 3; 11 comprising a modified amino acid sequence of the sequence shown in, the modified amino acid sequence having 0, 1, or 2 conservative amino acid substitutions at amino acid positions selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 3; 10 from the group consisting of; (f) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in, the modified amino acid sequence having 0, 1, or 2 conservative amino acid substitutions at amino acid positions selected from the group consisting of A1 to A of SEQ ID NO: 5; 11 comprising a modified amino acid sequence of the sequence shown in, the modified amino acid sequence having 0, 1, or 2 conservative amino acid substitutions at amino acid positions selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 5; 10 from the group consisting of; (g) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in, the modified amino acid sequence having 0, 1, or 2 conservative amino acid substitutions at amino acid positions selected from the group consisting of A1 to A of SEQ ID NO: 7; 11 comprising a modified amino acid sequence of the sequence shown in, the modified amino acid sequence having 0, 1, or 2 conservative amino acid substitutions at amino acid positions selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 7; 10 from the group consisting of; (h) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in, the modified amino acid sequence having 0, 1, or 2 conservative amino acid substitutions at amino acid positions selected from the group consisting of A1 to A of SEQ ID NO: 8; 11 comprising a modified amino acid sequence of the sequence shown in, the modified amino acid sequence having 0, 1, or 2 conservative amino acid substitutions at amino acid positions selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 8; 10 from the group consisting of; (i) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in, the modified amino acid sequence having 0, 1, or 2 conservative amino acid substitutions at amino acid positions selected from the group consisting of A1 to A of SEQ ID NO: 9; 11 comprising a modified amino acid sequence of the sequence shown in, the modified amino acid sequence having 0, 1, or 2 conservative amino acid substitutions at amino acid positions selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 9; 10 from the group consisting of; (j) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in, the modified amino acid sequence having 0, 1, or 2 conservative amino acid substitutions at amino acid positions selected from the group consisting of A1 to A of SEQ ID NO: 10; 11 comprising a modified amino acid sequence of the sequence shown in, the modified amino acid sequence having 0, 1, or 2 conservative amino acid substitutions at amino acid positions selected from the group consisting of A4, A6, A7, and A of SEQ ID NO: 10;10 at an amino acid position selected from the group consisting of 0, 1, or 2 conservative amino acid substitutions; (k) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 11, and the modified amino acid sequence is A1 to A of SEQ ID NO: 11 11 including the modified amino acid sequence shown in the sequence, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 11 10 at an amino acid position selected from the group consisting of 0, 1, or 2 conservative amino acid substitutions; (l) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 12, and the modified amino acid sequence is A1 to A of SEQ ID NO: 12 11 including the modified amino acid sequence shown in the sequence, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 12 10 at an amino acid position selected from the group consisting of 0, 1, or 2 conservative amino acid substitutions; (m) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 13, and the modified amino acid sequence is A1 to A of SEQ ID NO: 13 11 including the modified amino acid sequence shown in the sequence, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 13 10 at an amino acid position selected from the group consisting of 0, 1, or 2 conservative amino acid substitutions; (n) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 14, and the modified amino acid sequence is A1 to A of SEQ ID NO: 14 11 including the modified amino acid sequence shown in the sequence, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 14 10 at an amino acid position selected from the group consisting of 0, 1, or 2 conservative amino acid substitutions; (o) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 15, and the modified amino acid sequence is A1 to A of SEQ ID NO: 15 11 including the modified amino acid sequence shown in the sequence, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 15 10 at an amino acid position selected from the group consisting of 0, 1, or 2 conservative amino acid substitutions; (p) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 16, and the modified amino acid sequence is A1 to A of SEQ ID NO: 16 11 including the modified amino acid sequence shown in the sequence, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 16 10at the amino acid positions selected from the group consisting of 0, 1, or 2 conservative amino acid substitutions; (q) the modified amino acid sequence comprises the modified amino acid sequence of the sequence shown in SEQ ID NO: 17, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 17 11 including the modified amino acid sequence of the sequence shown in SEQ ID NO: 17, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 17 10 at the amino acid positions selected from the group consisting of 0, 1, or 2 conservative amino acid substitutions; (r) the modified amino acid sequence comprises the modified amino acid sequence of the sequence shown in SEQ ID NO: 18, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 18 11 including the modified amino acid sequence of the sequence shown in SEQ ID NO: 18, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 18 10 at the amino acid positions selected from the group consisting of 0, 1, or 2 conservative amino acid substitutions; (s) the modified amino acid sequence comprises the modified amino acid sequence of the sequence shown in SEQ ID NO: 19, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 19 11 including the modified amino acid sequence of the sequence shown in SEQ ID NO: 19, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 19 10 at the amino acid positions selected from the group consisting of 0, 1, or 2 conservative amino acid substitutions; (t) the modified amino acid sequence comprises the modified amino acid sequence of the sequence shown in SEQ ID NO: 20, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 20 11 including the modified amino acid sequence of the sequence shown in SEQ ID NO: 20, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 20 10 at the amino acid positions selected from the group consisting of 0, 1, or 2 conservative amino acid substitutions; (u) the modified amino acid sequence comprises the modified amino acid sequence of the sequence shown in SEQ ID NO: 21, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 21 11 including the modified amino acid sequence of the sequence shown in SEQ ID NO: 21, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 21 10 at the amino acid positions selected from the group consisting of 0, 1, or 2 conservative amino acid substitutions; (v) the modified amino acid sequence comprises the modified amino acid sequence of the sequence shown in SEQ ID NO: 22, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 22 11 including the modified amino acid sequence of the sequence shown in SEQ ID NO: 22, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 22 10at an amino acid position selected from the group consisting of 0, 1, or 2 conservative amino acid substitutions; (w) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 23, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 23 11 comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 23, and the modified amino acid sequence is A4, A6, A7, and A 10 at an amino acid position selected from the group consisting of 0, 1, or 2 conservative amino acid substitutions; (x) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 24, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 24 11 comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 24, and the modified amino acid sequence is A4, A6, A7, and A 10 at an amino acid position selected from the group consisting of 0, 1, or 2 conservative amino acid substitutions; (y) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 25, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 25 11 comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 25, and the modified amino acid sequence is A4, A6, A7, and A 10 at an amino acid position selected from the group consisting of 0, 1, or 2 conservative amino acid substitutions; (z) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 26, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 26 11 comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 26, and the modified amino acid sequence is A4, A6, A7, and A 10 at an amino acid position selected from the group consisting of 0, 1, or 2 conservative amino acid substitutions; (aa) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 27, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 27 11 comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 27, and the modified amino acid sequence is A4, A6, A7, and A 10 at an amino acid position selected from the group consisting of 0, 1, or 2 conservative amino acid substitutions; (bb) the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 28, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 28 11 comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 28, and the modified amino acid sequence is A4, A6, A7, and A 10at the amino acid positions selected from the group consisting of 0, 1, or 2 conservative amino acid substitutions; or (cc) the modified amino acid sequence comprises the modified amino acid sequence of the sequence shown in SEQ ID NO: 29, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 29 11 including the modified amino acid sequence of the sequence shown in SEQ ID NO: 29, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 29 10 at the amino acid positions selected from the group consisting of 0, 1, or 2 conservative amino acid substitutions; or (dd) the modified amino acid sequence comprises the modified amino acid sequence of the sequence shown in SEQ ID NO: 30, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 30 11 including the modified amino acid sequence of the sequence shown in SEQ ID NO: 30, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 30 10 at the amino acid positions selected from the group consisting of 0, 1, or 2 conservative amino acid substitutions; or (ee) the modified amino acid sequence comprises the modified amino acid sequence of the sequence shown in SEQ ID NO: 31, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 31 11 including the sequence shown in SEQ ID NO: 31 including the modified amino acid sequence of the sequence shown in SEQ ID NO: 31, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 31 10 at the amino acid positions selected from the group consisting of 0, 1, or 2 conservative amino acid substitutions; or (ff) the modified amino acid sequence comprises the modified amino acid sequence of the sequence shown in SEQ ID NO: 32, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 32 11 including the modified amino acid sequence of the sequence shown in SEQ ID NO: 32, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 32 10 at the amino acid positions selected from the group consisting of 0, 1, or 2 conservative amino acid substitutions; or (gg) the modified amino acid sequence comprises the modified amino acid sequence of the sequence shown in SEQ ID NO: 33, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 33 11 including the modified amino acid sequence of the sequence shown in SEQ ID NO: 33, and the modified amino acid sequence is A4, A6, A7, and A of SEQ ID NO: 33 10 at the amino acid positions selected from the group consisting of 0, 1, or 2 conservative amino acid substitutions.

[0028] In certain embodiments, one or more of amino acids A1 - A 11 substituted with another amino acid are A1 - A of SEQ ID NOs: 1 - 33 11It is present on the surface that does not interact with E1. In certain embodiments, the non-interacting surfaces of SEQ ID NOs: 1-33 are amino acids A1, A2, A5, A8, A9, and A 12 include.

[0029] In certain embodiments, any one of amino acids A1-A of SEQ ID NOs: 1-33 11 The 0-6 amino acids on the non-interacting surface are replaced with amino acids selected from the group consisting of alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives.

[0030] In certain embodiments, one or more of amino acids A4, A6, A7, and A 10 are replaced with naturally occurring amino acids that are alpha-methylated or alpha-ethylated. In certain embodiments, one or more of amino acids A4, A6, A7, and A 10 are replaced with amino acids selected from the group consisting of L-alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives.

[0031] In certain embodiments, one or more of amino acids A1, A2, A3, A5, A8, A9, and A 11 are replaced with naturally occurring amino acids that are alpha-methylated or alpha-ethylated. In certain embodiments, one or more of amino acids A1, A2, A3, A5, A8, A9, and A 11 are replaced with amino acids selected from the group consisting of L-alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives.

[0032] In certain embodiments, the modified amino acid sequence includes the modified amino acid sequence of the sequence shown for amino acids A1-A of SEQ ID NO: 4 11 .

[0033] In certain embodiments, the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in amino acids A1 to A of SEQ ID NO: 6 11 as shown in the sequence shown in 11 .

[0034] In certain embodiments, the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in any one of SEQ ID NOs: 1 to 33. In certain embodiments, one or more amino acids of SEQ ID NOs: 1 to 33 are replaced with one or more amino acids selected from the group consisting of L-alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives. In certain embodiments, 0 to 5 amino acids of SEQ ID NOs: 1 to 33 are removed from the C-terminus or removed and replaced with 1 to 6 amino acids from the group consisting of alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives. In certain embodiments, one or more amino acids of SEQ ID NOs: 1 to 33 replaced with another amino acid are present on a face that does not interact with E1 of the amino acids of SEQ ID NOs: 1 to 33. In certain embodiments, the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 4. In certain embodiments, the modified amino acid sequence comprises a modified amino acid sequence of the sequence shown in SEQ ID NO: 6.

[0035] In certain embodiments, (i) the overall hydrophobicity of the peptide is reduced compared to the peptides of SEQ ID NOs: 1 to 33; (ii) the overall positive charge of the peptide is reduced compared to the peptides of SEQ ID NOs: 1 to 33; or (iii) a combination of (i) and (ii).

[0036] In certain embodiments, the peptide comprises the sequence shown in SEQ ID NO: 132. In certain embodiments, the peptide comprises the sequence shown in SEQ ID NO: 133. In certain embodiments, the peptide comprises the sequence shown in SEQ ID NO: 107. In certain embodiments, the peptide comprises the sequence shown in SEQ ID NO: 50.

[0037] In yet another aspect, a peptide comprising a modified amino acid sequence of the sequence shown in SEQ ID NO: 35 or 36, wherein one or more amino acids of SEQ ID NO: 35 or 36 are replaced with another amino acid, and the modified amino acid sequence comprises a modification that stabilizes at least one peptide structure, and the peptide binds to and inhibits ubiquitin-activating enzyme 3 (UBA3), is provided herein.

[0038] In certain embodiments, the modification that stabilizes the peptide structure comprises substitution of at least two amino acids of the sequence of SEQ ID NO: 35 or 36 with non-natural amino acids, and the non-natural amino acids comprise olefin side chains. In certain embodiments, the modification that stabilizes the peptide structure is a hydrocarbon staple / stitch, a lactam staple / stitch; a UV cycloaddition staple / stitch; an oxime staple / stitch; a thioether staple / stitch; a double click staple / stitch; a bis-lactam staple / stitch; a bis-arylation staple / stitch; or any combination of two or more thereof. In certain embodiments, the modification that stabilizes the peptide structure is a stitch.

[0039] In certain embodiments, the modification that stabilizes the peptide structure is a staple. In certain embodiments, the staple is present at one or more of two positions in the amino acid sequence, and the two positions are i and i + 3, i and i + 4, or i and i + 7. In certain embodiments, the staple comprises an amino acid substitution at each of the two positions, and each of the amino acid substitutions is a substitution with a non-natural amino acid, and the non-natural amino acids comprise olefin side chains.

[0040] In certain embodiments where the peptide comprises non-natural amino acids containing olefin side chains, the non-natural amino acids containing olefin side chains are selected from the group consisting of S-pentenylalanine, R-octenylalanine; R-propenylalanine, S-pentenylalanine; R-pentenylalanine, S-pentenylalanine; Bis-pentenylglycine, S-pentenylalanine, R-octenylalanine; and Bis-pentenylglycine, S-octenylalanine, and R-octenylalanine.

[0041] In certain embodiments, (a) the modified amino acid sequence comprises the modified amino acid sequence of SEQ ID NO: 35, and the modified amino acid sequence contains 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of Arg-1, Arg-2, Ser-4, Val-5, Arg-6, Asp-7, Leu-9, Leu-10, Glu-13, Ala-15, and Glu-16 of SEQ ID NO: 35, or (b) the modified amino acid sequence comprises the modified amino acid sequence of SEQ ID NO: 36, and the modified amino acid sequence contains 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of Lys-1, Ser-4, Ala-5, Ala-6, Arg-9, Ile-10, and Asp-134 of SEQ ID NO: 36.

[0042] In certain embodiments, (a) the modified amino acid sequence comprises the modified amino acid sequence of SEQ ID NO: 35, and the modified amino acid sequence contains 0, 1, or 2 conservative amino acid substitutions at amino acid positions selected from the group consisting of Val-3, Lys-8, Val-11, Lys-12, and Val-14 of SEQ ID NO: 35, or (b) the modified amino acid sequence comprises the modified amino acid sequence of SEQ ID NO: 36, and the modified amino acid sequence contains 0, 1, or 2 conservative amino acid positions selected from the group consisting of Lys-2, Ala-3, Gln-7, Leu-8, Gln-11, Lys-12, Ile-14, Asn-15, and Glu-16 of SEQ ID NO: 36.

[0043] In certain embodiments, one or more amino acids substituted with another amino acid are present on a surface that does not interact with E1 of SEQ ID NO: 35 or 36. In certain embodiments, the non-interacting surface of SEQ ID NO: 35 includes the amino acids Arg-1, Arg-2, Ser-4, Val-5, Arg-6, Asp-7, Leu-9, Leu-10, Glu-13, Ala-15, and Glu-16 of SEQ ID NO: 25, and the non-interacting surface of SEQ ID NO: 36 includes the amino acids Lys-1, Ser-4, Ala-5, Ala-6, Arg-9, Ile-10, and Asp-13 of SEQ ID NO: 36.

[0044] In certain embodiments, 0 to 6 amino acids on the non-interacting surface of SEQ ID NO: 35 or 36 are substituted with an amino acid selected from the group consisting of alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives.

[0045] In certain embodiments, one or more of amino acids B7, B8, B 11 、B 12 、B 15 、およびB 16 are substituted with a natural amino acid that is alpha-methylated or alpha-ethylated. In certain embodiments, one or more of amino acids B7, B8, B 11 、B 12 、B 15 、およびB 16 are substituted with an amino acid selected from the group consisting of L-alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives.

[0046] In certain embodiments, one or more of amino acids B1, B2, B3, B4, B5, B6, B9, B 10 、B 13 、およびB 14 are substituted with a natural amino acid that is alpha-methylated or alpha-ethylated. In certain embodiments, one or more of amino acids B1, B2, B3, B4, B5, B6, B9, B 10, B 13 , and B 14 One or more of is substituted with an amino acid selected from the group consisting of L-alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives.

[0047] In certain embodiments, 0 to 5 amino acids of SEQ ID NO: 35 or 36 have been removed from the C-terminus or removed and replaced with 1 to 6 amino acids from the group consisting of alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives.

[0048] In certain embodiments, (i) the overall hydrophobicity of the peptide is reduced compared to the peptide of SEQ ID NO: 35 or 36; (ii) the overall positive charge of the peptide is reduced compared to the peptide of SEQ ID NO: 35 or 36; or (iii) a combination of (i) and (ii).

[0049] In a third aspect, a peptide comprising a modified amino acid sequence of the sequence shown in SEQ ID NO: 37, wherein one or more amino acids of SEQ ID NO: 37 are substituted with another amino acid, and the modified amino acid sequence comprises a modification that stabilizes at least one peptide structure, and the peptide binds to and inhibits ubiquitin-activating enzyme 2 (UBA2), the peptide is provided herein.

[0050] In certain embodiments, the modification to stabilize the peptide structure comprises substitution of at least two amino acids of the sequence of SEQ ID NO: 35 or 36 with non-natural amino acids, where the non-natural amino acids comprise olefin side chains. In certain embodiments, the modification to stabilize the peptide structure is a hydrocarbon staple / stitch, a lactam staple / stitch; a UV cycloaddition staple / stitch; an oxime staple / stitch; a thioether staple / stitch; a double click staple / stitch; a bis-lactam staple / stitch; a bis-arylation staple / stitch; or any combination of two or more thereof. In certain embodiments, the modification to stabilize the peptide structure is a stitch.

[0051] In certain embodiments, the modification to stabilize the peptide structure is a staple. In certain embodiments, the staple is present at one or more of two positions in the amino acid sequence, where the two positions are i and i + 3, i and i + 4, or i and i + 7. In certain embodiments, the staple comprises an amino acid substitution at each of the two positions, and each of the amino acid substitutions is a substitution with a non-natural amino acid, where the non-natural amino acid comprises an olefin side chain.

[0052] In certain embodiments where the peptide comprises a non-natural amino acid comprising an olefin side chain, the non-natural amino acid comprising an olefin side chain is selected from the group consisting of S-pentenylalanine, R-octenylalanine; R-propenylalanine, S-pentenylalanine; R-pentenylalanine, S-pentenylalanine; Bis-pentenylglycine, S-pentenylalanine, R-octenylalanine; and Bis-pentenylglycine, S-octenylalanine, and R-octenylalanine.

[0053] In certain embodiments, the modified amino acid sequence comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of Ala-1, Arg-4, Leu-5, Glu-8, Ala-11, Trp-12, and Asp-15 of SEQ ID NO: 37.

[0054] In certain embodiments, the modified amino acid sequence comprises 0, 1, or 2 conservative amino acid substitutions at amino acid positions selected from the group consisting of Leu-2, Ser-3, Ala-6, Gln-7, Arg-9, Lys-10, Arg-13, and Lys-14 of SEQ ID NO: 37.

[0055] In certain embodiments, one or more amino acids substituted with another amino acid are present on a face that does not interact with E1 of SEQ ID NO: 37. In certain embodiments, the non-interacting face of SEQ ID NO: 37 comprises the amino acids Ala-1, Arg-4, Leu-5, Glu-8, Ala-11, Trp-12, and Asp-15 of SEQ ID NO: 37.

[0056] In certain embodiments, 0 to 6 amino acids on the non-interacting face of SEQ ID NO: 37 are substituted with amino acids selected from the group consisting of alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives.

[0057] In certain embodiments, one or more of the amino acids Leu-2, Ser-3, Ala-6, Gln-7, Arg-9, Lys-10, Arg-13, and Lys-14 of SEQ ID NO: 37 are substituted with alpha-methylated or alpha-ethylated natural amino acids. In certain embodiments, one or more of the amino acids Leu-2, Ser-3, Ala-6, Gln-7, Arg-9, Lys-10, Arg-13, and Lys-14 of SEQ ID NO: 37 are substituted with amino acids selected from the group consisting of L-alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives.

[0058] In certain embodiments, one or more of the amino acids Ala-1, Arg-4, Leu-5, Glu-8, Ala-11, Trp-12, and Asp-15 of SEQ ID NO: 37 are substituted with alpha-methylated or alpha-ethylated natural amino acids. In certain embodiments, one or more of the amino acids Ala-1, Arg-4, Leu-5, Glu-8, Ala-11, Trp-12, and Asp-15 of SEQ ID NO: 37 are substituted with an amino acid selected from the group consisting of L-alanine, D-alanine, alpha-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives.

[0059] In certain embodiments, 0 to 5 amino acids of SEQ ID NO: 37 are removed from the C-terminus or removed and replaced with 1 to 6 amino acids from the group consisting of alanine, D-alanine, alpha-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives.

[0060] In certain embodiments, (i) the overall hydrophobicity of the peptide is decreased compared to the peptide of SEQ ID NO: 37; (ii) the overall positive charge of the peptide is decreased compared to the peptide of SEQ ID NO: 37; or (iii) a combination of (i) and (ii).

[0061] In a fourth aspect, a peptide comprising a modified amino acid sequence of the sequence shown in SEQ ID NO: 38, wherein one or more amino acids of SEQ ID NO: 38 are substituted with another amino acid, and the modified amino acid sequence comprises a modification that stabilizes at least one peptide structure, and the peptide binds to and inhibits ubiquitin-activating enzyme 6 (UBA6), is provided herein.

[0062] In certain embodiments, the modification to stabilize the peptide structure includes substitution of at least two amino acids of the sequence of SEQ ID NO: 38 with non-natural amino acids, and the non-natural amino acids include olefin side chains. In certain embodiments, the modification to stabilize the peptide structure is a hydrocarbon staple / stitch, a lactam staple / stitch; a UV cycloaddition staple / stitch; an oxime staple / stitch; a thioether staple / stitch; a double click staple / stitch; a bis-lactam staple / stitch; a bis-arylation staple / stitch; or any combination of two or more thereof. In certain embodiments, the modification to stabilize the peptide structure is a stitch.

[0063] In certain embodiments, the modification to stabilize the peptide structure is a staple. In certain embodiments, the staple is present at one or more of two positions in the amino acid sequence, and the two positions are i and i + 3, i and i + 4, or i and i + 7. In certain embodiments, the staple includes an amino acid substitution at each of the two positions, and each of the amino acid substitutions is a substitution with a non-natural amino acid, and the non-natural amino acid includes an olefin side chain.

[0064] In certain embodiments where the peptide includes a non-natural amino acid with an olefin side chain, the non-natural amino acid with an olefin side chain is selected from the group consisting of S-pentenylalanine, R-octenylalanine; R-propenylalanine, S-pentenylalanine; R-pentenylalanine, S-pentenylalanine; Bis-pentenylglycine, S-pentenylalanine, R-octenylalanine; and Bis-pentenylglycine, S-octenylalanine, and R-octenylalanine.

[0065] In certain embodiments, the modified amino acid sequence comprises 0, 1, 2, 3, 4, or 5 amino acid substitutions at amino acid positions selected from the group consisting of Met-1, Ala-2, Ala-3, Ser-4, Leu-6, Asn-9, Leu-10, Arg-12, Leu-13, Ser-15, Arg-16, and Cys-17 of SEQ ID NO: 38.

[0066] In certain embodiments, the modified amino acid sequence comprises 0, 1, or 2 conservative amino acid substitutions at amino acid positions selected from the group consisting of Arg-5, Glu-7, Leu-8, Val-11, and Leu-14 of SEQ ID NO: 38.

[0067] In certain embodiments, one or more amino acids substituted with another amino acid are present on a surface that does not interact with E1 of SEQ ID NO: 38. In certain embodiments, the non-interacting surface of SEQ ID NO: 38 comprises the amino acids Met-1, Ala-2, Ala-3, Ser-4, Leu-6, Asn-9, Leu-10, Arg-12, Leu-13, Ser-15, Arg-16, and Cys-17 of SEQ ID NO: 38.

[0068] In certain embodiments, 0 to 6 amino acids on the non-interacting surface of SEQ ID NO: 38 are substituted with amino acids selected from the group consisting of alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives.

[0069] In certain embodiments, one or more of the amino acids Arg-5, Glu-7, Leu-8, Val-11, and Leu-14 of SEQ ID NO: 38 are substituted with alpha-methylated or alpha-ethylated natural amino acids. In certain embodiments, one or more of the amino acids Arg-5, Glu-7, Leu-8, Val-11, and Leu-14 of SEQ ID NO: 38 are substituted with amino acids selected from the group consisting of L-alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives.

[0070] In certain embodiments, one or more of the amino acids Met-1, Ala-2, Ala-3, Ser-4, Leu-6, Asn-9, Leu-10, Arg-12, Leu-13, Ser-15, Arg-16, and Cys-17 of SEQ ID NO: 38 are replaced with alpha-methylated or alpha-ethylated natural amino acids. In certain embodiments, one or more of the amino acids Met-1, Ala-2, Ala-3, Ser-4, Leu-6, Asn-9, Leu-10, Arg-12, Leu-13, Ser-15, Arg-16, and Cys-17 of SEQ ID NO: 38 are replaced with an amino acid selected from the group consisting of L-alanine, D-alanine, alpha-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives.

[0071] In certain embodiments, 0 to 5 amino acids of SEQ ID NO: 38 are removed from the C-terminus or removed and replaced with 1 to 6 amino acids from the group consisting of alanine, D-alanine, alpha-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives.

[0072] In certain embodiments, (i) the overall hydrophobicity of the peptide is reduced compared to the peptide of SEQ ID NO: 38; (ii) the overall positive charge of the peptide is reduced compared to the peptide of SEQ ID NO: 38; or (iii) a combination of (i) and (ii).

[0073] In a fifth aspect, a peptide derivative of the peptide of the first aspect, the peptide derivative comprising an electrophilic warhead, is provided herein. In certain embodiments, the electrophilic warhead is present at amino acid position A7, A8, or A 11 which is present. In certain embodiments, the electrophilic warhead is present at the N-terminus of the peptide. In certain embodiments, the electrophilic warhead is not present at the N-terminus of the peptide.

[0074] In certain embodiments, the electrophilic warhead is a non-natural amino acid having an electrophilic group. In certain embodiments, the non-natural amino acid having an electrophilic group has an electrophilic acrylamide or a substituted acrylamide linked to the polypeptide backbone. In certain embodiments, the non-natural amino acid having an electrophilic group is (S)-1-acryloylpyrrolidine-3-carboxamide; 1-acrylopiperidine-4-carboxamide, (R)-1-acryloylpiperidine-3-carboxamide; (S)-1-acryloylpiperidine-3-carboxamide; (S)-1-acryloylpyrrolidine -2-carboxamide; (R)-1-acryloylpyrrolidine-2-carboxamide; (E)-4-(dimethylamino)but-2-enamide; acrylamide; aziridine, diaziridine, azetidine, pyrrolidine, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, piperidine, piperazine, morpholine, thiomorpholine, azepaneazirine, diazirine, azeto, pyrrole, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, pyridine, diazine, oxazine, thiazine, azepinephenyl (aniline); naphthalene, anthracene, phenanthrene, indole, isoindole, indolizine, quinoline, isoquinoline, quinoxaline, phthalzine, quinazoline, purine, carbazole, indazole, benzimidazole, azaindole, α-cyanoacrylamide, propiolamide, trans4-dimethylamino-2-butenamide, trans4-piperidinyl-2-butenamide, substituted acrylamide, and vinyl-sulfonamide. In certain embodiments, the electrophilic warhead is diaminobutyric acid with a terminal bromoacetyl or diaminobutyric acid with a terminal acrylamide. In certain embodiments, the electrophilic warhead is a cysteine-reactive D-nipetidic acid moiety. In certain embodiments, the electrophilic warhead is a cysteine-reactive moiety.

[0075] In certain embodiments, the derivative comprises the sequence shown in any one of SEQ ID NOs: 392, 428, 464, 500, 680, 716, 752, and 788. In certain embodiments, the derivative comprises the sequence shown in SEQ ID NO: 680. In certain embodiments, the derivative comprises the sequence shown in SEQ ID NO: 752. In certain embodiments, the derivative comprises the sequence shown in any one of SEQ ID NOs: 393, 429, 465, 501, 681, 717, 753, and 789. In certain embodiments, the derivative comprises the sequence shown in any one of SEQ ID NOs: 367, 403, 439, 655, 727, and 757. In certain embodiments, the derivative comprises the sequence shown in SEQ ID NO: 727. In certain embodiments, the derivative comprises the sequence shown in any one of SEQ ID NOs: 833 - 836 and 841 - 844. In certain embodiments, the derivative comprises the sequence shown in SEQ ID NO: 841. In certain embodiments, the derivative comprises the sequence shown in SEQ ID NO: 842. In certain embodiments, the derivative comprises the sequence shown in SEQ ID NO: 844.

[0076] In certain embodiments, the derivative covalently binds to UBA1.

[0077] In a sixth aspect, there is provided herein a pharmaceutical composition comprising a peptide as described in any one of the first to fourth aspects or a derivative peptide as described in the fifth aspect, and a pharmaceutically acceptable carrier.

[0078] In a seventh aspect, there is provided herein a method of treating a disease that expresses E1 or a disease that is dependent on E1 in a human subject in need thereof, the method comprising administering to the human subject a therapeutically effective amount of the peptide according to any one of the first to fourth aspects, the derivative peptide according to the fifth aspect, or the pharmaceutical composition according to the sixth aspect. In certain embodiments, the disease that expresses E1 or the disease that is dependent on E1 is cancer. In certain embodiments, the disease that expresses E1 or the disease that is dependent on E1 is a hematological malignancy, a solid tumor, an antibody-mediated transplant rejection, an autoimmune disorder, or an inflammatory disorder.

[0079] In an eighth aspect, the present disclosure features a composition comprising means for inhibiting the interaction between ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzyme (E2). In some examples, the means for inhibiting the interaction is a stabilized (e.g., stapled, stitched) peptide that inhibits the interaction between E1 and E2. In certain cases, the means for inhibiting the interaction is a stabilized peptide that mimics E2 hA (see, e.g., Tables 1-6). In some examples, the means for inhibiting the interaction is a peptide of Table 7, 8, or 9, or a variant thereof. In one case, the means for inhibiting the interaction is a stabilized peptide that is a stapled peptide based on UBE2A / UBE2B E2 hA (i.e., SEQ ID NO: 4). In one case, the means for inhibiting the interaction is a stabilized peptide that is SAH-UBE2A-11 (SEQ ID NO: 132) or a variant thereof. In one case, the means for inhibiting the interaction is a stabilized peptide comprising the sequence shown in SEQ ID NO: 65, 96, or 101, or a variant thereof. In some examples, the composition is a pharmaceutical composition and comprises a pharmaceutically acceptable carrier, excipient, or diluent.

[0080] In a ninth aspect, the present disclosure provides a method of treating a disease that expresses E1 or a disease that depends on E1 in a human subject in need thereof. The method includes administering to the human subject a therapeutically effective amount of a composition comprising a means for inhibiting the interaction between ubiquitin-activating enzyme (E1) and ubiquitin-conjugating enzyme (E2). In some examples, the means for inhibiting the interaction is a stabilized (e.g., stapled, stitched) peptide that inhibits the interaction between E1 and E2. In certain cases, the means for inhibiting the interaction is a stabilized peptide that mimics E2 hA (see, e.g., Tables 1-6). In some examples, the means for inhibiting the interaction is a peptide of Table 7, 8, or 9, or a variant thereof. In one case, the means for inhibiting the interaction is a stabilized peptide that is a stapled peptide based on UBE2A / UBE2B E2 hA (i.e., SEQ ID NO: 4). In one case, the means for inhibiting the interaction is a stabilized peptide that is SAH-UBE2A-11 (SEQ ID NO: 132), or a variant thereof. In one case, the means for inhibiting the interaction is a stabilized peptide comprising the sequence shown in SEQ ID NO: 65, 96, or 101, or a variant thereof. In some examples, the composition is a pharmaceutical composition and comprises a pharmaceutically acceptable carrier, excipient, or diluent. In certain examples, the disease that expresses E1 or the disease that depends on E1 is cancer. In certain examples, the disease that expresses E1 or the disease that depends on E1 is a hematological malignancy, a solid tumor, an antibody-mediated transplant rejection, an autoimmune disorder, an inflammatory disorder, or a disease involved in the survival of diseased cells. BRIEF DESCRIPTION OF THE DRAWINGS

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DETAILED DESCRIPTION OF THE INVENTION

[0110] The E1 enzyme first activates Ub by catalyzing the adenylation of the C-terminus of Ub and then forming a high-energy thioester bond between the C-terminus of Ub and the catalytic cysteine of E1. Then, E1 binds to E2 and transfers Ub from the catalytic cysteine of E1 to the catalytic cysteine of E2, thereby transferring it to E2. E2 then forms a complex with ubiquitin ligase (E3) and the substrate protein and transfers the carboxyl group at the C-terminus of Ub to the substrate protein by covalent bond. The interaction between E1 and E2 buries 3000 Å of the protein surface area 2 and 1000 Å of that 2is buried at the interface between helix A of E2 (E2 hA) and the E1 ubiquitin-fold domain (E1 UFD). Thus, the interaction between E1 and E2 is important in the formation of the E1-E2 encounter complex. The present disclosure provides, for example, structurally stabilized (e.g., stapled) alpha-helical peptides that mimic E2 hA and can act as direct inhibitors of E1 by competitive inhibition of the E1-E2 interaction. In certain embodiments, the structurally stabilized (e.g., stapled) E2 hA peptide binds to the E1 UFD. The present disclosure also provides versions having a warhead of the stabilized (e.g., stapled) E2 hA peptides described herein that can covalently bind to cognate E1. Without being bound by any theory, the stabilized E2 hA provided herein represents a mechanism for inhibiting E1 by preventing E1 from transferring to E2. The present disclosure features methods for using such stabilized peptides (or versions having a warhead thereof) alone or in combination with other therapeutic agents in the treatment of E1-dependent cancers and / or cancers expressing E1 (e.g., hematological malignancies, solid tumors, or other specific cancers described herein) as well as other diseases involved in the survival of diseased cells (e.g., antibody-mediated transplant rejection, autoimmune disorders, or inflammatory disorders). E1 protein The amino acid sequence of human UBA1 is provided below (GenBank accession number NP_695012):

Chemical formula

Chemical formula

[0111] The UFD of human UBA1 consists of amino acid residues 950 - 1058 of SEQ ID NO: 845.

[0112] The amino acid sequence of human UBA2 is provided below (GenBank accession number NP_005490):

Chemical formula

[0113] The UFD of human UBA2 consists of amino acid residues 444 to 552 of SEQ ID NO: 846.

[0114] The amino acid sequence of human UBA3 is provided below (GenBank accession number NP_003959): [Chemical formula] [Chemical formula]

[0115] The UFD of human UBA3 consists of amino acid residues 445 to 561 of SEQ ID NO: 847.

[0116] The amino acid sequence of human UBA6 is provided below (GenBank accession number NP_060697): [Chemical formula]

[0117] The UFD of human UBA6 consists of amino acid residues 950 to 1052 of SEQ ID NO: 848.

[0118] The amino acid sequence of S. pombe UBA1 is provided below (GenBank accession number CAA22354.2): [Chemical formula] [Chemical formula]

[0119] The UFD of S. pombe UBA1 consists of amino acid residues 911 to 1012 of SEQ ID NO: 849. E2 helix A peptide

[0120] Exemplary E2 hA peptides of the present disclosure are the peptides in Tables 1-5. Amino acids in the interacting surface of the E2 hA peptide contribute to the interaction between the E2 hA peptide and E1, for example, UBA1, UBA6, UBA2, or UBA3. In contrast, amino acids in the non-interacting surface of the E2 hA peptide are not involved in the direct interaction between the E2 hA peptide and E1, for example, UBA1, UBA6, UBA2, or UBA3. Residues involved in the direct interaction of the E2 hA peptide with its cognate E1 enzyme (for example, UBA1 related to SEQ ID NOs: 1-34, UBA6 related to SEQ ID NO: 38, UBA2 related to SEQ ID NO: 37, and UBA3 related to SEQ ID NOs: 35 and 36) are in bold in Tables 1-5, and all other amino acids do not participate in (or are predicted not to participate in) the direct interaction with its cognate E1 enzyme.

[0121] The present disclosure provides an E2 hA peptide that binds to and inhibits UBA1 E1. In certain embodiments, the E2 hA peptide binds to the UBA1 E1 UFD. Exemplary E2 hA peptides that bind to UBA1 of the present disclosure are provided in Table 1 (from S. pombe) and Table 2 (from H. sapiens). The numbering of the consensus positions (A # ) used for the peptides that bind to UBA1 are provided in Tables 1 and 2 below. Methods for evaluating UBA1 inhibition by the peptides are known in the art and are described herein.

[0122] Table 1. Peptides that bind to UBA1 from S. pombe. Residues that interact directly with UBA1 are in bold are.

Table 1

[0123] Residues of Ubc4 E2 hA (SEQ ID NO: 1) that interact directly with UBA1, for example, bind to UBA1, are amino acid consensus positions A1, A3, A4, A7, A 10, and A 11 It is. For example, in relation to the position numbering for SEQ ID NO: 1, the residues of Ubc4 E2 hA that directly interact with UBA1 are Met-1 (i.e., A1), Leu-3 (i.e., A3), Lys-4 (i.e., A4), Asn-7 (i.e., A7), Leu-10 (i.e., A 10 ), and Ala-11 (i.e., A 11 ) (see Lv et al., 2017, Mol. Cell, 65(4):699-714).

[0124] Residues of Ubc4 E2 hA (i.e., SEQ ID NO: 1) that do not participate in direct interaction with UBA1, for example, binding to UBA1, are A2, A5, A6, A8, A9, A 12 , A 13 , A 14 , A 15 , and A 16 It is. For example, in relation to the position numbering for SEQ ID NO: 1, the residues of Ubc4 E2 hA that do not participate in direct interaction with UBA1 are Ala-2 (i.e., A2), Arg-5 (i.e., A5), Ile-6 (i.e., A6), Arg-8 (i.e., A8), Glu-9 (i.e., A9), Asp-12 (i.e., A 12 ), Leu-13 (i.e., A 13 ), Gly-14 (i.e., A 14 ), Lys-15 (i.e., A 15 ), and Asp-16 (i.e., A 16 ).

[0125] Residues of Ubc15 E2 hA (SEQ ID NO: 2) that directly interact with UBA1, for example, bind to UBA1, are amino acid consensus positions A -4 , A -3 , A -2 , A -1 , A3, A7, A8, A 10 , A 11 , and A 14That is. For example, in relation to the positional numbering for SEQ ID NO: 2, the residues of Ubc15 E2 hA that directly interact with UBA1 are Met-1 (i.e., A -4 ), Pro-2 (i.e., A -3 ), Ser-3 (i.e., A -2 ), Ser-4 (i.e., A -1 ), Glu-7 (i.e., A3), Arg-11 (i.e., A7), Lys-12 (i.e., A8), Leu-14 (i.e., A 10 ), Lys-15 (i.e., A 11 ), and Gln-18 (i.e., A 14 ) (see Lv et al., 2017, Mol. Cell, 65(4):699-714) .

[0126] Residues of Ubc15 E2 hA (i.e., SEQ ID NO: 2) that do not participate in direct interaction with UBA1, for example, binding to UBA1, are A1, A2, A4, A5, A6, A9, A , A 12 , A 13 , A 15 , and A 16 . For example, in relation to the positional numbering for SEQ ID NO: 2, the residues of Ubc4 E2 hA that do not participate in direct interaction with UBA1 are Ala-5 (i.e., A1), Ser-6 (i.e., A2), Gln-8 (i.e., A4), Leu-9 (i.e., A5), Leu-10 (i.e., A6), Gln-13 (i.e., A9), Glu-16 (i.e., A 12 ), Ile-17 (i.e., A 13 ), Lys-19 (i.e., A 15 ), and Asn-20 (i.e., A 16 ).

[0127] The residues of E2 hA peptides of SEQ ID NOs: 3 to 20 and 22 to 34 that directly interact with UBA1, for example, are predicted to bind to UBA1, are at the amino acid consensus positions A4, A6, A7, A 10 , and optionally A 13is. For example, in relation to the position numbering for SEQ ID NO: 4, the residues of UBE2A E2 hA (SEQ ID NO: 4) predicted to directly interact with UBA1 are Arg-7 (i.e., A4), Leu-9 (i.e., A6), Met-10 (i.e., A7), Phe-13 (i.e., A 10 ), and optionally Leu-16 (i.e., A 13 ). In another example, in relation to the position numbering for SEQ ID NO: 6, the residues of UBE2G2 E2 hA (SEQ ID NO: 6) predicted to directly interact with UBA1 are Lys-7 (i.e., A4), Leu-9 (i.e., A6), Met-10 (i.e., A7), Tyr-13 (i.e., A 10 ), and optionally Leu-16 (i.e., A 13 ). In yet another example, in relation to the position numbering for SEQ ID NO: 10, the residues of UBE2D2 E2 hA (SEQ ID NO: 10) predicted to directly interact with UBA1 are Lys-4 (i.e., A4), Ile-6 (i.e., A6), His-7 (i.e., A7), Leu-10 (i.e., A 10 ), and optionally Leu-13 (i.e., A 13 ).

[0128] The residues of the E2 hA peptides of SEQ ID NOs: 3 - 20 and 22 - 34 predicted not to be involved in direct interaction with UBA1, e.g., binding to UBA1, are A1, A2, A3, A5, A8, A9, A 11 , A 12 , A 14 , A 15 , A 16 , and are at positions N-terminal to A1. For example, in relation to the position numbering for SEQ ID NO: 4, the residues of UBE2A E2 hA predicted not to be involved in direct interaction with UBA1 are Met-1 (i.e., A -3 ), Ser-2 (i.e., A -2 ), Thr-3 (i.e., A -1)、Pro-4 (i.e., A1), Ala-5 (i.e., A2), Arg-6 (i.e., A3), Arg-8 (i.e., A5), Arg-11 (i.e., A8), Asp-12 (i.e., A9), Lys-14 (i.e., A 11 )、Arg-15 (i.e., A 12 )、Gln-17 (i.e., A 14 )、Glu-18 (i.e., A 15 )、and Asp-19 (i.e., A 16 ). In another example, residues of UBE2G2 E2 hA predicted not to be involved in direct interaction with UBA1 in relation to the numbering of SEQ ID NO: 6 are Met-1 (i.e., A -3 ), Ala-2 (i.e., A -2 ), Gly-3 (i.e., A -1 ), Thr-4 (i.e., A1), Ala-5 (i.e., A2), Leu-6 (i.e., A3), Arg-7 (i.e., A5), Ala-10 (i.e., A8), Glu-11 (i.e., A9), Lys-13 (i.e., A 11 ), Gln-14 (i.e., A 12 ), Thr-16 (i.e., A 14 ), Leu-17 (i.e., A 15 ), and Asn-18 (i.e., A 16 ). In yet another example, residues of UBE2D2 E2 predicted not to be involved in direct interaction with UBA1 in relation to the numbering of SEQ ID NO: 10 are Met-1 (i.e., A1), Ala-2 (i.e., A2), Leu-3 (i.e., A3), Arg-5 (i.e., A5), Lys-8 (i.e., A8), Glu-9 (i.e., A9), Asn-11 (i.e., A 11 ), Asp-12 (i.e., A 12 ), Ala-14 (i.e., A 14 ), Arg-15 (i.e., A 15 ), and Asp-16 (i.e., A 16 ).

[0129] In one example, the present disclosure features a peptide having the sequence BPSSASRQLLRKQLKEIQZ (SEQ ID NO: 1340), where B is norleucine and Z is biotinylated lysine (Lys(biotin)). In some cases, the peptide includes at least 2 (e.g., 2, 3, 4, 5) amino acid substitutions, and the amino acids are substituted with non-natural amino acids having olefin side chains. In some cases, at least 2 of the substituted amino acids are separated by 2, 3, or 6 amino acids. These peptides inhibit the E1-E2 interaction.

[0130] In one example, the present disclosure features a peptide having the sequence BPSSASRQLLRKQLKEIQ (SEQ ID NO: 1341), where B is norleucine. In some cases, the peptide includes at least 2 (e.g., 2, 3, 4, 5) amino acid substitutions, and the amino acids are substituted with non-natural amino acids having olefin side chains. In some cases, at least 2 of the substituted amino acids are separated by 2, 3, or 6 amino acids. These peptides inhibit the E1-E2 interaction.

[0131] Table 2. Peptides that bind to UBA1 from H. sapiens. Residues predicted to interact directly with UBA1 are in bold (amino acid consensus positions A4, A6, A7, and A for SEQ ID NOs: 3 - 20 and 22 - 34, and optionally A 10 ; amino acid consensus positions A 13 for SEQ ID NO: 21, and A 2, A3, A4, A6, A7, A8, A -1 2, A 10 2, A 11 2, and A 14 2).

Table 2-1

Table 2-2

[0132] Residues of UBE2T E2 hA (SEQ ID NO: 21) that interact directly with UBA1, for example, bind to UBA1, are amino acid consensus positions A -1 , A2, A3, A4, A6, A7, A8, A 10 , A 11 , and A 14 . For example, in relation to the numbering of positions with respect to SEQ ID NO: 21, the residues of UBE2T E2 hA that interact directly with UBA1 are Met-1 (i.e., A -1 ), Arg-3 (i.e., A2), Ala-4 (i.e., A3), Ser-5 (i.e., A4), Leu-7 (i.e., A6), Lys-8 (i.e., A7), Arg-9 (i.e., A8), Leu-11 (i.e., A 10 ), His-12 (i.e., A 11 ), and Ala-15 (i.e., A 14 ) (see Lv et al., 2018, JBC, 293(47):18337-18352).

[0133] Residues of UBE2T E2 hA (SEQ ID NO: 21) that do not participate in direct interaction with UBA1, for example, binding to UBA1, are A1, A5, A9, A , A 12 , A 13 , A 15 , and A 16 . For example, in relation to the numbering of positions with respect to SEQ ID NO: 21, the residues of UBE2T E2 hA that do not participate in direct interaction with UBA1 are Gln-2 (i.e., A1), Arg-6 (i.e., A5), Glu-10 (i.e., A9), Met-13 (i.e., A 12 ), Leu-14 (i.e., A 13 ), Tyr-16 (i.e., A 15 ), and Glu-17 (i.e., A 16 ).

[0134] The present disclosure also provides an E2 hA peptide that binds to E1 UBA6 and inhibits E1 UBA6. In certain embodiments, the E2 hA peptide binds to UBA6 E1 UFD. Exemplary E2 hA peptides that bind to UBA6 of the present disclosure are provided in Table 3 (i.e., SEQ ID NO: 38). Methods for assessing UBA6 inhibition by the peptide are known in the art and are described herein.

[0135] In one example, the present disclosure features a peptide having the sequence BSTPARRRLBRDFKRLQZ (SEQ ID NO: 1342), where B is norleucine and Z is biotinylated lysine (Lys(biotin)). In some cases, the peptide includes at least two (e.g., 2, 3, 4, 5) amino acid substitutions, and the amino acids are substituted with unnatural amino acids having olefin side chains. In some cases, at least two substituted amino acids are separated by 2, 3, or 6 amino acids. These peptides inhibit the E1-E2 interaction.

[0136] In one example, the present disclosure features a peptide having the sequence BSTPARRRLBRDFKRLQ (SEQ ID NO: 1343), where B is norleucine. In some cases, the peptide includes at least two (e.g., 2, 3, 4, 5) amino acid substitutions, and the amino acids are substituted with unnatural amino acids having olefin side chains. In some cases, at least two substituted amino acids are separated by 2, 3, or 6 amino acids. These peptides inhibit the E1-E2 interaction.

[0137] Table 3. Peptides that bind to UBA6. Residues predicted to interact directly with UBA6 are in bold (amino acids Arg-5, Glu-7, Leu-8, Val-11, and Leu-14).

Table 3

[0138] In relation to the position numbering for SEQ ID NO: 38, the residues of USE1 E2 hA (SEQ ID NO: 38) that directly interact with UBA6, for example, are predicted to bind to UBA6, are Arg-5, Glu-7, Leu-8, Val-11, and Leu-14.

[0139] In relation to the position numbering for SEQ ID NO: 38, the residues of USE1 E2 hA (SEQ ID NO: 38) that are predicted not to be involved in direct interaction with UBA6, for example, binding to UBA6, are Met-1, Ala-2, Ala-3, Ser-4, Leu-6, Asn-9, Leu-10, Arg-12, Leu-13, Ser-15, Arg-16, and Cys-17.

[0140] The present disclosure also provides an E2 hA peptide that binds to and inhibits E1 UBA3. In certain embodiments, the E2 hA peptide binds to UBA3 E1 UFD. Exemplary human E2 hA peptides of the present disclosure that are capable of binding to UBA3 are provided in Table 4. Methods for assessing UBA3 inhibition by peptides are known in the art and are described herein

[0141] Table 4. Peptides that bind to UBA3. Residues that directly interact with UBA3 are in bold.

Table 4

[0142] In relation to the position numbering for SEQ ID NO: 35, the residues of UBE2F E2 hA (SEQ ID NO: 35) that directly interact with UBA3, for example, are predicted to bind to UBA3, are Val-3, Lys-8, Val-11, Lys-12, and Val-14 (see Huang et al., 2009, Mol. Cell, 33(4):483-95).

[0143] In relation to the positional numbering for SEQ ID NO: 35, the residues of UBE2F E2 hA (SEQ ID NO: 35) that do not participate in direct interaction with UBA3 are Arg-1, Arg-2, Ser-4, Val-5, Arg-6, Asp-7, Leu-9, Leu-10, Glu-13, Ala-15, and Glu-16.

[0144] In relation to the positional numbering for SEQ ID NO: 36, the residues of UBE2M E2 hA (SEQ ID NO: 36) that interact directly with UBA3, for example, bind to UBA3, are Lys-2, Ala-3, Gln-7, Leu-8, Gln-11, Lys-12, Ile-14, Asn-15, and Glu-16 (see Huang et al., 2005, Mol. Cell, 17(3):341-350).

[0145] In relation to the positional numbering for SEQ ID NO: 36, the residues of UBE2M E2 hA (SEQ ID NO: 36) that do not participate in direct interaction with UBA3 are Lys-1, Ser-4, Ala-5, Ala-6, Arg-9, Ile-10, and Asp-13.

[0146] The present disclosure also provides an E2 hA peptide that binds to and inhibits UBA2. In certain embodiments, the E2 hA peptide binds to UBA2 E1 UFD. Exemplary human E2 hA peptides of the present disclosure that are capable of binding to UBA2 are provided in Table 5 (i.e., SEQ ID NO: 37). Methods for assessing UBA2 inhibition by the peptide are known in the art and are described herein.

[0147] Table 5. Peptides that bind to UBA2. Residues that interact directly with UBA2 are in bold (amino acids Leu-2, Ser-3, Ala-6, Gln-7, Arg-9, Lys-10, Arg-13, and Lys-14).

Table 5

[0148] In relation to the position numbering for SEQ ID NO: 37, the residues of UBE2I E2 hA (SEQ ID NO: 37) that directly interact with UBA2, for example, bind to UBA2, are Leu-2, Ser-3, Ala-6, Gln-7, Arg-9, Lys-10, Arg-13, and Lys-14 (see Wang et al., 2010, PLoS ONE, 5(12):e15805). etc.).

[0149] In relation to the position numbering for SEQ ID NO: 37, the residues of UBE2I E2 hA (SEQ ID NO: 37) that are not involved in the direct interaction with UBA2 are Ala-1, Arg-4, Leu-5, Glu-8, Ala-11, Trp-12, and Asp-15.

[0150] In certain embodiments, peptides comprising modified amino acid sequences of the E2 hA peptides described herein are also provided herein. In some cases, the peptides are modified to introduce structural stabilization into the peptide (e.g., to maintain the alpha-helicity of the peptide). The structural stabilization may be, for example, by "stapling" or "stitching" of the peptide. In some cases, the staple or stitch is a hydrocarbon staple or stitch. Modifications that introduce structural stabilization (e.g., internal cross-linking, e.g., stapling, stitching) into the E2 hA peptides described herein may be located on (i) a face of E2 hA that is not involved in direct interaction with the cognate E1 enzyme of E2 hA, (ii) the interface between the polar and non-polar faces of E2 hA, and / or (iii) a face of E2 hA that directly interacts with the cognate E1 enzyme of E2 hA. In some cases, the E2 hA peptide is stabilized by introducing a staple or stitch (e.g., a hydrocarbon staple or stitch) at the interface between the polar and non-polar faces of E2 hA. In some cases, the E2 hA peptide is stabilized by introducing a staple or stitch at a face of E2 hA that directly interacts with the cognate E1 enzyme of E2 hA, e.g., the staple may "mimic" a hydrophobic patch at the interaction face of the helix interaction face. In certain examples, the structurally stabilized (e.g., internally cross-linked, e.g., stapled) E2 hA peptides described herein may contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7) additional amino acid substitutions (compared to the wild-type E2 hA peptide sequence), e.g., one or more (e.g., 1, 2, 3, 4, 5, 6, 7) conservative and / or non-conservative amino acid substitutions (i.e., in addition to the amino acid substitutions made to confer structural stabilization to E2 hA, one or more amino acid substitutions). In certain examples, these additional substitutions are amino acid substitutions that directly interact with the cognate E1 enzyme of E2 hA. In certain examples, these additional substitutions are amino acid substitutions that are not involved in direct interaction with the cognate E1 enzyme of E2 hA.In certain examples, these additional substitutions are substitutions of both amino acids that directly interact with the cognate E1 enzyme of E2 hA and amino acids that do not participate in the direct interaction with the cognate E1 enzyme of E2 hA. In certain examples, the structurally stabilized (e.g., internally cross-linked, e.g., stapled) E2 hA peptides described herein may contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions from the N-terminus and / or C-terminus of E2 hA. For example, the structurally stabilized (e.g., internally cross-linked, e.g., stapled) E2 hA peptide may be 5 or longer (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 23, 27) amino acids in length. In certain examples, the structurally stabilized (e.g., internally cross-linked, e.g., stapled) E2 hA peptide is 5 to 11 amino acids in length. In certain examples, the structurally stabilized (e.g., internally cross-linked, e.g., stapled) E2 hA peptide is 5 to 17 amino acids in length. In certain examples, the structurally stabilized (e.g., internally cross-linked, e.g., stapled) E2 hA peptide is 11 to 17 amino acids in length. In certain examples, the structurally stabilized (e.g., internally cross-linked, e.g., stapled) E2 hA peptide is 5 to 27 amino acids in length. In certain examples, the structurally stabilized (e.g., internally cross-linked, e.g., stapled) E2 hA peptide is 11 to 27 amino acids in length. In certain examples, the structurally stabilized (e.g., internally cross-linked, e.g., stapled) E2 hA peptide is 17 to 27 amino acids in length.

[0151] In certain embodiments, the E2 hA peptides of the present disclosure may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acid substitutions at any one of SEQ ID NOs: 1-38 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acids are conservatively or non-conservatively substituted). For example, in certain embodiments, the E2 hA peptides of the present disclosure are modified amino acid sequences of the sequence shown in SEQ ID NO: 4, comprising a modified amino acid sequence comprising SEQ ID NO: 4 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acid substitutions in the sequence of SEQ ID NO: 4 (e.g., the modified amino acid sequence comprises SEQ ID NO: 4 except that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acids of SEQ ID NO: 4 are amino acids that are conservatively or non-conservatively substituted). In another example, in certain embodiments, the E2 hA peptides of the present disclosure are modified amino acid sequences of the sequence shown in SEQ ID NO: 6, comprising a modified amino acid sequence comprising SEQ ID NO: 6 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acid substitutions in the sequence of SEQ ID NO: 6 (e.g., the modified amino acid sequence comprises SEQ ID NO: 6 except that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acids of SEQ ID NO: 6 are amino acids that are conservatively or non-conservatively substituted). In another example, in certain embodiments, the E2 of the present disclosure The hA peptide is a modified amino acid sequence of the sequence shown in SEQ ID NO: 10, and includes a modified amino acid sequence that includes SEQ ID NO: 10 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acid substitutions in the sequence of SEQ ID NO: 10 (for example, the modified amino acid sequence includes SEQ ID NO: 10 except that 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acids of SEQ ID NO: 10 are substituted with conservative or non-conservative amino acids). "Conservative amino acid substitution" means that by this substitution, one amino acid is replaced with another amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids having beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). In some examples, 1 to 3 amino acids of any one of SEQ ID NOs: 1 to 38 are substituted. The amino acid substitutions in any one of SEQ ID NOs: 1 to 38 can be amino acid substitutions that directly interact (or are predicted to directly interact) with the cognate E1 enzyme of E2 hA, or do not participate (or are predicted not to participate) in the direct interaction with the cognate E1 enzyme of E2 hA. Even more variability is tolerated in the amino acids of E2 hA that do not participate (or are predicted not to participate) in the direct interaction with the cognate E1 enzyme of E2 hA than in the amino acids of E2 hA that directly interact (or are predicted to directly interact) with the cognate E1 enzyme of E2 hA.In fact, almost all amino acids that do not participate in (or are predicted not to participate in) the direct interaction with the cognate E1 enzyme of E2 hA can be substituted (e.g., conservative or non-conservative amino acid substitutions or substitutions with alanine). In certain embodiments, 1, 2, or 3 amino acids of E2 hA that interact directly with (or are predicted to interact directly with) the cognate E1 enzyme of E2 hA are substituted with another amino acid. In some examples, the substitution(s) are conservative amino acid substitutions. In other examples, the substitution(s) are non-conservative amino acid substitutions. In some examples, when there are more than one amino acid substitution, the substitutions are both conservative and non-conservative amino acid substitutions. In some examples, when there are more than one amino acid substitution, each of the substitutions is a conservative amino acid substitution. In some cases, when 1 to 3 amino acids (e.g., 1, 2, or 3) of any one of SEQ ID NOs: 1-38 are substituted, the substitutions are all amino acid substitutions of E2 hA that do not participate in (or are predicted not to participate in) the direct interaction with the cognate E1 enzyme of E2 hA. In some cases, when 1 to 3 amino acids (e.g., 1, 2, or 3) of any one of SEQ ID NOs: 1-38 are substituted, the substitutions are all amino acid substitutions of E2 hA that interact directly with (or are predicted to interact directly with) the cognate E1 enzyme of E2 hA. In some cases, when 1 to 3 amino acids (e.g., 1, 2, or 3) of any one of SEQ ID NOs: 1-38 are substituted, the substitutions are both amino acid substitutions of E2 hA that interact directly with (or are predicted to interact directly with) the cognate E1 enzyme of E2 hA and amino acid substitutions of E2 hA that do not participate in (or are predicted not to participate in) the direct interaction with the cognate E1 enzyme of E2 hA. In certain embodiments, each of amino acid positions 8 and 11 of SEQ ID NO: 132, or the stabilized version of SEQ ID NO: 4, or the truncated version thereof (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 amino acids are removed from the N-terminus and / or C-terminus) is substituted with another amino acid.In certain embodiments, each of amino acid positions 8 and 11 of SEQ ID NO: 132, or a stabilized version of SEQ ID NO: 4, or a truncated version thereof (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 amino acids have been removed from the N-terminus and / or C-terminus) is substituted with glutamic acid. In certain embodiments, each of amino acid positions 4, 6, 11, 14, and 15 of SEQ ID NO: 132, or a stabilized version of SEQ ID NO: 4, or a truncated version thereof (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 amino acids have been removed from the N-terminus and / or C-terminus) is substituted with alanine. In certain embodiments, each of amino acid positions 4, 14, and 15 of SEQ ID NO: 132, or a stabilized version of SEQ ID NO: 4, or a truncated version thereof (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 amino acids have been removed from the N-terminus and / or C-terminus) is substituted with alanine, and each of amino acid positions 6, 8, and 11 of SEQ ID NO: 132, or a stabilized version of SEQ ID NO: 4, or a truncated version thereof (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 amino acids have been removed from the N-terminus and / or C-terminus) is substituted with glutamic acid.

[0152] In certain examples, the substituted amino acid(s) is selected from the group consisting of L-Ala, D-Ala, Aib, Sar, Ser, substituted alanine, or a substituted glycine derivative. In certain examples, the modified Ubc15 E2 hA peptide comprises an E7 residue (numbered according to SEQ ID NO: 2) substituted with arginine.

[0153] In certain embodiments, the E2 hA peptide of the present disclosure may have 1, 2, 3, 4, or 5 amino acids removed / deleted from the C-terminus of the sequence shown in any one of SEQ ID NOs: 1-38. For example, in certain embodiments, the E2 hA peptide of the present disclosure comprises or consists of a modified amino acid sequence of the amino acid sequence shown in SEQ ID NO: 4, wherein 2 amino acids have been removed / deleted from the C-terminus of the sequence of SEQ ID NO: 4 (i.e., the E2 hA peptide comprises or consists of the amino acid sequence of SEQ ID NO: 39). In another example, in certain embodiments, the E2 hA peptide of the present disclosure comprises or consists of a modified amino acid sequence of the amino acid sequence shown in SEQ ID NO: 6, wherein 5 amino acids have been removed / deleted from the C-terminus of the sequence of SEQ ID NO: 6 (i.e., the E2 hA peptide comprises or consists of the amino acids of SEQ ID NO: 55). In certain embodiments, the E2 hA peptide of the present disclosure may have 1, 2, 3, 4, or 5 amino acids removed / deleted from the N-terminus of the sequence shown in any one of SEQ ID NOs: 1-38. In certain embodiments, the E2 hA peptide that interacts with UBA1 of the present disclosure has amino acid positions A 12 ~A 16 removed / deleted from the C-terminus of the sequence shown in any one of SEQ ID NOs: 1-34. For example, in certain embodiments, the E2 hA peptide of the present disclosure comprises or consists of a modified amino acid sequence of the amino acid sequence shown in SEQ ID NO: 6, wherein amino acid positions A 12 ~A 16is removed / deleted from the C-terminus of the sequence of SEQ ID NO: 6 (i.e., the E2 hA peptide comprises or consists of the amino acid sequence of SEQ ID NO: 55). In certain embodiments, the E2 hA peptide that binds to UBA1 of the present disclosure may have 1, 2, 3, or all of the amino acid positions N-terminal to amino acid position A1 that is removed / deleted from the N-terminus of the sequence shown in any one of SEQ ID NOs: 1-34. In certain embodiments, the E2 hA peptide of the present disclosure may have 1, 2, 3, 4, or 5 amino acids removed / deleted from both the N-terminus and the C-terminus of the sequence shown in any one of SEQ ID NOs: 1-38. In certain embodiments, the E2 hA peptide that binds to UBA1 of the present disclosure may have 1, 2, 3, 4, or 5 amino acids removed / deleted from the C-terminus of the sequence shown in any one of SEQ ID NOs: 1-34, and 1, 2, 3, or all of the amino acids N-terminal to amino acid position A1 that is removed / deleted from the N-terminus of that sequence. For example, in certain embodiments, the E2 hA peptide that binds to UBA1 of the present disclosure comprises or consists of a modified amino acid sequence of the amino acid sequence shown in SEQ ID NO: 6, wherein amino acids A -3 ~A -1 and A 12 ~A 16 are removed / deleted from SEQ ID NO: 6 (i.e., the peptide comprises or consists of amino acids A1-A 11 of SEQ ID NO: 6). In another example, in certain embodiments, the E2 hA peptide that binds to UBA1 of the present disclosure comprises or consists of a modified amino acid sequence of the amino acid sequence shown in SEQ ID NO: 6, wherein amino acids A -3 ~A -1 and A 12 ~A 16 are removed / deleted from SEQ ID NO: 4 (i.e., the peptide comprises or consists of amino acids A1-A 11comprises or consists of). In yet another example, in certain embodiments, the E2 hA peptide that binds to UBA1 of the present disclosure comprises or consists of a modified amino acid sequence of the amino acid sequence set forth in SEQ ID NO: 6, wherein amino acid A 12 ~A 16 is removed / deleted from SEQ ID NO: 10 (i.e., the peptide comprises or consists of amino acids A1 - A of SEQ ID NO: 10 11 ). In certain examples, these removed amino acids can be replaced with 1 - 6 (e.g., 1, 2, 3, 4, 5, or 6) amino acids selected from the group consisting of L - Ala, D - Ala, Aib, Sar, Ser, substituted alanine, or substituted glycine derivatives.

[0154] The present disclosure also encompasses E2 hA peptides that are at least 14% (e.g., 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%, at least 20% - 50%, at least 20% - 45%, at least 20% - 40%, at least 20% - 35%, at least 20% - 30%, at least 20% - 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%, at least 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 100%) identical to any one of SEQ ID NOs: 1 - 38. The variability in the amino acid sequence of any one of SEQ ID NOs: 1 - 38 can be present in one or both of the sides of the alpha - helix that do or do not directly interact. E2 that is not involved (or is predicted not to be involved) in the direct interaction with the cognate E1 enzyme of E2 Almost all of the amino acids of hA can be changed. Amino acids of E2 hA that directly interact (or are predicted to directly interact) with the cognate E1 enzyme of E2 hA can also be changed. In a specific embodiment, the E2 hA peptide comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 1-38. In a specific embodiment, the E2 hA peptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to any one of SEQ ID NOs: 1-38. In a specific embodiment, the E2 hA peptide consists of the amino acid sequence of any one of SEQ ID NOs: 1-38, 99, and 55. Methods for determining the percent identity between amino acid sequences are known in the art. For example, sequences are aligned for optimal comparison (e.g., gaps may be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences may be ignored for comparison). In a preferred embodiment, the length of the reference sequence aligned for comparison is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, even more preferably at least 70%, 80%, 90%, or 100% of the length of the reference sequence. Then, the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are compared. If the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The determination of the percent identity between two amino acid sequences is achieved using the BLAST2.0 program. The sequence comparison is performed using an alignment without gaps and using default parameters (Blossom62 matrix, gap existence cost 11, gap cost per residue 1, and lambda ratio 0.85). The mathematical algorithms used in the BLAST program are described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997).

[0155] In some embodiments, the present disclosure features any one variant of SEQ ID NOs: 1-38, wherein the variant of the peptide binds to E1 by non-covalent bonding (e.g., UBA1 for SEQ ID NOs: 1-34, UBA6 for SEQ ID NO: 38, UBA2 for SEQ ID NO: 37, and UBA3 for SEQ ID NOs: 35 and 36).

[0156] In certain examples, the E2 hA peptide has the amino acid sequence shown in Table 6 below. The present disclosure also features stabilized versions of these variant E2 hA peptides (e.g., internally cross-linked versions). For example, two (or more) residues of these variants separated by, for example, 3 or 6 amino acids are replaced with non-natural amino acids that can form cross-links by olefin metathesis. The cross-links are placed within these variants at locations that do not disrupt the binding of the E2 hA peptide to its cognate E1 enzyme. In some examples, the variant E2 hA peptides are stabilized by hydrocarbon staples or stitches, lactam staples or stitches; UV-cycloaddition staples or stitches; oxime staples or stitches; thioether staples or stitches; double-crick staples or stitches; bis-lactam staples or stitches; bis-arylation staples or stitches; or combinations of any two or more of these. Table 6. Exemplary Variant E2 hA Peptides

Table 6-1

Table 6-2

[0157] The E2 hA peptides described herein can be optimized for therapeutic use. For example, if any of the above E2 hA peptides causes membrane disruption (cytolysis), the peptide can be optimized by reducing the overall hydrophobicity of the peptide. For example, this can be achieved, in particular, by substituting hydrophobic residues with amino acids having lower hydrophobicity (e.g., alanine). Membrane disruption can also be reduced by reducing the overall positive charge of the peptide. This can also be achieved by substituting basic residues with uncharged or acidic residues. In certain examples, both the overall hydrophobicity of the peptide and the overall positive charge of the peptide are reduced.

[0158] In certain embodiments, the E2 hA peptides described herein are between 5 and 35 amino acids in length, between 5 and 25 amino acids in length, between 5 and 20 amino acids in length, between 5 and 18 amino acids in length, between 10 and 35 amino acids in length, between 10 and 25 amino acids in length, between 10 and 20 amino acids in length, between 10 and 18 amino acids in length, between 15 and 26 amino acids in length, between 15 and 18 amino acids in length, or between 10 and 28 amino acids in length. In certain embodiments, the E2 hA peptides described herein are 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 amino acids in length.

[0159] In certain examples, the E2 hA peptides described herein are structurally stabilized. To stabilize these E2 hA peptides, the peptide can include two or more substitutions, for example, replacing the amino acids of the E2 hA peptide with non-natural amino acids such that the peptide can be stapled and / or stitched. Stabilized peptide

[0160] Peptide helices are important mediators of protein-protein interactions that are key to regulating many important biological processes such as apoptosis. However, when such helices are removed from their environment (context) within a protein and prepared in isolation, they typically adopt a random coil conformation, resulting in a significant loss of biological activity and thus a reduction in therapeutic potential. The present disclosure provides structurally stabilized peptides of E2 hA. The present disclosure includes at least two modified amino acids joined by internal (intramolecular) crosslinks (or staples), and in certain examples, a reactive group (such as a "warhead" which is a non-natural amino acid having an electrophilic group) capable of forming a covalent bond with a cysteine (Cys) residue within a target protein (e.g., E1) to which the structurally stabilized peptide binds. The structurally stabilized E2 hA peptides (such as those described above) are included. The stabilized peptides described herein include stapled peptides and stitched peptides, as well as peptides containing a mixture of multiple stitches, multiple staples or staples and stitches, or other chemical strategies for structural enhancement (e.g., Balaram P. Cur. Opin. Struct. Biol. 1992;2:845; Kemp DS, et al., J. See Am. Chem. Soc. 1996;118:4240; Orner BP, et al., J. Am. Chem. Soc. 2001;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., Org. Biomol. Chem., 2016,14:8002-8013, all of which are hereby incorporated by reference in their entirety).

[0161] In certain embodiments, one or more of the E2 hA peptides described herein can be stabilized by peptide stapling (see, e.g., Walensky, J. Med. Chem., 57:6275-6288 (2014), the contents of which are hereby incorporated by reference in their entirety). A peptide is "stabilized" in that it maintains its native secondary structure. For example, stapling enables a polypeptide that is prone to having an α-helical secondary structure to maintain its native α-helical conformation. This secondary structure can increase the protein cleavage and heat resistance of the polypeptide, and can also increase target binding affinity, hydrophobicity, and cell permeability. Accordingly, the stapled (cross-linked) polypeptides described herein have improved biological activity compared to the corresponding non-stapled (non-cross-linked) polypeptides.

[0162] ​"Peptide stapling" is a neologism from synthetic methodology, where two olefin-containing side chains (e.g., cross-linkable side chains) present in a polypeptide chain are covalently joined (e.g., "stapled together") using a ring-closing metathesis (RCM) reaction to form a cross-linked ring (see, e.g., Blackwell et al., J. Org. Chem., 66: 5291-5302, 2001; Angew et al., Chem. Int. Ed. 37:3281, 1994). As used herein, the term "peptide stapling" includes using any of a number of reaction conditions and / or catalysts that facilitate such reactions to join two double-bond-containing side chains, two triple-bond-containing side chains, or a double-bond-containing side chain and a triple-bond-containing side chain (e.g., at least one pair thereof) present in a polypeptide chain to obtain a "stapled" polypeptide alone. The term "multi-stapled" polypeptide refers to these polypeptides that contain more than one individual staple and may contain two, three, or more independent staples at various intervals. Further, the term "peptide stitching" as used herein refers to multiple and tandem "stapling" events in a single polypeptide chain, e.g., providing a "stitched" (e.g., tandem or multi-stapled) polypeptide where two staples are linked to a common residue. Peptide stitching is disclosed, for example, in WO2008 / 121767 and WO2010 / 068684, both of which are hereby incorporated by reference in their entirety. In some examples, the staples can, or can be reduced to, retain an unsaturated bond as used herein.

[0163] In certain embodiments, one or more of the peptides described herein may be stabilized. In some examples, the E2 hA peptides of the present disclosure are stabilized by hydrocarbon staples or stitches, lactam staples or stitches; UV-cycloaddition staples or stitches; oxime staples or stitches; thioether staples or stitches; double-crossover staples or stitches; bis-lactam staples or stitches; bis-arylation staples or stitches; or combinations of any two or more thereof. In one example, the peptides described herein are stabilized by hydrocarbon stapling. In some embodiments, the stapled peptide is a cross-linked version of a polypeptide comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 1-39, 55-63, or 792-806. In some examples, the stapled peptide is a hydrocarbon stapled version of a polypeptide comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 1-39, 55-63, or 792-806. In some examples, the stapled peptide is a polypeptide comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 1-39, 55-63, or 792-806, except that at least 2 (e.g., 2, 3, 4, 5, 6) amino acids are replaced with non-natural amino acids (e.g., S5, R8). In some embodiments, the stapled peptide comprises or consists of any one of the amino acid sequences of SEQ ID NOs: 1-38, or comprises 1-13 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13) amino acid substitutions, deletions and / or insertions therein (e.g., as described in the examples above and below, e.g., SEQ ID NOs: 39, 55, or 47). In certain examples, the stapled peptide comprises at least 2 (e.g., 2, 3, 4, 5, 6) amino acid substitutions, where the substituted amino acids are separated by 2, 3, or 6 amino acids and are non-natural amino acids having olefin side chains.There are many known non-natural or unnatural amino acids, any of which may be included in the peptides of the present disclosure. Some examples of unnatural amino acids are 4-hydroxyproline, desmosine, gamma-aminobutyric acid, beta-cyanoalanine, 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-aminocyclobutanecarboxylic acid, 4-aminocyclopentene carboxylic acid, 3-aminocyclohexanecarboxylic acid, 4-piperidylacetic acid, 4-amino-l-methylpyrrole-2-carboxylic acid, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2-aminoheptanedioic acid, 4-(aminomethyl)benzoic acid, 4-aminobenzoic acid, ortho-, meta- and / or para-substituted phenylalanine (e.g., substituted with -C(=O)C6H5; -CF3; -CN; -halo; -NO2; CH3), disubstituted phenylalanine, substituted tyrosine (e.g., further substituted with -C=O)C6H5; -CF3; -CN; -halo; -NO2; CH3), and statins. Further, the amino acids may be derivatized and include hydroxylated, phosphorylated, sulfonated, acylated, or glycosylated amino acid residues.

[0164] Hydrocarbon stapled polypeptide has two Contains one or more tethers (linkages) between non-natural amino acids, which significantly enhance the α-helix secondary structure of the polypeptide. Generally, the tether extends across the length of one or two helical turns (i.e., approximately 3.4 or approximately 7 amino acids). Thus, amino acids located at i and i + 3, i and i + 4, or i and i + 7 are ideal candidates for chemical modification and cross-linking. Thus, for example, when a peptide has the sequence...X1, X2, X3, X4, X5, X6, X7, X8, X9..., a cross-link between X1 and X4, or between X1 and X5, or between X1 and X8 is a useful hydrocarbon stapled form of that peptide, as are cross-links between X2 and X5, or between X2 and X6, or between X2 and X9. The use of multiple cross-links (e.g., 2, 3, 4, or more) is also contemplated. The use of multiple cross-links is highly effective, particularly for the stabilization and optimization of peptides with increased peptide length. Thus, the present disclosure encompasses incorporating more than one cross-link within a polypeptide sequence to further stabilize the sequence or to promote enhanced structural stability, proteolytic resistance, acid stability, thermal stability, cell permeability, and / or biological activity of longer polypeptide stretches. Further descriptions regarding the production and use of hydrocarbon stapled polypeptides can be found, for example, in U.S. Patent Application Publication Nos. 2012 / 0172285, 2010 / 0286057, and 2005 / 0250680, the entire contents of all of which are incorporated herein by reference in their entirety.

[0165] In certain embodiments, when staples are present at residues i and i + 3, R-propenylalanine and S-pentenylalanine; or R-pentenylalanine and S-pentenylalanine replace the amino acids at those positions. In certain embodiments, when staples are present at residues i and i + 4, S-pentenylalanine replaces the amino acids at those positions. In certain embodiments, when staples are present at residues i and i + 7, S-pentenylalanine and R-octenylalanine replace the amino acids at those positions. In some examples, when the peptide is stitched, the amino acids of the peptide involved in the "stitch" are replaced with bis-pentenylglycine, S-pentenylalanine, and R-octenylalanine; or bis-pentenylglycine, S-octenylalanine, and R-octenylalanine.

[0166] In a peptide to be stapled, amino acids that interfere with the stapling reaction (e.g., inhibit it or reduce its efficiency) should be replaced with amino acids that do not interfere with the stapling reaction (e.g., do not inhibit it or do not substantially reduce its efficiency). For example, methionine (Met, M) can interfere with the stapling reaction, and thus, in certain embodiments, methionine(s) in the peptide to be stapled is / are replaced with, for example, norleucine(s).

[0167] In certain embodiments where the peptide is an E2 hA that binds to UBA1 (e.g., any one of SEQ ID NOs: 1-34 or a modified version thereof (e.g., SEQ ID NO: 39, 55, or 57)), the staple and / or stitch is at positions A2 and A9, A5 and A9, A8 and A 12 、A9 and A 13 、A1 and A8, A4 and A 11 、or A5 and A 12It is prepared in. In certain embodiments where the peptide is an E2 hA that binds to UBA1 (e.g., any one of SEQ ID NOs: 1-34 or a modified version thereof (e.g., SEQ ID NO: 39, 55, or 57, or a modified version thereof)), staples and / or stitches are made at positions A2 and A9 (see Table 7 for exemplary staple peptides). In certain embodiments where the peptide is an E2 hA that binds to UBA1 (e.g., any one of SEQ ID NOs: 1-34 or a modified version thereof (e.g., SEQ ID NO: 39, 55, or 57, or a modified version thereof)), staples and / or stitches are made at positions A1 and A8. The staple positions can be altered by testing the locations of different staples in a staple walk.

[0168] In certain embodiments where the peptide comprises or consists of SEQ ID NO: 35 or a modified version thereof, staples and / or stitches are made at positions Arg-6 and Glu-13. In certain embodiments where the peptide comprises or consists of SEQ ID NO: 36 or a modified version thereof, staples and / or stitches are made at positions Ala-6 and Asp-13. In certain embodiments where the peptide comprises or consists of SEQ ID NO: 37 or a modified version thereof, staples and / or stitches are made at positions Leu-5 and Trp-12. In certain embodiments where the peptide comprises or consists of SEQ ID NO: 38 or a modified version thereof, staples and / or stitches are made at positions Ala-3 and Leu-10. See Tables 7 and 8 for exemplary staple peptides.

[0169] Table 7. Exemplary staple peptides. SEQ ID NOs: 64-99 and 850 (top to bottom, respectively): "B" is norleucine, and "X1" and "X2" are closed-ring metas to form a crosslinked ring Can be joined by covalent bonds using a ring-closing metathesis (RCM) reaction (“stapled together”) Are unnatural amino acids. SEQ ID NOs: 100-135 and 851 (top to bottom, respectively): “B” is norleucine, “X1” is R-octenylalanine, and “X2” is S-pentenylalanine. “tr” = truncated; “m” = mutant Are unnatural amino acids. SEQ ID NOs: 100-135 and 851 (top to bottom, respectively): “B” is norleucine, “X1” is R-octenylalanine, and “X2” is S-pentenylalanine. “tr” = truncated; “m” = mutant [Table 7]

[0170] Table 8. Exemplary stapled peptides. SEQ ID NOs: 136-139 (top to bottom, respectively): “B” is norleucine, and “X1” and “X2” are unnatural amino acids that can be joined by covalent bonds using a ring-closing metathesis (RCM) reaction to form a crosslinked ring (“stapled together”). SEQ ID NOs: 140-143 (top to bottom, respectively): “B” is norleucine, “X1” is R-octenylalanine, and “X2” is S-pentenylalanine. [Table 8]

[0171] The upper panel of Figure 26 shows exemplary chemical structures of unnatural amino acids that can be used to generate various crosslinked compounds. The middle panel of Figure 26 illustrates peptides having hydrocarbon crosslinks between residues at positions i and i + 3, i and i + 4, and i and i + 7. The lower panel of Figure 26 illustrates a staple walk along a peptide sequence. Figure 27 shows double and triple stapling strategies, as well as various peptide sequences having exemplary staple walks. Figure 28 illustrates an exemplary staple walk using branched stitched moieties of various lengths. Figure 29 illustrates point mutants and staple scans, as well as variants of peptides based on N- and C-terminal deletions, additions, and / or derivatizations.

[0172] In one aspect, the stabilized E2 hA peptide has the formula (I),

Chemical formula

Chemical formula

[0173] Table 9. N-terminal [Xaa] regarding constructs 1 - 225 of formula (I) y , [Xaa] x , and the C-terminal [Xaa] y sequence .

Table 9 - 1

Table 9 - 2

Table 9 - 3

Table 9 - 4

Table 9 - 5

Table 9 - 6

Table 9 - 7

Table 9 - 8

Table 9 - 9

Table 9 - 10

Table 9 - 11

Table 9-12

Table 9-13

[0174] In certain examples, the methionine (M) in the sequences shown above in Table 9 is replaced with norleucine (B). In certain examples, the sequences shown above in Table 9 may have substitutions or deletions of at least 1 (e.g., 1, 2, 3, 4, 5, 6) amino acids. The E2 hA peptide may comprise any amino acid sequence described herein.

[0175] The tether may comprise an alkyl, alkenyl, or alkynyl moiety (e.g., C5, C8, or C 11 alkyl, C5, C8, or C 11 alkenyl, or C5, C8, or C 11 alkynyl). The tethered amino acid may be alpha-disubstituted (e.g., C1-C3 or methyl).

[0176] In some examples, x is 2, 3, or 6. In some examples, each y is independently an integer between 0 and 15, or between 3 and 15. In some examples, R1 and R2 are each independently H or C1-C6 alkyl. In some examples, R1 and R2 are each independently C1-C3 alkyl. In some examples, at least one of R1 and R2 is methyl. For example, R1 and R2 may both be methyl. In some examples, R3 is alkyl (e.g., C8 alkyl) and x is 3. In some examples, R3 is C 11 alkyl and x is 6. In some examples, R3 is alkenyl (e.g., C8 alkenyl) and x is 3. In some examples, x is 6 and R3 is C 11It is alkenyl. In some examples, R3 is linear alkyl, alkenyl, or alkynyl. In some examples, R3 is -CH2-CH2-CH2-CH=CH-CH2-CH2-CH2-.

[0177] In another aspect, the two alpha, alpha-disubstituted stereocenters are either both in the R configuration or the S configuration (e.g., i, i+4 bridging), or one stereocenter is R and the other is S (e.g., i, i+7 bridging). Thus, here, formula (I) is shown as follows: [Chemical formula]

[0178] The two-substituted stereocenters of C’ and C’’ may both be in the R configuration, or they may both be in the S configuration (e.g., when x is 3). When x is 6, the two-substituted stereocenter of C’ is in the R configuration and the two-substituted stereocenter of C’’ is in the S configuration. The double bond of R3 may be in the E or Z stereochemical configuration.

[0179] In some examples, R3 is [R4-K-R4] n and R4 is linear alkyl, alkenyl, or alkynyl.

[0180] In some embodiments, the present disclosure provides an internally crosslinked ( "stapled" or "stitched") peptide comprising an amino acid sequence of any one of SEQ ID NOs: 1-38 (or a modified version thereof, such as SEQ ID NO: 39, 55, or 57, or a modified version thereof), wherein the side chains of two amino acids separated by 2, 3, or 6 amino acids are replaced by internal staples; the side chains of three amino acids are replaced by internal stitches; the side chains of four amino acids are replaced by two internal staples; or the side chains of five amino acids are replaced by a combination of internal staples and internal stitches. In certain examples, the amino acids at one or more of positions Met-1, Leu-3, Lys-4, Asn-7, Leu-10, and Ala-11 of SEQ ID NO: 1 are not replaced by staples or stitches. In certain examples, the amino acids at one or more of positions Met-1, Pro-2, Ser-3, Ser-4, Glu-7, Arg-11, Lys-12, Leu-14, Lys-15, and Gln-18 of SEQ ID NO: 2 are not replaced by staples or stitches. In certain examples, at any one position A4, A6, A7, A 10 , and optionally A 13The amino acids in one or more of them are not replaced by staples or stitches. In certain examples, the amino acids in one or more of positions Met-1, Arg-3, Ala-4, Ser-5, Leu-7, Lys-8, Arg-9, Leu-11, His-12, and Ala-15 of SEQ ID NO: 21 are not replaced by staples or stitches. In certain examples, the amino acids in one or more of positions Val-3, Lys-8, Val-11, Lys-12, and Val-14 of SEQ ID NO: 35 are not replaced by staples or stitches. In certain examples, the amino acids in one or more of any single position Lys-2, Ala-3, Gln-7, Leu-8, Gln-11, Lys-12, Ile-14, Asn-15, and Glu-16 of SEQ ID NO: 36 are not replaced by staples or stitches. In certain examples, the amino acids in one or more of positions Leu-2, Ser-3, Ala-6, Gln-7, Arg-9, Lys-10, Arg-13, and Lys-14 of SEQ ID NO: 37 are not replaced by staples or stitches. In certain examples, the amino acids in one or more of positions Arg-5, Glu-7, Leu-8, Val-11, and Leu-14 of SEQ ID NO: 38 are not replaced by staples or stitches. The stapled / stitched peptide can be 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, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. In a specific embodiment, the stapled / stitched peptide is 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In a specific embodiment, the stapled / stitched peptide is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 amino acids in length. In a specific embodiment, the stapled / stitched peptide is 11 amino acids in length. In a specific embodiment, the stapled / stitched peptide is 12 amino acids in length.Exemplary E2 hA staple peptides are shown in Tables 7, 8, and 11 - 15. In one embodiment, the E2 hA staple peptide comprises or consists of the amino acid sequences shown in SEQ ID NOs: 65, 96, 101, and 132. In one embodiment, the E2 hA staple peptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 132. In one embodiment, the E2 hA staple peptide comprises or consists of the amino acid sequences shown in SEQ ID NOs: 67, 97, 103, and 133. In one embodiment, the E2 hA staple peptide comprises or consists of the amino acid sequences shown in SEQ ID NOs: 71 and 107. In certain embodiments, the stapled polypeptide comprises or consists of the amino acid sequence shown in any one of SEQ ID NOs: 1 - 39, 55 - 63, 792 - 806, wherein at least two amino acids separated by 2, 3, or 6 amino acids are modified to structurally stabilize the peptide (e.g., by substituting them with non - natural amino acids to enable hydrocarbon stapling). In certain embodiments, the stapled polypeptide comprises or consists of a variant of the amino acid sequence shown in any one of SEQ ID NOs: 1 - 39, 55 - 63, 792 - 806, wherein at least two amino acids separated by 2, 3, or 6 amino acids are modified to structurally stabilize the peptide (e.g., by substituting them with non - natural amino acids to enable hydrocarbon stapling). In certain embodiments, the stapled polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 4 (or a modified version thereof), wherein at least two amino acids separated by 2, 3, or 6 amino acids are modified to structurally stabilize the peptide (e.g., by substituting them with non - natural amino acids to enable hydrocarbon stapling).In certain embodiments, the staple polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 39 (or a modified version thereof), wherein at least two amino acids separated by 2, 3, or 6 amino acids are modified (e.g., by substituting them with non-natural amino acids to enable hydrocarbon stapling) to structurally stabilize the peptide. In certain embodiments, the staple polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 6 (or a modified version thereof), wherein at least two amino acids separated by 2, 3, or 6 amino acids are modified (e.g., by substituting them with non-natural amino acids to enable hydrocarbon stapling) to structurally stabilize the peptide. In certain embodiments, the staple polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 55 (or a modified version thereof), wherein at least two amino acids separated by 2, 3, or 6 amino acids are modified (e.g., by substituting them with non-natural amino acids to enable hydrocarbon stapling) to structurally stabilize the peptide. In certain embodiments, the staple polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 10 (or a modified version thereof), wherein at least two amino acids separated by 2, 3, or 6 amino acids are modified (e.g., by substituting them with non-natural amino acids to enable hydrocarbon stapling) to structurally stabilize the peptide. In certain embodiments, the staple polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57 (or a modified version thereof), wherein at least two amino acids separated by 2, 3, or 6 amino acids are modified (e.g., by substituting them with non-natural amino acids to enable hydrocarbon stapling) to structurally stabilize the peptide.

[0181] Hydrocarbon tethers are common, but in the E2 hA peptides described herein, other tethers can also be used. For example, the tether may include one or more of an ether, thioether, ester, amine, or amide, or a triazole moiety. In some cases, naturally occurring amino acid side chains may be incorporated into the tether. For example, the tether may be linked to a functional group such as the hydroxyl of serine, the thiol of cysteine, the primary amine of lysine, the acid of aspartic acid or glutamic acid, or the amide of asparagine or glutamine. Thus, it is possible to create tethers using naturally occurring amino acids rather than using tethers made by linking two non-naturally occurring amino acids. It is also possible to use a single non-naturally occurring amino acid together with a naturally occurring amino acid. Cross-links containing triazole (e.g., 1,4-triazole or 1,5-triazole) can be used (see, e.g., Kawamoto et al. 2012 Journal of Medicinal Chemistry 55:1137; WO 2010 / 060112). Furthermore, other methods of performing various types of stapling are well known in the art and can be used with the E2 hA peptides described herein (e.g., lactam stapling: Shepherd et al., J. Am. Chem. Soc., 127:2974-2983 (2005); UV-cycloaddition stapling: Madden et al., Bioorg. Med. Chem. Lett., 21:1472-1475 (2011); disulfide stapling: Jackson et al., Am. Chem. Soc., 113:9391-9392 (1991); oxime st apling: Haney et al., Chem. Commun., 47:10915-10917 (2011); thioether stapling: Brunel and Dawson, Chem. Commun., 552-2554 (2005); pho Switchable stapling: J. R. Kumita et al., Proc. Natl. Acad. Sci. U. S. A., 97:3803-3808 (2000); Double click stapling: Lau et al., Chem. Sci., 5:1804-1809 (2014); Bis-lactam stapling: J. C. Phelan et al., J. Am. Chem. Soc., 119:455-460 (1997); and Bis-arylation Stapling: A. M. Spokoyny et al., J. Am. Chem. Soc., 135:5946-5949 (2013). See also).

[0182] It is further recalled that the length of the tether can be varied. For example, a shorter length tether can be used if it is desirable to provide a relatively high degree of constraint with respect to the secondary alpha-helix structure, while in some instances a longer tether may be desirable as it is not desirable to provide as much constraint with respect to the secondary alpha-helix structure.

[0183] Furthermore, tethers spanning amino acids i-i+3, i-i+4, and i-i+7 are common to provide tethers that predominantly reside on a single face of the alpha helix, but the tethers can be synthesized and extended to any combination of multiple amino acids, or the tethers can be used in combination to attach multiple tethers.

[0184] In some examples, the hydrocarbon tethers (i.e., crosslinks) described herein may be further manipulated. In one example, the double bond of a hydrocarbon alkenyl tether (e.g., synthesized using ruthenium-catalyzed ring-closing metathesis (RCM)) can be oxidized (e.g., by epoxidation, aminohydroxylation or dihydroxylation) to yield one of the following compounds. [Chemical formula]

[0185] Either the epoxide moiety or one of the free hydroxyl moieties may be further functionalized. For example, the epoxide may be treated with a nucleophile to provide additional functionality that can be used, for example, to attach a therapeutic agent. Alternatively, such derivatization may be accomplished by synthetic manipulation of the amino or carboxy terminus of the polypeptide or by amino acid side chains. Other agents, such as agents that facilitate entry of the polypeptide into cells may be attached to the functionalized tether.

[0186] In some examples, alpha-disubstituted amino acids are used in the polypeptide to improve the stability of the alpha-helical secondary structure. However, examples are also envisioned where alpha-disubstituted amino acids are not required and mono-alpha substituents (e.g., in tether amino acids) are used.

[0187] The stapled polypeptide may include a drug, toxin, derivative of polyethylene glycol, a second polypeptide, a carbohydrate, and the like. It may be desirable for a polymer or other agent to be linked to the stapled polypeptide in a composition that is substantially homogeneous.

[0188] The pharmacokinetic and pharmacodynamic properties of the polypeptide can be improved by the addition of polyethylene glycol (PEG) molecules. For example, PEGylation may result in reduced renal clearance and a more stable plasma concentration. PEG is a water-soluble polymer and may be represented as linked to a polypeptide of the following formula:

[0189] XO--(CH2CH2O) n--CH2CH2--Y (wherein n is from 2 to 10,000, X is H or a terminal modification, for example, C1-4 alkyl; Y is an amide, carbamate or urea bond to an amine group of a polypeptide (including but not limited to the epsilon amine of lysine or the N-terminus). Y may also be a maleimide bond to a thiol group (including but not limited to the thiol group of cysteine)). Other methods for directly or indirectly linking PEG to a polypeptide are known to those skilled in the art. PEG may be linear or branched. Various forms of PEG, including various functionalized derivatives, are commercially available.

[0190] PEG having a bond cleavable in the main chain may be used. For example, PEG may be prepared by an ester bond that is subject to hydrolysis. Conjugates having a cleavable PEG bond are described in WO99 / 34833; WO99 / 14259, and U.S. Patent No. 6,348,558.

[0191] In certain embodiments, a polymeric polymer (e.g., PEG) is attached to an agent described herein via an intermediate linker. In certain embodiments, the linker is composed of 1 to 20 amino acids linked by a peptide bond, where the amino acids are selected from the 20 naturally occurring amino acids. Some of these amino acids may be glycosylated, as is well understood by those skilled in the art. In other embodiments, the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In other embodiments, the linker is composed of sterically unhindered major amino acids, such as glycine and alanine. Non-peptide linkers are also possible. For example, -NH(CH2) nAn alkyl linker such as C(O)-(where n = 2 to 20) may be used. These alkyl linkers may be further substituted by any non - steric hindrance group such as, for example, lower alkyl (e.g., C1 - C6), lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc. U.S. Patent No. 5,446,090 describes its use in forming a conjugate having a bifunctional PEG linker and a peptide at each end of the PEG linker.

[0192] The staple peptide may also be modified, for example, in some embodiments, to further promote cell uptake or to further increase in vivo stability. For example, by acylating or PEGylating the peptidomimetic macrocyclic molecule, cell uptake is promoted, bioavailability is increased, blood circulation is increased, pharmacokinetics are altered, immunogenicity is reduced, and / or the required dosing frequency is decreased.

[0193] In some embodiments, the staple peptides disclosed herein have an enhanced ability to penetrate cell membranes (e.g., as compared to staple - free peptides). See, for example, International Publication No. WO 2017 / 147283, which is hereby incorporated by reference in its entirety.

[0194] Methods for synthesizing the stabilized peptides described herein are known in the art. Nevertheless, the following exemplary methods may be used. It is recognized that the various steps may be carried out in alternative sequences or orders to obtain the desired compound. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and are described, for example, in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 3d. Ed., John Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), as well as their revisions.

[0195] The peptides of the present invention can be made by chemical synthesis methods well known to those skilled in the art. See, for example, Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, W. H. Freeman & Co., New York, N.Y., 1992, p. 77. Thus, for example, on an Applied Biosystems Peptide Synthesizer Model 430A or 431, peptides are synthesized using an automated Merrifield technique of solid phase synthesis with α-NH2 protected by either t-Boc or Fmoc chemistry using side chain protected amino acids.

[0196] One way to produce the peptides described in this specification is to use solid-phase peptide synthesis (SPPS). The C-terminal amino acid is attached to a cross-linked polystyrene resin via an acid-labile bond to a linker molecule. This resin is insoluble in the solvents used for synthesis, whereby excess reagents and by-products are washed away relatively easily and quickly. The N-terminus is protected with an Fmoc group, which is stable in acid but removable with base. All side-chain functional groups are protected using base-stable and acid-labile groups.

[0197] Longer peptides can be produced by joining individual synthetic peptides using native chemical ligation. Alternatively, longer synthetic peptides can be synthesized by well-known recombinant DNA techniques. Such techniques are provided in well-known standard manuals with detailed protocols. To construct the gene encoding the peptide of the present invention, the amino acid sequence is reverse-translated to obtain a nucleic acid sequence encoding an amino acid sequence that preferably has codons optimal for the organism in which the gene is to be expressed. The synthetic gene is then typically prepared by synthesizing oligonucleotides encoding the peptide and, if necessary, any regulatory elements. The synthetic gene is inserted into a suitable cloning vector and transfected into a host cell. The peptide is then expressed under suitable conditions appropriate for the selected expression system and host. The peptide is purified and characterized by standard methods.

[0198] Peptides can be made in a high-throughput combinatorial fashion using, for example, a high-throughput multi-channel combinatorial synthesizer available from Advanced Chemtech. Peptide bonds can be replaced, for example, with retro-inverso bonds (C(O)-NH); reduced amide bonds (NH-CH2); thiomethylene bonds (S-CH2 or CH2-S); oxymethylene bonds (O-CH2 or CH2-O); ethylene bonds (CH2-CH2); thioamide bonds (C(S)-NH); trans-olefin bonds (CH=CH); fluoro-substituted trans-olefin bonds (CF=CH); ketomethylene bonds (C(O)-CHR) or CHR-C(O) (where R is H or CH3); and fluoro-ketomethylene bonds (C(O)-CFR or CFR-C(O) (where R is H or F or CH3)) to increase the physiological stability of the peptide.

[0199] The polypeptide can be further modified by acetylation, amidation, biotinylation, cinnamoylation, farnesylation, fluoresceinylation, formylation, myristoylation, palmitoylation, phosphorylation (Ser, Tyr or Thr), stearoylation, succinylation and sulfurylation. As described above, the peptide can be conjugated, for example, with polyethylene glycol (PEG); an alkyl group (e.g., a C1-C20 straight or branched chain alkyl group); a fatty acid radical; and combinations thereof. α,α-disubstituted unnatural amino acids containing olefin side chains of various lengths can be synthesized by known methods (Williams et al. J. Am. Chem. Soc., 113:9276, 1991 ; Schafmeister et al., J. Am. Chem Soc., 122:5891, 2000; and Bird et al., Methods Enzymol., 446:369, 2008; Bird et al, Current Protocols in Chemical Biology, 2011). For peptides, staples that link i and i + 7 are used (two turns of a stabilized helix), a) one S5 amino acid and one R8 are used, or b) one S8 amino acid and one R5 amino acid are used. R8 is synthesized using the same route except that the starting chiral auxillary gives an R-al kil-stereoisomer. Also, 8-iodooctene is used instead of 5-iodopentene. The inhibitor is synthesized on a solid support using solid-phase peptide synthesis (SPPS) in MBHA resin (see, for example, WO2010 / 148335).

[0200] Fmoc-protected α-amino acids (other than the olefinic amino acids Fmoc-S5-OH, Fmoc-R8-OH, Fmoc-R8-OH, Fmoc-S8-OH and Fmoc-R5-OH), 2-(6-chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU), and Rink Amide MBHA are commercially available, for example, from Novabiochem (San Diego, CA). Dimethylformamide (DMF), N-methyl-2-pyrrolidinone (NMP), N,N-diisopropylethylamine (DIEA), trifluoroacetic acid (TFA), 1,2-dichloroethane (DCE), fluorescein isothiocyanate (FITC), and piperidine are commercially available, for example, from Sigma-Aldrich. Olefinic amino acid synthesis has been reported in the art (Williams et al., Org. Synth., 80:31, 2003).

[0201] Also, methods suitable for obtaining (e.g., synthesizing), stapling, and purifying the peptides disclosed herein are also known in the art (see, e.g., Bird et. al., Methods in Enzymol., 446:369-386 (2008); Bird et al, Current Protocols in Chemical Biology, 2011; Walensky et al., Science, 305:1466-1470 (2004); Schafmeister et al., J. Am. Chem. Soc., 122:5891-5892 (2000); U.S. Patent Application No. 12 / 525,123, filed Mar. 18, 2010; and U.S. Patent No. 7,723,468, issued May 25, 2010, each of which is incorporated herein by reference in its entirety).

[0202] In some embodiments, the peptide is substantially free of or isolated from stapleless peptide contaminants. Methods for purifying the peptide include, for example, synthesizing the peptide on a solid support. After cyclization, the solid support may be isolated and suspended in a solution of a solvent such as DMSO, a DMSO / dichloromethane mixture, or a DMSO / NMP mixture. The DMSO / dichloromethane or DMSO / NMP mixture may contain about 30%, 40%, 50% or 60% DMSO. In a specific embodiment, a 50% / 50% DMSO / NMP solution is used. The solution may be incubated for a period of 1, 6, 12 or 24 hours, after which the resin may be washed, for example, with dichloromethane or NMP. In one embodiment, the resin is washed with NMP. Shaking and bubbling of an inert gas into the solution may be performed.

[0203] Also provided herein is a method for generating a stabilized peptide, comprising: (a) stapling or stitching an E2 hA peptide; and (b) isolating the staple peptide or stitch peptide.

[0204] The properties of the stapled (crosslinked) polypeptides of the present invention can be assayed using, for example, the methods described below and in the examples.

[0205] Assay for determining α-helicity: Dissolve the compound in an aqueous solution (e.g., 5 mM potassium phosphate solution (pH 7), or distilled H2O, to a concentration of 25 - 50 μM). Using standard measurement parameters (e.g., temperature, 20 °C; wavelength, 190 - 260 nm; step resolution, 0.5 nm; speed, 20 nm / sec; accumulation, 10; response, 1 sec; bandwidth, 1 nm; optical path length, 0.1 cm), obtain a circular dichroism (CD) spectrum on a spectropolarimeter (e.g., Jasco J-710, Aviv). Calculate the α-helix content of each peptide by dividing the mean residue ellipticity by the value reported for the model helical decapeptide (Yang et al., Methods Enzymol. 130:208 (1986)).

[0206] Assay for determining the melting temperature (Tm): Dissolve the crosslinked or unmodified template peptide in distilled H2O or other buffer or solvent (e.g., at a final concentration of 50 μM), and using standard parameters (e.g., wavelength 222 nm; step resolution, 0.5 nm; speed, 20 nm / sec; accumulation, 10; response, 1 sec; bandwidth, 1 nm; temperature increase rate: 1 °C / min; optical path length, 0.1 cm) on a spectropolarimeter (e.g., Jasco J-710, Aviv), determine the Tm by measuring the change in ellipticity over a temperature range (e.g., 4 - 95 °C).

[0207] In Vitro Protease Resistance Assay: The amide bonds of the peptide backbone are susceptible to hydrolysis by proteases, which causes peptide-based compounds to rapidly degrade in vivo. However, peptide helix formation can typically bury and / or distort and / or protect the amide backbone and thus can prevent or substantially delay protein cleavage. The peptidomimetic macrocyclic molecules of the present invention can be subjected to in vitro enzymatic proteolysis (e.g., trypsin, chymotrypsin, pepsin) to evaluate any change in the degradation rate compared to the corresponding uncrosslinked polypeptide or stapled polypeptide. For example, the peptidomimetic macrocyclic molecule and the corresponding uncrosslinked polypeptide can be incubated with trypsin agarose, the reaction can be quenched at various time points by centrifugation, and then the remaining substrate can be quantified by HPLC injection and ultraviolet absorption at 280 nm. Briefly, the peptidomimetic macrocyclic molecule and the peptidomimetic precursor (5 mcg) are incubated with trypsin agarose (Pierce) (S / E approximately 125) for 0, 10, 20, 90, and 180 minutes. The reaction is quenched by high-speed benchtop centrifugation, and the substrate remaining in the isolated supernatant is quantified by peak detection by HPLC at 280 nm. The proteolysis reaction exhibits first-order kinetics, and the rate constant, k, is determined from a plot of ln[S] versus time.

[0208] Peptide-mimetic macrocyclic molecules and / or the corresponding non-crosslinked polypeptides can each be incubated with fresh serum (e.g., 1 - 2 mL) from mice, rats, and / or humans at 37 °C for, for example, 0, 1, 2, 4, 8, and 24 hours. Samples with different macrocyclic molecule concentrations can be prepared by serial dilution with serum. To determine the level of intact compound, the following procedure can be used: for example, transfer 100 μL of serum to a 2 mL centrifuge tube, then add 10 μL of 50% formic acid and 500 μL of acetonitrile, and extract the sample by centrifuging at 14,000 RPM for 10 minutes at 4 ± 2 °C. Then transfer the supernatant to a new 2 mL tube and evaporate it at 37 °C with N2 < 10 psi in a Turbovap. Reconstitute the sample with 100 μL of 50:50 acetonitrile:water and perform LC-MS / MS analysis. Equivalent or similar procedures for testing ex vivo stability are known and can be used to determine the stability of macrocyclic molecules in serum.

[0209] In vivo protease resistance assay: The key benefit of peptide stapling is the transfer of in vitro protease resistance to significantly improved in vivo pharmacokinetics.

[0210] In vitro binding assay: To evaluate the binding and affinity of peptide-mimetic macrocyclic molecules and peptide-mimetic precursors to acceptor proteins, for example, a fluorescence polarization assay (FPA) can be used. The FPA technique measures molecular orientation and motion using polarization and a fluorescent tracer. When excited by polarization, a fluorescent tracer (e.g., FITC) bound to a molecule with a high apparent molecular weight (e.g., a FITC-labeled peptide bound to a large protein) emits a higher level of polarized fluorescence due to their slower rotation rates compared to a fluorescent tracer bound to a small molecule (e.g., a FITC-labeled peptide free in solution).

[0211] In vitro enzyme inhibition assay: To evaluate the inhibition of E1 enzyme by the E2 hA peptide, for example, an in vitro enzyme inhibition assay can be used. In the in vitro enzyme inhibition assay, the ability of the E2 hA peptide to inhibit the conjugation of ubiquitin to E2 by E1 is measured. Briefly, for example, in a buffer containing 50 mM NaCl, 50 mM HEPES (pH 7.5), at room temperature for a certain period of time (e.g., 30 minutes), in the presence of the E2 hA peptide or a vehicle control (e.g., 1% DMSO), recombinant E1 (e.g., UBA1), recombinant E2 (e.g., UBE2A), ubiquitin, and Mg-ATP are combined. The control reaction does not contain E1. The reaction is quenched by the addition of a loading dye containing SDS, and the samples are separated on a gel (e.g., a 4-12% Bis-Tris protein gel) under non-reducing conditions. The proteins on the gel are visualized, for example, by silver staining. The conjugation of ubiquitin to E2 by E1 is monitored by the conversion of free E2 (approximately 17 kDa) to the E2-ubiquitin conjugate (approximately 26 kDa). Variants of the stabilized E2 hA peptide

[0212] In some embodiments, an internally cross-linked peptide can be made by modifying any one of the peptides of SEQ ID NOs: 1-38 or a modified version thereof (i.e., a variant thereof such as SEQ ID NO: 39, 55, or 57) (e.g., by amino acid substitution). In some embodiments, an internal staple replaces the side chains of two amino acids, i.e., each staple is between two amino acids separated by, for example, 2, 3, or 6 amino acids. In some embodiments, an internal stitch replaces the side chains of three amino acids, i.e., the stitch is a pair of cross-links between three amino acids separated by, for example, 3 and 6 amino acids. In some embodiments, the internal staples and / or internal stitches include at least two internal staples (replacing the side chains of four amino acids, i.e., each staple is between two amino acids separated by, for example, 3 amino acids). In some embodiments, the internal staples and / or internal stitches include a combination of at least one internal staple and an internal stitch. In some embodiments, an internal stitch replaces the side chains of a first amino acid and second and third amino acids, thereby cross-linking the first amino acid (which is between the second and third amino acids) to the second and third amino acids by an internal cross-link, where the first and second amino acids are separated by 2, 3, or 6 amino acids, the first and third amino acids are separated by 2, 3, or 6 amino acids, and the second and third amino acids are separate amino acids. In some embodiments, the side chains of four amino acids of the internally cross-linked polypeptide of the present disclosure are replaced by two separate internal staples. In some embodiments, the first of the two separate internal staples cross-links a first pair of amino acids separated by 2, 3, or 6 amino acids, and the second of the at least two separate internal staples cross-links a second pair of amino acids separated by 2, 3, or 6 amino acids.

[0213] The staple polypeptide contains at least two modified amino acids joined by an internal intramolecular bridge (or “staple”), and these at least two amino acids are separated by 2, 3, or 6 amino acids. As used herein, stabilized peptides include staple peptides, including peptides having two staples and / or stitch peptides. The at least two modified amino acids may be unnatural alpha-amino acids (including but not limited to α,α-disubstituted amino acids and N-alkylated amino acids). There are many known unnatural amino acids, any of which may be included in the peptides of the present invention. Some examples of unnatural amino acids are 4-hydroxyproline, desmosine, gamma-aminobutyric acid, beta-cyanoalanine, 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-aminocyclobutanecarboxylic acid, 4-aminocyclopentene carboxylic acid, 3-aminocyclohexanecarboxylic acid, 4-piperidylacetic acid, 4-amino-l-methylpyrrole-2-carboxylic acid, 2,4-diaminobutyric acid, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid, 2-aminoheptanedioic acid, 4-(aminomethyl)benzoic acid, 4-aminobenzoic acid, ortho-, meta- and / para-substituted phenylalanine (e.g., substituted with -C(=O)C6H5; -CF3; -CN; -halo; -NO2; CH3), disubstituted phenylalanine, substituted tyrosine (e.g., further substituted with -Q=O)C6H5; -CF3; -CN; -halo; -NO2; CH3), and statins.

[0214] In some embodiments, variants of the internally cross-linked E2 hA peptides of the present disclosure are prepared from any one of the polypeptides of SEQ ID NOs: 1-38 and have, for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids are conservatively or non-conservatively substituted), and / or have, for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid deletions from the N-terminus and / or C-terminus (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids are deleted from the N-terminus and / or C-terminus). Exemplary E2 hA peptides containing the variants are provided in Tables 1-6. For example, in certain embodiments, variants of the internally cross-linked E2 hA peptides of the present disclosure have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acid substitutions in any one of SEQ ID NOs: 1-38 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acids are conservatively or non-conservatively substituted). In some examples, 1-3 amino acids in any one of SEQ ID NOs: 1-38 are substituted. The amino acid substitutions in any one of SEQ ID NOs: 1-38 can be substitutions of amino acids that directly interact (or are predicted to directly interact) with the cognate E1 enzyme of E2 hA or that do not participate (or are predicted not to participate) in direct interaction with it. Even more variability is tolerated in amino acids that do not participate (or are predicted not to participate) in direct interaction with the cognate E1 enzyme of E2 hA than in amino acids that directly interact (or are predicted to directly interact) with the cognate E1 enzyme of E2 hA. In fact, almost all (e.g., 5, 4, 3, 2, or 1 amino acid of those that do not directly interact (or are predicted not to directly interact)) of the amino acids that do not participate (or are predicted not to participate) in direct interaction with the cognate E1 enzyme of E2 hA can be substituted (e.g., by conservative or non-conservative amino acid substitutions or substitution with alanine).In certain embodiments, 1, 2, or 3 amino acids that directly interact (or are predicted to directly interact) with the cognate E1 enzyme of E2 hA are substituted with another amino acid. In some examples, the substitution(s) are conservative amino acid substitutions. In other examples, the substitution(s) are non-conservative amino acid substitutions. In some examples, when there are more than 1 amino acid substitution, the substitutions are both conservative amino acid substitutions and non-conservative amino acid substitutions. In some examples, when there are more than 1 amino acid substitution, each of the substitutions is a conservative amino acid substitution. In some cases, when 1 to 3 amino acids (e.g., 1, 2, or 3) of any one of SEQ ID NOs: 1-38 are substituted, the substitutions are all substitutions of amino acids that do not participate (or are predicted not to participate) in the direct interaction with the cognate E1 enzyme of E2 hA. In some cases, when 1 to 3 amino acids (e.g., 1, 2, or 3) of any one of SEQ ID NOs: 1-38 are substituted, the substitutions are all substitutions of amino acids that directly interact (or are predicted to directly interact) with the cognate E1 enzyme of E2 hA. In some cases, when 1 to 3 amino acids (e.g., 1, 2, or 3) of any one of SEQ ID NOs: 1-38 are substituted, the substitutions are both substitutions of amino acids that directly interact (or are predicted to directly interact) with the cognate E1 enzyme of E2 hA and substitutions of amino acids that do not participate (or are predicted not to participate) in the direct interaction with the cognate E1 enzyme of E2 hA. In certain examples, the substituted amino acid(s) are selected from the group consisting of L-Ala, D-Ala, Aib, Sar, Ser, substituted alanine, or substituted glycine derivatives. In certain examples, the modified Ubc15 E2 hA peptide comprises an E7 residue substituted with arginine (numbering according to SEQ ID NO: 2).

[0215] In certain embodiments, variants of the internally cross-linked E2 hA peptides of the present disclosure can have 1, 2, 3, 4, or 5 amino acids removed / deleted from the C-terminus of the sequences shown in any one of SEQ ID NOs: 1-38. For example, in certain embodiments, variants of the internally cross-linked E2 hA peptides of the present disclosure comprise or consist of a modified amino acid sequence of the amino acid sequence shown in SEQ ID NO: 4, wherein 2 amino acids have been removed / deleted from the C-terminus of the sequence of SEQ ID NO: 4 (e.g., the variant of the internally cross-linked E2 hA peptide comprises or consists of the amino acid sequence of SEQ ID NO: 39). In another example, in certain embodiments, variants of the internally cross-linked E2 hA peptides of the present disclosure comprise or consist of a modified amino acid sequence of the amino acid sequence shown in SEQ ID NO: 6, wherein 5 amino acids have been removed / deleted from the C-terminus of the sequence of SEQ ID NO: 6 (e.g., the variant of the internally cross-linked E2 hA peptide comprises or consists of the amino acid sequence of SEQ ID NO: 55). In certain embodiments, variants of the internally cross-linked E2 hA peptides of the present disclosure can have 1, 2, 3, 4, or 5 amino acids removed / deleted from the N-terminus of the sequences shown in any one of SEQ ID NOs: 1-38. In certain embodiments, the E2 hA peptide that binds to UBA1 of the present disclosure has amino acid positions A removed / deleted from the C-terminus of the sequences shown in any one of SEQ ID NOs: 1-34 12 ~A 16It may have. In certain embodiments, a variant of the internally cross-linked E2 hA peptide that binds to UBA1 of the present disclosure may have 1, 2, 3, or all of the amino acid positions N-terminal to the amino acid position A1 removed / deleted from the N-terminus of the sequence shown in any one of SEQ ID NOs: 1-34. In certain embodiments, a variant of the internally cross-linked E2 hA peptide of the present disclosure may have 1, 2, 3, 4, or 5 amino acids removed / deleted from both the N-terminus and the C-terminus of the sequence shown in any one of SEQ ID NOs: 1-38. In certain embodiments, a variant of the internally cross-linked E2 hA peptide that binds to UBA1 of the present disclosure may have 1, 2, 3, 4, or 5 amino acids removed / deleted from the C-terminus of the sequence shown in any one of SEQ ID NOs: 1-34, and 1, 2, 3, or all of the amino acids N-terminal to the amino acid position A1 removed / deleted from the N-terminus of that sequence. In certain examples, these removed amino acids may be replaced with 1 to 6 (e.g., 1, 2, 3, 4, 5, or 6) amino acids selected from the group consisting of L-Ala, D-Ala, Aib, Sar, Ser, substituted alanine, or substituted glycine derivatives.

[0216] In certain examples, the internally cross-linked E2 hA peptide or variant has the amino acid sequence shown in any one of Tables 7, 8, and 11-15.

[0217] Variants of the internally cross-linked E2 hA peptides described herein can be optimized for therapeutic use. For example, if any of the variants of the internally cross-linked E2 hA peptides described above cause membrane disruption (cell lysis), the peptide can be optimized by reducing the overall hydrophobicity of the peptide. For example, this can be achieved, in particular, by substituting hydrophobic residues with amino acids having lower hydrophobicity (e.g., alanine). Membrane disruption can also be reduced by reducing the overall positive charge of the peptide. This can be achieved by substituting basic residues with uncharged or acidic residues. In certain examples, both the overall hydrophobicity of the peptide and the overall positive charge of the peptide are reduced.

[0218] In certain embodiments, variants of the internally cross-linked E2 hA peptides described herein have an amino acid length between 5 and 35, between 5 and 25, between 5 and 20, between 5 and 18, between 10 and 35, between 10 and 25, between 10 and 20, between 10 and 18, between 15 and 26, or between 15 and 18. In certain embodiments, variants of the internally cross-linked E2 hA peptides described herein are 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids in length.

[0219] In certain embodiments, variants of the stapled E2 hA peptides comprise or consist of the amino acid sequences shown in any one of Tables 7, 8, and 11-15. Non-limiting examples of the structural stabilization of these peptides are achieved by hydrocarbon stapling by introducing unnatural amino acids at positions separated by 2, 3, or 6 amino acids in these sequences. Variants of stabilized E2 hA peptides having a warhead

[0220] The present disclosure features an E2 hA peptide that binds to stabilized UBA1 and includes a warhead, i.e., a reactive group such as a non-natural amino acid having an electrophilic group. Importantly, due to the peptides containing these warheads, the non-covalent interaction between the E2 hA peptide that binds to UBA1 and E1 is joined by a covalent bond between an electrophilic agent and the cysteine at position Cys-1039 of UBA1 (i.e., position 1039 of SEQ ID NO: 845), allowing for significant (much greater than in the absence of the warhead) variability in the amino acid sequence of the relevant E2 hA peptide. Thus, even when there are substitutions and / or deletions in any one of SEQ ID NOs: 1-34 that include amino acid substitutions that directly interact (or are predicted to directly interact) with the cognate E1 enzyme of E2 hA (e.g., positions A4, A5, A7, and A10 for SEQ ID NOs: 3-20 and 22-34), the peptides having these warheads tend to be effective in binding to UBA1. Furthermore, the presence of the warhead can reduce the size of the E2 hA peptide (e.g., any one of the sequences in Tables 1, 2, 6, and 7) to lengths that can be reduced (e.g., down to 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acids).

[0221] The electrophilic agent can be introduced not only in the context of non-natural amino acids but also as a chemical cap on the N-terminus or C-terminus of a stabilized (e.g., cross-linked) E2 hA peptide. In some examples, the electrophilic agent can be introduced into the stabilized peptide. Such stabilized peptides having warheads can form a covalent bond with at least a portion of the protein with which they interact. For example, an E2 hA peptide having such a warhead can modify UBA1 by covalent bond (e.g., by covalent bond with Cys1039 of UBA1 and non-covalent interaction with UBA1).

[0222] The warhead may be present at the N-terminus, C-terminus, or within the polypeptide sequence. In certain embodiments, the warhead is present at position A7. In certain embodiments, the warhead is present at position A8. In certain embodiments, the warhead is present at position A 11 In some cases, the warhead is an amino acid with a non-natural electrophile. In certain embodiments, the warhead is selected from the group consisting of diaminobutyric acid with a bromoacetyl terminus, diaminobutyric acid with an acrylamide terminus; 3S-1-pyrrolidine-3-carboxylic acid with an acrylamide terminus; D-homoproline with an acrylamide terminus; L-homoproline with an acrylamide terminus; isonipecotic acid with an acrylamide terminus; D-nipecotic acid with an acrylamide terminus; L-nipecotic acid with an acrylamide terminus; D-proline with an acrylamide terminus; L-proline with an acrylamide terminus; trans-4-dimethylaminocrotonic acid; and acrylic acid. In certain embodiments, the warhead is diaminobutyric acid with a bromoacetyl terminus. In certain embodiments, the warhead is diaminobutyric acid with an acrylamide terminus.

[0223] In some embodiments, the electrophilic warhead is a cysteine-reactive D-nipecotic acid moiety. In other embodiments, the electrophilic warhead is a cysteine-reactive moiety.

[0224] In certain embodiments, the warhead is a non-natural amino acid having an electrophilic group selected from the group consisting of (S)-1-acryloylpyrrolidine-3-carboxamide; 1-acrylopiperidine-4-carboxamide, (R)-1 acryloylpiperidine-3-carboxamide; (S)-1-acryloylpiperidine-3-carboxamide; (S)-1-acryloylpyrrolidine-2-carboxamide; (R)-1-acryloylpyrrolidine-2-carboxamide; (E)-4-(dimethylamino)but-2-enamide; and acrylamide. In other embodiments, the warhead is not an amino acid. For example, the electrophilic moiety and the peptide are linked by a heterocycle containing saturated (aziridine, diaziridine, azetidine, pyrrolidine, imidazolidine, pyrazolidine, oxazolidine, isoxazolidine, thiazolidine, isothiazolidine, piperidine, piperazine, morpholine, thiomorpholine, azepane) or unsaturated (azirine, diazirine, azeto, pyrrole, imidazole, pyrazole, oxazole, isoxazole, thiazole, isothiazole, pyridine, diazine, oxazine, thiazine, azepine) nitrogen. The peptide and the electrophile can also be linked by a substituted amino-functionalized ring such as phenyl (aniline) (e.g., N-arylacrylamide), or by a more complex bicyclic or polycyclic ring such as naphthalene, anthracene, phenanthrene, indole, isoindole, indolizine, quinolone, isoquinoline, quinoxaline, phthalazine, quinazoline, purine, carbazole, indazole, benzimidazole, azaindole. In some embodiments, the electrophilic warhead is acrylamide or, more generally, an α,β-unsaturated carbonyl such as α-cyanoacrylamide, propiolamide, trans4-dimethylamino-2-butenamide, or trans4-piperidinyl-2-butenamide, or any other substituted acrylamide, or an N-functionalized vinylsulfonyl, alpha-fluoroacetyl, alpha-chloroacetyl, alpha-bromoacetyl, and alpha-iodoacetyl or other electrophilic moiety as defined.The electrophilic agent can be introduced not only in relation to non-natural amino acids, but also as a chemical cap on the N-terminus or C-terminus of a cross-linked (e.g., stapled, stitched) polypeptide.

[0225] In one aspect, the E2 hA that binds to UBA1 and has a warhead comprises or consists of an amino acid sequence shown in any one of SEQ ID NOs: 1-34, or a modified version thereof described herein or in the examples (e.g., SEQ ID NO: 39, 55, or 57), and is modified to include a warhead. In another aspect, the E2 hA peptide that binds to UBA1 and has a warhead comprises or consists of an amino acid sequence shown in any one of SEQ ID NOs: 1-34, or a modified version thereof (e.g., SEQ ID NO: 39, 55, or 57), and is modified to include a warhead. In another aspect, the E2 hA peptide that binds to UBA1 and has a warhead comprises or consists of an amino acid sequence containing 5 or more (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) amino acids of the sequence shown in any one of SEQ ID NOs: 1-34, and is modified to include a warhead. In another aspect, the E2 hA peptide that binds to UBA1 and has a warhead comprises or consists of an amino acid sequence containing 1-8 deletions (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) at the C-terminus or N-terminus of the sequence shown in any one of SEQ ID NOs: 1-34, and is modified to include a warhead. In another aspect, the E2 hA peptide that binds to UBA1 and has a warhead comprises or consists of an amino acid sequence shown in any one of Tables 1, 2, 6, and 7, which is modified to include a warhead. In some examples, the electrophilic warhead is present at the N-terminus of the peptide. In other examples, the electrophilic warhead is present within the peptide, e.g., at position A7, A8, or A 11 is present.

[0226] In one embodiment, the E2 hA peptide that binds to UBA1 and has a warhead has a sequence selected from the sequences shown in Table 10 below.

[0227] Table 10. Peptides with exemplary warheads. SEQ ID NOs: 144-251 and 827-829 (top to bottom, respectively): "J" is a non-natural electrophile containing an amino acid or a moiety that is not an amino acid and is an electrophilic warhead presented in relation to (the electrophile can act as a chemical cap). SEQ ID NOs: 252-359 and 830-832 (top to bottom, respectively): "J" is diaminobutyric acid with an acrylamide terminus or diaminobutyric acid with a bromoacetyl terminus. "tr" = truncate; "m" = mutant

Table 10-1

Table 10-1

Table 10-2

Table 10-3

[0228] In certain examples, the sequences having the above warheads can be structurally stabilized by any method known in the art or by any method described herein. For example, at least two amino acids (e.g., 2, 3, 4, 5) of a sequence separated by 2, 3, or 6 amino acids may be replaced with non-natural amino acids that can form staples and / or stitches.

[0229] In some embodiments, the stapled polypeptide having a warhead comprises or consists of an amino acid sequence selected from Table 11.

[0230] Table 11. Staple peptides with exemplary warheads. SEQ ID NOs: 360-503 and 833-836 (top to bottom, respectively): "B" is norleucine, and "X1" and "X2" are crosslinked rings They are unnatural amino acids that can be covalently joined (``stapled together'') using a ring-closing metathesis (RCM) reaction for formation. "J" is an electrophilic warhead presented in relation to a non-natural electrophile containing an amino acid or a moiety that is not an amino acid (the electrophile can act as a chemical cap). SEQ ID NOs: 504-647 and 837-840 (top to bottom, respectively): "B" is norleucine, "X1" is R-octenylalanine, "X2" is S-pentenylalanine, and "J" is an electrophilic warhead presented in relation to a non-natural electrophile containing an amino acid or a moiety that is not an amino acid (the electrophile can act as a chemical cap). The electrophile can act as a chemical cap). SEQ ID NOs: 648-791 and 841-844 (top to bottom, respectively): "B" is norleucine, "X1" is R-octenylalanine, "X2" is S-pentenylalanine, and "J" is a diamino butyric acid with an acrylamide at the end or a diamino butyric acid with a bromoacetyl at the end. "tr" = truncate; "m" = mutant or a moiety that is not an amino acid (the electrophile can act as a chemical cap). SEQ ID NOs: 648-791 and 841-844 (top to bottom, respectively): "B" is norleucine, "X1" is R-octenylalanine, "X2" is S-pentenylalanine, and "J" is a diamino butyric acid with an acrylamide at the end or a diamino butyric acid with a bromoacetyl at the end. "tr" = truncate; "m" = mutant or a moiety that is not an amino acid (the electrophile can act as a chemical cap). SEQ ID NOs: 648-791 and 841-844 (top to bottom, respectively): "B" is norleucine, "X1" is R-octenylalanine, "X2" is S-pentenylalanine, and "J" is a diamino butyric acid with an acrylamide at the end or a diamino butyric acid with a bromoacetyl at the end. "tr" = truncate; "m" = mutant or a diamino butyric acid with a bromoacetyl at the end. "tr" = truncate; "m" = mutant [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4]

[0231] In some embodiments, the staple peptide having a bullet head comprises or consists of the amino acid sequences listed above in Table 11, and a plurality (e.g., 1, 2, 3, 4, 5, 6) of residues that do not participate in the direct interaction with the cognate E1 enzyme of E2 hA are replaced with residues selected from the group consisting of Ala (alanine), D-Ala (D-alanine), Aib (α-aminoisobutyric acid), Sar (N-methylglycine), and Ser (serine), other substituted alanines, and glycine derivatives. Further, one or more (e.g., 1, 2, 3, 4, 5, 6) of these residues can be added to the C-terminus of the peptide. In some examples, 0 to 5 amino acids at the C-terminus of the above peptides in Table 11 are replaced with residues (one or more) selected from the group consisting of Ala (alanine), D-Ala (D-alanine), Aib (α-aminoisobutyric acid), Sar (N-methylglycine), and Ser (serine), other substituted alanines, and glycine derivatives.

[0232] In some embodiments, the present disclosure features an E2 hA staple peptide having a bullet head that is at least 14%, at least 15%, at least 20%, at least 27%, at least 34%, at least 40%, at least 47%, at least 50%, at least 53%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequences shown in Table 10 or 11, and the modified peptide binds to UBA1 by covalent bond. In some embodiments, these modified peptides have a reduced hydrophobicity and / or overall positive charge compared to the stapled E2 hA peptide prior to the amino acid variation. In some embodiments, the hydrophobicity or positive charge is independently enhanced to optimize cell permeability.

[0233] In certain embodiments, the present disclosure features an E2 hA staple peptide having a warhead with 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions as compared to the amino acid sequences shown in Table 10 or 11, and the modified peptide covalently binds to UBA1. In some embodiments, these modified peptides have a reduced hydrophobicity and / or overall positive charge as compared to the stapled E2 hA peptide prior to the amino acid substitutions. In some embodiments, the hydrophobicity or positive charge is independently enhanced to optimize cell permeability. Variants of exemplary stabilized E2 hA peptides and variants of stabilized E2 hA peptides having a warhead

[0234] In a specific embodiment, the stabilized peptide is based on the amino acid sequence of SEQ ID NO: 4 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions. In a specific embodiment, the stabilized peptide is based on the amino acid sequence of SEQ ID NO: 39 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions. In a specific embodiment, the stabilized peptide comprises the amino acid sequence of BSTPX1RRRLBRX2FKRLQ, where "B" is norleucine, "X1" is S-pentenylalanine, "X2" is R-octenylalanine (SEQ ID NO: 132), and has 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions. In certain embodiments, the stapled peptide further comprises one or more of the modifications described in the "E2 hA peptide" and "stabilized peptide" items above. In another embodiment, the stabilized peptide consists of the amino acid sequence of SEQ ID NO: 132. In certain specific embodiments, 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acids of SEQ ID NO: 132 substituted with another amino acid are present on the face that does not interact with E1 of the helix of SEQ ID NO: 132. In some embodiments, 0 to 3 amino acids of SEQ ID NO: 132 are removed from the C-terminus or removed and replaced with 1 to 6 amino acids from the group consisting of alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives. In some embodiments, 0 to 6 amino acids on the non-interacting face of SEQ ID NO: 132 are substituted with amino acids selected from the group consisting of alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives. In some embodiments, one or more of the following amino acids of SEQ ID NO: 132, Arg-7 (i.e., A4), Leu-9 (i.e., A6), norleucine-10 (i.e., A7), Phe-13 (i.e., A10), and optionally Leu-16 (i.e., A13) are substituted with alpha-methylated or alpha-ethylated natural amino acids.In some embodiments, one or more of the following amino acids of SEQ ID NO: 132, Arg-7 (i.e., A4), Leu-9 (i.e., A6), norleucine-10 (i.e., A7), Phe-13 (i.e., A10), and optionally Leu-16 (i.e., A13) are substituted with an amino acid selected from the group consisting of L-alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives. In some embodiments, one or more of the following amino acids of SEQ ID NO: 132, norleucine-1 (i.e., A-3), Ser-2 (i.e., A-2), Thr-3 (i.e., A-1), Pro-4 (i.e., A1), Ala-5 (i.e., A2), Arg-6 (i.e., A3), Arg-8 (i.e., A5), Arg-11 (i.e., A8), Asp-12 (i.e., A9), Lys-14 (i.e., A11), Arg-15 (i.e., A12), and Gln-17 (i.e., A14) are substituted with an alpha-methylated or alpha-ethylated natural amino acid. In certain embodiments, one or more of the following amino acids of SEQ ID NO: 132, norleucine-1 (i.e., A-3), Ser-2 (i.e., A-2), Thr-3 (i.e., A-1), Pro-4 (i.e., A1), Ala-5 (i.e., A2), Arg-6 (i.e., A3), Arg-8 (i.e., A5), Arg-11 (i.e., A8), Asp-12 (i.e., A9), Lys-14 (i.e., A11), Arg-15 (i.e., A12), and Gln-17 (i.e., A14) are substituted with an amino acid selected from the group consisting of L-alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives. In certain examples, 1 to 6 amino acids of SEQ ID NO: 132 substituted with another amino acid are present on the surface that interacts with E1 of the helix of SEQ ID NO: 132. In other examples, 1 to 6 amino acids of SEQ ID NO: 132 substituted with another amino acid are present on the surface that does not interact and the surface that interacts with E1 of the helix of SEQ ID NO: 132.In certain embodiments, one to six amino acids of SEQ ID NO: 132 are substituted with one or more amino acids selected from the group consisting of L-alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives.

[0235] In a specific embodiment, the stabilized peptide is based on the amino acid sequence of SEQ ID NO: 6 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions. In a specific embodiment, the stabilized peptide is based on the amino acid sequence of SEQ ID NO: 55 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions. In a specific embodiment, the stabilized peptide comprises the amino acid sequence of BAGTX1LKRLBAX2YK, wherein "B" is norleucine, "X1" is S-pentenylalanine, "X2" is R-octenylalanine (SEQ ID NO: 133), and has 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions. In a particular embodiment, the stapled peptide further comprises one or more of the modifications described in the above "E2 hA peptide" and "stabilized peptide" items. In another embodiment, the stabilized peptide consists of the amino acid sequence of SEQ ID NO: 133. In a certain specific embodiment, 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acids of SEQ ID NO: 133 substituted with another amino acid are present on the surface that does not interact with E1 of the helix of SEQ ID NO: 133. In some embodiments, 0 to 3 amino acids of SEQ ID NO: 133 are removed from the C-terminus or removed and replaced with 1 to 6 amino acids from the group consisting of alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives. In some embodiments, 0 to 6 amino acids on the non-interacting surface of SEQ ID NO: 133 are replaced with amino acids selected from the group consisting of alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives. In some embodiments, one or more of the following amino acids of SEQ ID NO: 133, Lys-7 (i.e., A4), Leu-9 (i.e., A6), norleucine-10 (i.e., A7), Tyr-13 (i.e., A10), and optionally Leu-16 (i.e., A13) are replaced with alpha-methylated or alpha-ethylated natural amino acids.In some embodiments, one or more of the following amino acids of SEQ ID NO: 133, Lys-7 (i.e., A4), Leu-9 (i.e., A6), norleucine-10 (i.e., A7), Tyr-13 (i.e., A10), and optionally Leu-16 (i.e., A13), are substituted with an amino acid selected from the group consisting of L-alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives. In some embodiments, one or more of the following amino acids of SEQ ID NO: 133, norleucine-1 (i.e., A-3), Ala-2 (i.e., A-2), Gly-3 (i.e., A-1), Thr-4 (i.e., A1), Ala-5 (i.e., A2), Leu-6 (i.e., A3), Arg-7 (i.e., A5), Ala-10 (i.e., A8), Glu-11 (i.e., A9), and Lys-13 (i.e., A11), are substituted with a natural amino acid that is alpha-methylated or alpha-ethylated. In certain embodiments, the following amino acids of SEQ ID NO: 107, norleucine-1 (i.e., A-3), Ala-2 (i.e., A-2), Gly-3 (i.e., A-1), Thr-4 (i.e., A1), Ala-5 (i.e., A2), Leu-6 (i.e., A3), Arg-7 (i.e., A5), Ala-10 (i.e., A8), Glu-11 (i.e., A9), and Lys-13 (i.e., A11), are substituted with an amino acid selected from the group consisting of L-alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives. In certain examples, the 1 to 6 amino acids of SEQ ID NO: 133 substituted with another amino acid are present on the face that interacts with E1 of the helix of SEQ ID NO: 133. In other examples, the 1 to 6 amino acids of SEQ ID NO: 133 substituted with another amino acid are present on the face that does not interact with and the face that interacts with E1 of the helix of SEQ ID NO: 133. In certain embodiments, the 1 to 6 amino acids of SEQ ID NO: 133 are substituted with one or more amino acids selected from the group consisting of L-alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives.

[0236] In a specific embodiment, the stabilized peptide is based on the amino acid sequence of SEQ ID NO: 10 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions. In a specific embodiment, the stabilized peptide comprises the amino acid sequence of BX1LKRIHKX2LNDLARD, where "B" is norleucine, "X1" is S-pentenylalanine, "X2" is R-octenylalanine (SEQ ID NO: 107), and has 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions. In certain embodiments, the stapled peptide further comprises one or more of the modifications described in the "E2 hA peptide" and "stabilized peptide" items above. In another embodiment, the stabilized peptide consists of the amino acid sequence of SEQ ID NO: 107. In certain specific embodiments, 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acids of SEQ ID NO: 107 substituted by another amino acid are present on the face that does not interact with E1 of the helix of SEQ ID NO: 107. In some embodiments, 0 to 3 amino acids of SEQ ID NO: 107 are removed or removed and replaced with 1 to 6 amino acids from the group consisting of alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives. In some embodiments, 0 to 6 amino acids on the non-interacting face of SEQ ID NO: 107 are substituted with amino acids selected from the group consisting of alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives. In some embodiments, one or more of the following amino acids of SEQ ID NO: 107, Lys-4 (i.e., A4), Ile-6 (i.e., A6), His-7 (i.e., A7), Leu-10 (i.e., A10), and optionally Leu-13 (i.e., A13) are substituted with alpha-methylated or alpha-ethylated natural amino acids.In some embodiments, one or more of the following amino acids of SEQ ID NO: 107, Lys-4 (i.e., A4), Ile-6 (i.e., A6), His-7 (i.e., A7), Leu-10 (i.e., A10), and optionally Leu-13 (i.e., A13) are substituted with an amino acid selected from the group consisting of L-alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives. In some embodiments, one or more of the following amino acids of SEQ ID NO: 107, norleucine-1 (i.e., A1), Ala-2 (i.e., A2), Leu-3 (i.e., A3), Arg-5 (i.e., A5), Lys-8 (i.e., A8), Glu-9 (i.e., A9), Asn-11 (i.e., A11), Asp-12 (i.e., A12), Ala-14 (i.e., A14), Arg-15 (i.e., A15), and Asp-16 (i.e., A16) are substituted with an alpha-methylated or alpha-ethylated natural amino acid. In certain embodiments, one or more of the following amino acids of SEQ ID NO: 107, norleucine-1 (i.e., A1), Ala-2 (i.e., A2), Leu-3 (i.e., A3), Arg-5 (i.e., A5), Lys-8 (i.e., A8), Glu-9 (i.e., A9), Asn-11 (i.e., A11), Asp-12 (i.e., A12), Ala-14 (i.e., A14), Arg-15 (i.e., A15), and Asp-16 (i.e., A16) are substituted with an amino acid selected from the group consisting of L-alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives. In certain examples, 1 to 6 amino acids of SEQ ID NO: 107 substituted with another amino acid are present on the surface that interacts with E1 of the helix of SEQ ID NO: 107. In other examples, 1 to 6 amino acids of SEQ ID NO: 107 substituted with another amino acid are present on the surface that does not interact and the surface that interacts with E1 of the helix of SEQ ID NO: 107.In certain embodiments, one to six amino acids of SEQ ID NO: 107 are substituted with one or more amino acids selected from the group consisting of L-alanine, D-alanine, α-aminoisobutyric acid, N-methylglycine, serine, substituted alanine, and glycine derivatives.

[0237] In another specific embodiment, the peptide is an E2 hA peptide having a warhead that binds to E1 as described herein. For example, the peptides of SEQ ID NOs: 4, 6, 10, 39, 55, or variants thereof are modified to include an electrophilic warhead presented in the context of a non-natural electrophile containing an amino acid or a moiety that is not an amino acid (the electrophile acts as a chemical cap) (e.g., a cysteine-reactive moiety such as diaminobutyric acid with an acrylamide terminus or diaminobutyric acid with a bromoacetyl terminus).

[0238] In a specific embodiment, the E2 hA peptide having a warhead that binds to E1 includes the sequence of SEQ ID NO: 176 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In certain embodiments, the E2 hA peptide having a warhead that binds to E1 includes the sequence of SEQ ID NO: 392 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In another specific embodiment, the E2 hA peptide having a warhead that binds to E1 includes the sequence of SEQ ID NO: 680 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In certain embodiments, the E2 hA peptide having a warhead that binds to E1 (e.g., SEQ ID NO: 176, 392, or 680) further includes one or more modifications described in the "E2 hA Peptide" and "Stabilized Peptide" sections above.

[0239] In a specific embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 212 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In a particular embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 428 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In another particular embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 716 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In a particular embodiment, the E2 hA peptide having a warhead and binding to E1 (e.g., SEQ ID NO: 212, 428, or 716) further comprises one or more modifications described in the above "E2 hA Peptide" and "Stabilized Peptide" sections.

[0240] In a specific embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 248 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In a particular embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 464 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In another particular embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 752 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In a particular embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 500 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In another particular embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 788 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In a particular embodiment, the E2 hA peptide having a warhead and binding to E1 (e.g., SEQ ID NO: 248, 464, 500, 752, or 788) further comprises one or more of the modifications described in the above "E2 hA Peptide" and "Stabilized Peptide" sections.

[0241] In a specific embodiment, the E2 hA peptide having a bullet head and binding to E1 comprises the sequence of SEQ ID NO: 177 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In certain embodiments, the E2 hA peptide having a bullet head and binding to E1 comprises the sequence of SEQ ID NO: 393 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In another specific embodiment, the E2 hA peptide having a bullet head and binding to E1 comprises the sequence of SEQ ID NO: 681 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In certain embodiments, the E2 hA peptide having a bullet head and binding to E1 (e.g., SEQ ID NO: 177, 393, or 681) further comprises one or more modifications described in the above "E2 hA Peptide" and "Stabilized Peptide" sections.

[0242] In a specific embodiment, the E2 hA peptide having a bullet head and binding to E1 comprises the sequence of SEQ ID NO: 213 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In certain embodiments, the E2 hA peptide having a bullet head and binding to E1 comprises the sequence of SEQ ID NO: 429 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In another specific embodiment, the E2 hA peptide having a bullet head and binding to E1 comprises the sequence of SEQ ID NO: 717 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In certain embodiments, the E2 hA peptide having a bullet head and binding to E1 (e.g., SEQ ID NO: 213, 429, or 717) further comprises one or more modifications described in the above "E2 hA Peptide" and "Stabilized Peptide" sections.

[0243] In a specific embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 249 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In a particular embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 465 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In another particular embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 753 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In a particular embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 501 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In another particular embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 789 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In a particular embodiment, the E2 hA peptide having a warhead and binding to E1 (e.g., SEQ ID NO: 249, 465, 501, 753, or 789) further comprises one or more of the modifications described in the above "E2 hA Peptide" and "Stabilized Peptide" sections.

[0244] In a specific embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 151 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In a particular embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 367 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In another particular embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 655 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In a particular embodiment, the E2 hA peptide having a warhead and binding to E1 (e.g., SEQ ID NO: 151, 367, or 655) further comprises one or more modifications described in the above "E2 hA peptide" and "stabilized peptide" sections.

[0245] In a specific embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 187 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In a particular embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 403 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In another particular embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 691 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In a particular embodiment, the E2 hA peptide having a warhead and binding to E1 (e.g., SEQ ID NO: 187, 403, or 691) further comprises one or more modifications described in the above "E2 hA peptide" and "stabilized peptide" sections.

[0246] In a specific embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 223 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In a particular embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 439 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In another particular embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 727 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In a particular embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 475 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In another particular embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 763 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In a particular embodiment, the E2 hA peptide having a warhead and binding to E1 (e.g., SEQ ID NO: 223, 439, 727, 757, or 763) further comprises one or more of the modifications described in the above "E2 hA Peptide" and "Stabilized Peptide" sections.

[0247] In a specific embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 827 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In certain embodiments, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 835 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In another specific embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 843 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions. In certain embodiments, the E2 hA peptide having a warhead and binding to E1 (e.g., SEQ ID NO: 827, 835, or 843) further comprises one or more modifications described in the above "E2 hA peptide" and "stabilized peptide" sections.

[0248] In a specific embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 828 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In certain embodiments, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 834 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In another specific embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 842 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions. In certain embodiments, the E2 hA peptide having a warhead and binding to E1 (e.g., SEQ ID NO: 828, 834, or 842) further comprises one or more modifications described in the above "E2 hA peptide" and "stabilized peptide" sections.

[0249] In a specific embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 829 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In certain embodiments, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 836 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In another specific embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 844 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In certain embodiments, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 833 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In another specific embodiment, the E2 hA peptide having a warhead and binding to E1 comprises the sequence of SEQ ID NO: 841 having 0 to 6 (i.e., 0, 1, 2, 3, 4, 5, 6) amino acid substitutions, insertions, and / or deletions (as described above). In certain embodiments, the E2 hA peptide having a warhead and binding to E1 (e.g., SEQ ID NO: 829, 833, 836, 841, or 844) further comprises one or more of the modifications described in the above "E2 hA Peptide" and "Stabilized Peptide" sections. Method of treatment

[0250] The present disclosure features a method of using any of the stabilized peptides described herein (or a pharmaceutical composition comprising said stabilized peptide) for the prevention and / or treatment of a disease that expresses E1 or a disease that is dependent on E1. Non-limiting examples of diseases that express E1 or are dependent on E1 include cancer, hematological malignancies, solid tumors, antibody-mediated transplant rejection, autoimmune disorders, inflammatory disorders, and other diseases involved in the survival of diseased cells. The term "treating" or "treatment" as used herein refers to alleviating, inhibiting, or improving a disease or condition from which a subject suffers.

[0251] The peptides (or compositions comprising the peptides) described herein may be useful for treating a human subject having a cancer that expresses E1. The peptides (or compositions comprising the peptides) described herein may also be useful for treating a human subject having a cancer that is dependent on E1. In certain embodiments, the cancer is a solid tumor or a liquid tumor. In certain embodiments, the solid tumor is a bladder cancer tumor, a bile duct cancer tumor, a bone cancer tumor, a soft tissue sarcoma, a brain tumor, a spinal cord tumor, a breast cancer tumor, a pancreatic cancer tumor, a colorectal cancer tumor, a rectal cancer tumor, a small intestine cancer tumor, a prostate cancer tumor, a kidney cancer tumor, a hepatocellular cancer tumor, a gallbladder cancer tumor, a lung cancer tumor, a bronchial cancer tumor, an ovarian cancer tumor, a cervical cancer tumor, a vaginal cancer tumor, an endometrial cancer tumor, a gastric cancer tumor, an esophageal cancer tumor, a head and neck cancer (e.g., nasopharyngeal cancer, oropharyngeal cancer, salivary gland cancer, and thyroid cancer) tumor, a melanoma tumor, an intraocular melanoma tumor, or a neuroendocrine tumor. In certain embodiments, the cancer is a melanoma, leukemia, lymphoma, or other hematologic malignancy or solid tumor. In certain embodiments, the hematologic malignancy is acute myeloid leukemia, acute lymphoblastic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome, multiple myeloma, chronic myelomonocytic leukemia, lymphoma (e.g., Hodgkin's disease, follicular lymphoma, mantle cell lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma, and T-cell lymphoma), myeloproliferative syndrome, Waldenström's macroglobulinemia. In certain embodiments, the cancer is breast cancer, ganglionic cancer, pancreatic cancer, skin cancer, CNS cancer, hematopoietic or lymphatic cancer, lung cancer, colorectal cancer, stomach cancer, soft tissue sarcoma, or bone cancer. In certain instances, the solid tumor is melanoma, breast cancer or lung cancer.In certain embodiments, the autoimmune or inflammatory disorder is a gastrointestinal disorder (e.g., celiac disease, Crohn's disease, or ulcerative colitis), a skin and musculoskeletal condition (e.g., alopecia areata, scleroderma, psoriasis, pemphigoid, rheumatoid arthritis, osteoarthritis, psoriatic arthritis, fibromyalgia, polymyalgia rheumatica, ankylosing spondylitis, Behçet's disease, CREST syndrome, lupus erythematosus, or vitiligo), an airway and lung disease (e.g., asthma or COPD), an autoimmune neuropathy (e.g., chronic inflammatory demyelinating polyneuropathy, acute motor axonal neuropathy, multiple sclerosis, or restless legs syndrome), vasculitis, nephritis, hepatitis, biliary cirrhosis, primary sclerosing cholangitis, myocarditis, Addison's disease, antiphospholipid syndrome, aplastic anemia, encephalitis, chronic fatigue syndrome, diabetes, endometriosis, Graves' disease, Guillain - Barré syndrome, sarcoidosis, infection - related inflammation, ischemia - related inflammation, or a neurodegenerative disorder (e.g., Alzheimer's disease).

[0252] In certain embodiments, a human subject in need thereof is administered a peptide selected from the group consisting of the sequences of Tables 7, 8, and 11 - 15. In certain embodiments, a human subject in need thereof is administered a stapled E2 hA peptide comprising or consisting of SEQ ID NO: 4 or a modified version thereof, e.g., a peptide comprising or consisting of any one of the amino acid sequences of SEQ ID NO: 65, 96, 101, and 132. In certain embodiments, a human subject in need thereof is administered a stapled E2 hA peptide comprising or consisting of SEQ ID NO: 6 or a modified version thereof, e.g., a peptide comprising or consisting of any one of the amino acid sequences of SEQ ID NO: 67, 97, 103, and 133. In certain embodiments, a human subject in need thereof is administered a stapled E2 hA peptide comprising or consisting of SEQ ID NO: 10, or a modified version thereof, e.g., a peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 71 or SEQ ID NO: 107. In certain embodiments, a human subject in need thereof is administered a stapled E2 comprising or consisting of SEQ ID NO: 2 The hA peptide, or a modified version thereof, for example, a peptide comprising or consisting of the amino acid sequences of SEQ ID NOs: 99, 135, 850, and 851 is administered.

[0253] In certain embodiments, a human subject in need thereof is administered a peptide having a warhead selected from the group consisting of the sequences of Tables 10 and 14. In certain embodiments, a human subject in need thereof is administered a staple peptide having a warhead based on SEQ ID NO: 4, e.g., a peptide comprising or consisting of the amino acid sequences of SEQ ID NOs: 361, 392, 397, 428, 433, 464, 469, 500, 505, 536, 541, 572, 577, 608, 613, 644, 649, 680, 685, 716, 721, 752, 757, and 788. In certain embodiments, a human subject in need thereof is administered a staple peptide having a warhead based on SEQ ID NO: 6, e.g., a peptide comprising or consisting of the amino acid sequences of SEQ ID NOs: 363, 393, 399, 429, 435, 465, 471, 501, 507, 537, 543, 573, 579, 609, 615, 645, 651, 681, 687, 717, 723, 753, 759, and 789. In certain embodiments, a human subject in need thereof is administered a staple peptide having a warhead based on SEQ ID NO: 10, e.g., a peptide comprising or consisting of the amino acid sequences of SEQ ID NOs: 367, 403, 439, 475, 511, 547, 583, 619, 655, 691, 727, and 763. In certain embodiments, a human subject in need thereof is administered a staple peptide having a warhead based on SEQ ID NO: 2, e.g., a peptide comprising or consisting of the amino acid sequences of SEQ ID NOs: 395, 431, 467, 502, 503, 539, 575, 611, 646, 647, 683, 719, 755, 790, 791, and 833 - 844.In certain embodiments, a human subject in need thereof is administered a peptide that is at least 14%, at least 15%, at least 20%, at least 27%, at least 34%, at least 40%, at least 47%, at least 50%, at least 53%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of the sequences of Tables 10 and 14.

[0254] In some examples for the treatment of cancer, autoimmune disorders, or inflammatory disorders, a human subject in need thereof is co-administered radiation therapy, immunotherapy, or chemotherapy. Non-limiting examples of chemotherapy include alkylating agents (e.g., busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mephelan, oxaliplatin, procarbazine hydrochloride, temozolomide, or thiotepa), antimetabolites (e.g., azathioprine, capecitabine, cladrabine, clofarabine, cytarabine, decitabine, floxuradine, fluorouracil, hydro (drozyurea), mercaptopurine, methotrexate, pralatrexate, thioguanine, pentostatin, or vidarabine), antitumor antibiotics (e.g., dactinomycin, bleomycin, mitomycin C, adriamycin, daunorubicin, idarubicin, doxorubicin, or mitoxantrone), mitotic inhibitors (e.g., paclitaxel, docetaxel, vinorelbine, vincristine, vinblastine), platinum agents (e.g., cisplatin, carboplatin, or oxaliplatin), proteasome inhibitors (e.g., bortezomib), topoisomerase inhibitors (e.g., etoposide, teniposide, camptothecin, topotecan, irinotecan, doxorubicin, or daunorubicin), thalidomide and related analogs, steroids (e.g., dexamethasone or prednisone), and antibodies (e.g., trastuzumab, rituximab, cetuximab, and bevacizumab). In some examples, a human subject in need thereof is co-administered with an immunomodulatory agent or an anti-inflammatory agent. Non-limiting examples of immunomodulatory agents include methotrexate, leflunomide, cyclophosphamide, cyclosporin A, mycophenolate mofetil, rapamycin, mizoribine, deoxyspergualin ), and brequinar. Non-limiting examples of anti-inflammatory agents include non-steroidal anti-inflammatory drugs (e.g., salicylic acid, acetylsalicylic acid, methyl salicylate, diflunisal, salsalate, olsalazine, sulfasalazine, acetaminophen, indomethacin, sulindac, etodolac, mefenamic acid, sodium meclofenamate, tolmetin, ketorolac, diclofenac, ibuprofen, naproxen, sodium naproxen, fenoprofen, ketoprofen, flurbinprofen, oxa prozin, piroxicam, meloxicam, ampiroxicam, droxicam, pivoxicam, tenoxicam, nabumetome, phenylbutazone, ox Corticosteroids, antipyrine, aminopyrine, apazone and nimesulide; leukotriene antagonists containing diroton, aurothioglucose, sodium aurothiomalate or auranofin), steroids (e.g., alclometasone diproprionate, amcinonide, beclometasone diproprionate, betamethasone, betamethasone benzoate, betamethasone diproprionate, sodium betamethasone phosphate, betamethasone valerate, clobetasol proprionate, clocortolone pivalate, hydrocortisone, hydrocortisone derivatives, desonide, desoximatasone, dexamethasone, fluocinonide, flucoxinolide, flurandrenolide, halcinocide, medrysone, methylprednisolone, methylprednisolone acetate, methyl succinate prednisolone sodium, mometasone furoate, paramethasone acetate, prednisolone, prednisolone acetate, prednisolone sodium phosphate, prednisolone tebuatate, prednisone, triamcinolone, triamcinolone acetonide , triamcinolone diacetate, or triamcinolone hexacetonide); and other anti-inflammatory agents (e.g., methotrexate, colchicine, allopurinol, probenecid, sulfinpyrazone or benzbromarone).

[0255] In some embodiments, the human subject has cancer, a hematologic malignancy, a solid tumor, antibody-mediated transplant rejection, an autoimmune disorder, or an inflammatory disorder.

[0256] Generally, the method includes the steps of selecting a subject, administering to the subject, e.g., in or as a pharmaceutical composition, one or more effective amounts of one of the peptides herein, and, if necessary, repeating the administration if required for cancer prevention or treatment, and can be administered orally, intravenously or topically. For example, based on the determination that the subject has a cancer that expresses E1, the subject can be selected for treatment. Using the peptides of the present disclosure, it is possible to determine whether the subject's cancer expresses E1 or whether the subject's cancer is dependent on E1.

[0257] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors including the activity of the particular compound used, age, weight, general health, sex, diet, time of administration, rate of excretion, drug combination, disease, state or severity and course of the symptoms, predisposition of the patient to the disease, state or symptoms, and the judgment of the physician treating the patient.

[0258] An effective amount can be administered in one or more administrations, applications or dosages. The therapeutically effective amount (i.e., the effective dosage) of the therapeutic compound depends on the therapeutic compound selected. The composition can be administered from once or more a day to once or more a week (including once every other day). One of ordinary skill in the art will recognize that certain factors, including but not limited to the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present, can affect the dosage and timing required to effectively treat the subject. Further, treatment of a subject with a therapeutically effective amount of the therapeutic compounds described herein can include a single treatment or a series of treatments. For example, an effective amount can be administered at least once. Pharmaceutical composition

[0259] One or more of the stabilized peptides described herein can be formulated as, or for use in, a pharmaceutical composition. The pharmaceutical composition may be used in the treatment methods described herein (see above). In certain embodiments, the pharmaceutical composition comprises or consists of a peptide having an amino acid sequence that is identical to the amino acid sequence shown in any one of Tables 7-14, except for one or more substitutions, insertions, or deletions of 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid. These changes to the amino acid sequence are made to the alpha-helix face that does not interact with E1 of these peptides (i.e., to amino acids not predicted to be involved in / directly interact with E1) and / or to the alpha-helix face that interacts with E1 (i.e., to amino acids predicted to directly interact with E1). Such compositions can be formulated or applied for administration to a subject by any route, such as any route approved by the U.S. Food and Drug Administration (FDA). Exemplary methods are described in the FDA's CDER Data Standards Manual, version number 004 (available at fda.give / cder / dsm / DRG / drg00301.htm). For example, the composition can be formulated or applied for administration by inhalation (e.g., oral and / or nasal inhalation (e.g., by nebulizer or spray)), injection (e.g., intravenously, intraarterially, subdermally, intraperitoneally, intramuscularly, and / or subcutaneously); and / or for oral administration, transmucosal administration, and / or topical administration (including topical (e.g., nasal) sprays and / or solutions). (available at fda.give / cder / dsm / DRG / drg00301.htm). For example, the composition can be formulated or applied for administration by inhalation (e.g., oral and / or nasal inhalation (e.g., by nebulizer or spray)), injection (e.g., intravenously, intraarterially, subdermally, intraperitoneally, intramuscularly, and / or subcutaneously); and / or for oral administration, transmucosal administration, and / or topical administration (including topical (e.g., nasal) sprays and / or solutions).

[0260] In some examples, the pharmaceutical composition may comprise an effective amount of one or more stabilized peptides. As used herein, the terms “effective amount” and “effective to treat” refer to an amount or concentration of one or more of the compounds or pharmaceutical compositions described herein that, in relation to its administration, is effective to bring about the intended effect or physiological outcome (e.g., treatment of an infectious disease) over a period of time (including acute or chronic administration and intermittent or continuous administration).

[0261] The pharmaceutical compositions of the invention may comprise one or more peptides and any pharmaceutically acceptable carrier and / or vehicle. In some examples, the pharmaceutical may further comprise one or more additional therapeutic agents in an amount effective to achieve modulation of a disease or disease symptom. For example, the pharmaceutical composition may comprise a radiation therapy agent, an immunotherapy agent, a chemotherapy agent, an immunomodulatory agent, or an anti-inflammatory agent.

[0262] The term “pharmaceutically acceptable carrier or adjuvant” refers to a carrier or adjuvant that can be administered to a patient together with a compound of the invention and that, when administered in a dosage sufficient to deliver a therapeutically effective amount of the compound, does not destroy the physiological activity of the compound and is non-toxic.

[0263] Pharmaceutically acceptable carriers, adjuvants and vehicles that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-α-tocopherol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tween® or other similar polymer delivery matrices, serum proteins such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, for example, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylate, wax, polyethylene-polyoxypropylene-block polymer, polyethylene glycol and lanolin. Cyclodextrins such as α-, β-, and γ-cyclodextrins can also be advantageously used to enhance the delivery of the compounds of the formulas described herein.

[0264] The pharmaceutical compositions of the present invention may contain any conventional non-toxic pharmaceutically acceptable carrier, adjuvant or vehicle. In some cases, the pH of the formulation can be adjusted with a pharmaceutically acceptable acid, base or buffer to enhance the stability of the formulated compound or its delivery form. The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-arterial, intra-synovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

[0265] The pharmaceutical composition can be in the form of a solution or a powder for inhalation and / or nasal administration. Such a composition may be formulated according to techniques known in the art using suitable dispersants or wetting agents (such as Tween® 80, etc.) and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3 - butanediol. Acceptable vehicles and solvents that can be used include mannitol, water, Ringer's solution, and isotonic sodium chloride solution. Further, sterile fixed oils have conventionally been used as solvents or suspending media. For this purpose, any non-irritating fixed oil containing synthetic monoglycerides or diglycerides can be used. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils such as olive oil or castor oil (especially their polyoxyethylenated versions). These oil solutions or suspensions can also contain long-chain alcohol diluents or dispersants, or carboxymethyl cellulose or similar dispersants, which are commonly used in the formulation of pharmaceutically acceptable dosage forms such as emulsions and suspensions. Other commonly used surfactants such as Tween® or Span® and / or other similar emulsifiers or bioavailability enhancers commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for formulation.

[0266] The pharmaceutical composition can be orally administered in any orally acceptable dosage form, including but not limited to capsule, tablet, emulsion, aqueous suspension, dispersion, and solution. In the case of tablets for oral use, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are also typically added. For oral administration in capsule form, useful diluents include lactose and dried corn starch. When an aqueous suspension and / or emulsion is orally administered, the active ingredient that can be suspended or dissolved in the oil layer is combined with an emulsifier and / or suspending agent. If desired, certain sweetening agents and / or flavoring agents and / or coloring agents may be added.

[0267] Alternatively or additionally, the pharmaceutical composition can be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation and can be prepared as a solution in saline using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents known in the art.

[0268] In some examples, one or more of the peptides disclosed herein can be conjugated, for example, to a carrier protein. Such conjugated compositions can be monovalent or multivalent. For example, the conjugated composition may include one peptide disclosed herein conjugated to a carrier protein. Alternatively, the conjugated composition may include two or more peptides disclosed herein conjugated to a carrier.

[0269] As used herein, when two entities are "conjugated" to each other, they are linked by a direct or indirect covalent or non-covalent interaction. In certain embodiments, the association is by covalent bond. In other embodiments, the association is by non-covalent bond. Non-covalent interactions include hydrogen bonding, van der Waals interactions, hydrophobic interactions, magnetic interactions, electrostatic interactions, and the like. An interaction by an indirect covalent bond is the case where two entities are covalently connected via a linker group, if necessary.

[0270] A carrier protein can include any protein that increases or enhances immunogenicity in a subject. Exemplary carrier proteins are described in the art (see, for example, Fattom et al., Infect. Immun., 58:2309-2312, 1990; Devi et al., 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., Infect. Immun. 62:4440-4444, 1994). A polymeric carrier can be a natural or synthetic material containing one or more primary and / or secondary amino groups, azide groups, or carboxyl groups. The carrier can be water-soluble. Method for making stabilized peptides

[0271] The present disclosure features a method of making stabilized (e.g., singly stapled, doubly stapled, or stitched) peptides. The method involves obtaining an uncrosslinked version of any of the peptides described herein that contain two or more unnatural amino acids, and crosslinking the peptide. In one example, the crosslinking is by a ring-closing metathesis (RCM) reaction.

[0272] Synthesis of stapled peptides: Stapled peptide fusion inhibitors can be synthesized according to reported methods for generating all hydrocarbon stapled peptides using Fmoc-based solid-phase peptide synthesis. For example, to achieve various staple lengths, α-methyl, α-alkenyl amino acids were introduced at the i, i + 4 (or i, i + 7) positions using, for example, two S-2-(4'-pentenyl)alanine (S5) residues; two (R)-2-(((9H-fluoren-9-yl)methoxy)carbonylamino)-2-methyl-dec-9-enoic acid (R8) residues; or one S5 residue and one S8 residue. In some examples, to achieve various staple lengths, α-methyl, α-alkenyl amino acids were introduced at the i, i + 4 (or i, and i + 7) positions using, for example, R-octenylalanine (e.g., (R)-α-(7'-octenyl)alanine); R-pentenylalanine (e.g., (R)-α-(4'-pentenyl)alanine); one bis-pentenylglycine (e.g., α,α-bis(4'-pentenyl)glycine); one one bis-octenylglycine (e.g., α,α-bis(7'-octenyl)glycine); or S-octenylalanine (e.g., (S)-α-(7'-octenyl)alanine). For the stapling reaction, a Grubbs first-generation ruthenium catalyst dissolved in dichloroethane is added to the resin-bound peptide. To ensure maximum conversion, 3 - 5 stapling runs are performed. The peptide is then cleaved from the resin using trifluoroacetic acid, precipitated using a hexane:ether (1:1) mixture, air dried, and purified by LC-MS. The peptide can be quantified by amino acid analysis.

[0273] Synthesis of Stitch Peptides: Methods for synthesizing stitch peptides are known in the art. Nevertheless, the following exemplary methods may be used. Synthetic chemical transformations and protecting group methodologies (protection and deprotection) useful for synthesizing the compounds described herein are known in the art and are described, for example, in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 3d. Ed., John Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), as well as methods as described in their revisions.

[0274] The peptides of the present invention can be made by chemical synthesis methods well known to those skilled in the art. For example, see Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, W. H. Freeman & Co., New York, N.Y., 1992, p. 77. Therefore, for example, on an Applied Biosystems Peptide Synthesizer Model 430A or 431, the peptides can be synthesized using an automated Merrifield technique of solid-phase synthesis with α-NH2 protected by either t-Boc or Fmoc chemistry using side-chain protected amino acids.

[0275] One way to produce the peptides described herein is to use solid-phase peptide synthesis (SPPS). The C-terminal amino acid is attached to a cross-linked polystyrene resin via an acid-labile bond to a linker molecule. This resin is insoluble in the solvents used for synthesis, whereby excess reagents and by-products are washed away relatively easily and quickly. The N-terminus is protected with an Fmoc group, which is stable to acid but removable by base. All side-chain functional groups are protected using base-stable and acid-labile protecting groups.

[0276] Longer peptides can be made by joining individual synthetic peptides using native chemical ligation. Insertion of the stitching amino acids may be carried out as described, for example, in Young and Schultz, J Biol Chem. 2010 Apr 9; 285(15): 11039-11044. Alternatively, longer synthetic peptides can be synthesized by well-known recombinant DNA techniques. Such techniques are provided in well-known standard manuals with detailed protocols. To construct a gene encoding the peptide of the invention, the amino acid sequence is reverse-translated to obtain a nucleic acid sequence encoding an amino acid sequence that preferably has codons optimal for the organism in which the gene is to be expressed. The synthetic gene is then typically made by synthesizing oligonucleotides encoding the peptide and, if necessary, any regulatory elements. The synthetic gene is inserted into a suitable cloning vector and transfected into a host cell. The peptide is then expressed under suitable conditions appropriate for the selected expression system and host. The peptide is purified and characterized by standard methods.

[0277] Peptides can be prepared in a high-throughput combinatorial fashion, for example, using a high-throughput multiple channel combinatorial synthesizer available from, for example, Advanced Chemtech or Symphony X. Peptide bonds may be replaced by, for example, retro-inverso bonds (C(O)-NH); reduced amide bonds (NH-CH2); thiomethylene bonds (S-CH2 or CH2-S); oxymethylene bonds (O-CH2 or CH2-O); ethylene bonds (CH2-CH2); thioamide bonds (C(S)-NH); trans-olefin bonds (CH=CH); fluoro-substituted trans-olefin bonds (CF=CH); ketomethylene bonds (C(O)-CHR) or CHR-C(O) (wherein R is H or CH3); and fluoroketomethylene bonds (C(O)-CFR or CFR-C(O) (wherein R is H or F or CH3)) in order to increase the physiological stability of the peptide.

[0278] The peptide can be further modified by acetylation, amidation, biotinylation, cinnamoylation, farnesylation, fluoresceinylation, formylation, myristoylation, palmitoylation, phosphorylation (Ser, Tyr or Thr), stearoylation, succinylation and sulfurylation. As described above, the peptide can be conjugated with, for example, polyethylene glycol (PEG); an alkyl group (e.g., a C1-C20 straight or branched chain alkyl group); a fatty acid radical; and combinations thereof. α,α-disubstituted unnatural amino acids containing olefin side chains of various lengths can be synthesized by known methods (Williams et al. J. Am. Chem. Soc., 113:9276, 1991; Schafmeister et al., J. Am. Chem Soc., 122:5891, 2000; and Bird et al., Methods Enzymol., 446:369, 2008; Bird et al, Current Protocols in Chemical Biology, 2011). i linked to i+7 and i+7 stitched linked to i+14 are used In some examples of the (stabilized 4-turn helix) peptides being made, one R-octenylalanine (e.g., (R)-α-(7’-octenyl)alanine), one bis-pentenylglycine (e.g., α,α-bis(4’-pentenyl)glycine), and one R-octenylalanine (e.g., (R)-α-(7’-octenyl)alanine) are used. In some examples of the (stabilized 4-turn helix) peptides where i is linked to i+7 and i+7 is linked to i+14 stitches, one S-octenylalanine (e.g., (S)-α-(7’-octenyl)alanine), one bis-pentenylglycine (e.g., α,α-bis(4’-pentenyl)glycine), and one R-octenylalanine (e.g., (R)-α-(7’-octenyl)alanine) are used. In some examples of the (stabilized 4-turn helix) peptides where i is linked to i+7 and i+7 is linked to i+14 stitches, one S-octenylalanine (e.g., (S)-α-(7’-octenyl)alanine), one bis-pentenylglycine (e.g., α,α-bis(4’-pentenyl)glycine), and one S-octenylalanine (e.g., (S)-α-(7’-octenyl)alanine) are used. In some examples of the (stabilized 4-turn helix) peptides where i is linked to i+7 and i+7 is linked to i+14 stitches, one R-pentenylalanine (e.g., (R)-α-(4’-pentenyl)alanine), one bis-octenylglycine (e.g., α,α-bis(7’-octenyl)glycine), and one S-pentenylalanine (e.g., (S)-α-(4’-pentenyl)alanine) are used. In some examples of the (stabilized 4-turn helix) peptides where i is linked to i+7 and i+7 is linked to i+14 stitches, one R-pentenylalanine (e.g., (R)-α-(4’-pentenyl)alanine), one bis-octenylglycine (e.g., α,α-bis(7’-octenyl)glycine), and one R-pentenylalanine (e.g., (R)-α-(4’-pentenyl)alanine) are used.In some examples of peptides that use i connected to i+7 and i+7 stitches connected to i+14 (4 turns of a stabilized helix), one S-pentenylalanine (e.g., (S)-α-(4’-pentenyl)alanine), one bis-octenylglycine (e.g., α,α-bis(7’-octenyl)glycine), and one R-pentenylalanine (e.g., (R)-α-(4’-pentenyl)alanine) are used. In some examples of peptides that use i connected to i+7 and i+7 stitches connected to i+14 (4 turns of a stabilized helix), one S-pentenylalanine (e.g., (S)-α-(4’-pentenyl)alanine), one bis-octenylglycine (e.g., α,α-bis(7’-octenyl)glycine), and one S-pentenylalanine (e.g., (S)-α-(4’-pentenyl)alanine) are used. R-octenylalanine is synthesized using the same route except that the starting chiral auxiliary gives the R-alkyl-stereoisomer. Also, 8-iodooctene is used instead of 5-iodopentene. The inhibitor is synthesized on a solid support using solid-phase peptide synthesis (SPPS) in MBHA resin (see, for example, WO2010 / 148335).

[0279] Fmoc-protected α-amino acids (other than olefinic amino acids N-Fmoc-α,α-bis(4'-pentenyl)glycine, (S)-N-Fmoc-α-(4'-pentenyl)alanine, (R)-N-Fmoc-α-(7'-octenyl)alanine, (R)-N-Fmoc-α-(7'-octenyl)alanine, and (R)-N-Fmoc-α-(4'-pentenyl)alanine), 2-(6-chloro-1-H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU), and Rink Amide MBHA are commercially available, for example, from Novabiochem (San Diego, CA). Dimethylformamide (DMF), N-methyl-2-pyrrolidinone (NMP), N,N-diisopropylethylamine (DIEA), trifluoroacetic acid (TFA), 1,2-dichloroethane (DCE), fluorescein isothiocyanate (FITC), and piperidine are commercially available, for example, from Sigma-Aldrich. Olefinic amino acid synthesis has been reported in the art (Williams et al., Org. Synth., 80:31, 2003).

[0280] Also, methods suitable for obtaining (e.g., synthesizing), stitching, and purifying the peptides disclosed herein are also known in the art (e.g., Bird et. al., Methods in Enzymol., 446:369-386 (2008); Bird et al, Current Protocols in Chemical Biology, 2011; Walensky et al., Science, 305:1466-1470 (2004); Schafmeister et al., J. Am. Chem. Soc., 122:5891-5892 (2000); U.S. Patent Application No. 12 / 525,123, filed Mar. 18, 2010; and U.S. Patent No. 7,723,468, issued May 25, 2010 (each of which is hereby incorporated by reference in its entirety)).

[0281] In some examples, the peptide is substantially free of or isolated from non-stapled peptide contaminants. Methods for purifying the peptide include, for example, the step of synthesizing the peptide on a solid support. After cyclization, the solid support may be isolated and suspended in a solution of a solvent such as DMSO, a DMSO / dichloromethane mixture, or a DMSO / NMP mixture. The DMSO / dichloromethane or DMSO / NMP mixture may contain about 30%, 40%, 50% or 60% DMSO. In a specific example, a 50% / 50% DMSO / NMP solution is used. The solution may be incubated for a period of 1, 6, 12 or 24 hours, after which the resin may be washed, for example, with dichloromethane or NMP. In one example, the resin is washed with NMP. Shaking and bubbling of an inert gas into the solution may be carried out.

[0282] The properties of the stitched or stapled peptides of the present disclosure can be assayed, for example, using the methods described below and in the examples.

Examples

[0283] The following examples are provided to more fully illustrate the claimed invention and should not be construed as limiting the scope of the invention. To the extent specific materials are recited, this is for illustration only and is not intended to limit the invention. One of ordinary skill in the art can develop equivalent means or reactants without exercising the capabilities of the present invention and without departing from the scope of the present invention. (Example 1) Preparation of the E2 hA staple peptide of S. pombe

[0284] In the ubiquitin system, the interaction between E1 and E2 buries 3000 Å of the protein surface area 2 and 1000 Å of that 2is buried at the interface between helix A of E2 (E2 hA) and the E1 ubiquitin-fold domain (E1 UFD). The Kd of the E1-E2 interaction is in the range from sub-nanomolar to single-digit nanomolar concentrations, and E2 hA accounts for at least 40% of the binding energy of that interaction. Furthermore, the interaction between E1 and E2 hA is important in the formation of the E1-E2 encounter complex. Thus, a stabilized alpha-helix peptide was designed and prepared that mimics E2 hA and can act as a competitive inhibitor of the E1-E2 interaction, preventing E1 from transferring to E2, and thus representing a novel mechanism for inhibiting E1 activity.

[0285] The crystal structure of the binding of Ubc15 to Ube1 of S. pombe (PDB ID: 5KNL) provided a structural basis for the design of UBA1 inhibitor peptides (Figure 1A). Structurally stabilized alpha-helix E2-related peptides were prepared by substituting unnatural amino acids with olefin side chains at the [i, i+4] or [i, i+7] positions of a peptide having a modified sequence of helix A (hA) of Ubc15 of S. pombe that includes the E7R amino acid substitution. The E7R amino acid substitution has been shown to result in a decrease of approximately 80-fold in K m (Lv et al., 2017, Molecular Cell, 65(4):699-714). Table 12 provides the sequences of the prepared peptides. Figures 1B and 1C illustrate the wheel diagrams of the peptide portion of SEQ ID NO: 40, which is expected to be an alpha helix, and show the [i, i+4] (Figure 1B) and [i, i+7] (Figure 1C) amino acid staple positions introduced into SEQ ID NO: 40 to generate the staple peptides of SEQ ID NOs: 41-54 (Table 12 below).

[0286] Table 12. E2 hA peptides. "B" is norleucine, "X1" is R-octenylalanine, and "X2" is S-pentenylalanine.

Table 12-1

[0287] The ability of E2 hA stapled peptides (SEQ ID NOs: 41-54) to inhibit E1-mediated ubiquitin transfer to E2 was evaluated. Human E1 activating enzyme UBA1 (10 nM), UbcH5b E2 enzyme (150 nM), ubiquitin (1 μM), and Mg-ATP (20 μM) were combined for 45 minutes at room temperature in a buffer containing 50 mM NaCl, 50 mM HEPES, 1 mM TCEP pH 7.5 in the presence of 100 μM E2hA non-stapled control peptide (SEQ ID NO: 40), a stapled peptide (any one of SEQ ID NOs: 41-54), or vehicle control (1% DMSO). Control reactions did not contain the E1 enzyme. The reaction was quenched by adding a loading dye containing SDS, and the samples were separated on a 4-12% Bis-Tris protein gel under non-reducing conditions. Proteins were visualized by silver staining. E1-mediated conjugation of ubiquitin to E2 was monitored by the conversion of free E2 (17 kDa) to the E2-ubiquitin conjugate (26 kDa).

[0288] Peptides containing staples at amino acid positions A2 and A9 (SEQ ID NO: 50) were the most excellent in this panel for inhibiting E1-mediated ubiquitin transfer to E2 (Figure 2). Peptides containing staples at amino acid positions A5 and A9 (SEQ ID NO: 44), A8 and A 12 (SEQ ID NO: 46), A9 and A 13 (SEQ ID NO: 47), A1 and A8 (SEQ ID NO: 49), A4 and A 11 (SEQ ID NO: 52), and A5 and A 12 (SEQ ID NO: 53) also inhibited the conversion of E2 to E2-ubiquitin by E1 (Figure 2).

[0289] This study revealed that not all staple positions introduced into the Ubc15 E2 hA peptide of S. pombe inhibit E1-mediated ubiquitin transfer to the E2. Without being bound by any particular theory, peptides containing staples at positions A1 and A5 (SEQ ID NO: 41), A2 and A6 (SEQ ID NO: 42), A4 and A8 (SEQ ID NO: 43), A6 and A 10 (SEQ ID NO: 45), A 10 and A 14 (SEQ ID NO: 48), A3 and A 10 (SEQ ID NO: 51), and A6 and A 13 (SEQ ID NO: 54) were hypothesized to be unable to inhibit E1-mediated ubiquitin transfer to the E2 because the introduction of their staples disrupted the E2 / E1 binding interface or blocked important residues of the E2 helix A peptide. (Example 2) Preparation of E2 hA staple peptides of H. sapiens

[0290] Based on the strong inhibition of E1-mediated ubiquitin transfer to the E2 by the S. pombe Ubc15 E2hA peptide containing staples at positions A2 and A9 (SEQ ID NO: 50) and the high homology between human E1 and S. pombe E1, E2 hA staple peptides were designed and generated based on the human E2 hA sequence. Table 2 above provides sequences for E2 hA encoded by 32 different human genes. Peptides containing the truncated forms of the E2 hA of UBE2D2, UBE2G2, or UBE2A with staples at positions A2 and A9 (peptides "SAH-UBE2G2-11", "SAH-UBED2-11", and "SAH-UBE2A-11", respectively; see Table 13; see also Figure 3 providing a diagram of the helix-wheel portion of these peptides) were generated.

[0291] Table 13. Stapled E2 hA peptide. “B” is norleucine, where “X1” is R-octenylalanine, and “X2” is S-pentenylalanine. [Table 13]

[0292] The ability of peptides to inhibit E1-mediated thioester transfer to E2 was evaluated. Human E1-activating enzyme UBA1 (10 nM), UbcH5b E2 enzyme (150 nM), ubiquitin (1 μM), and Mg-ATP (20 μM) were combined for 45 minutes at room temperature in a buffer containing 50 mM NaCl, 50 mM HEPES, 1 mM TCEP pH 7.5 in the presence of a range of concentrations of stapled peptide or vehicle control (1% DMSO). The reaction was quenched by adding a loading dye containing SDS, and the samples were separated on a 4–12% Bis-Tris protein gel under non-reducing conditions. Proteins were visualized by silver staining. E1-mediated conjugation of ubiquitin to E2 was monitored by conversion of free E2 (17 kDa) to the E2–ubiquitin conjugate (26 kDa). The percentage of inhibition was calculated from densitometry of the silver-stained images. Each of the stapled peptides SAH-Ubc15-11 (SEQ ID NO: 50), SAH-UBE2G2-11 (SEQ ID NO: 133), SAH-UBE2D2-11 (SEQ ID NO: 107), and SAH-UBE2A-11 (SEQ ID NO: 132) inhibited E1-mediated thioester transfer to E2 in a dose-dependent manner (Figure 4), and SAH-UBE2A-11 had the most potent activity. (Example 3) The E2 hA stapled peptide is a pan-active inhibitor of the E1-E2 interaction in the ubiquitin pathway

[0293] Considering the efficacy of SAH-UBE2A-11 (SEQ ID NO: 132), this peptide was selected for further evaluation. Humans encode at least 33 human E2 enzymes that are activated by the human E1 enzyme UBA1. To test the pan-inhibitory activity of SAH-UBE2A-11 (SEQ ID NO: 132), human E1 activating enzyme UBA1, SAH-UBE2A-11 or point mutant negative control SAH-UBE2A-11-R7E (BSTPX1RERLBRX2FKRLQ, where "X1" is S-pentenylalanine and "X2" is R-octenylalanine; SEQ ID NO: 852), and various different human E2 enzymes (UBE2A, UBE2B, UBE2C, UBE2D1, UBE2D2, UBE2D3, UBE2D4, UBE2E1, UBE2E3, UBE2G1, UBE2L3, UBE2J1, UBE2J2, UBE2R1, UBE2R2, UBE2S, UBE2T, UBE3Q1, UBE2Q1, and UBE2W2) were used to perform in vitro enzyme inhibition. SAH-UBE2A-11 inhibited E1-mediated thioester transfer to all human E2 enzymes tested, while the point mutant control SAH-UBE2A-11-R7E lost its inhibitory ability, highlighting the specificity of the action (Figure 5). These studies revealed that SAH-UBE2A-11 is pan-active in its ability to inhibit E1-mediated thioester transfer to E2: SAH-UBE2A-11 inhibits not only the transfer to the E2 from which it is derived (i.e., UBE2A), but also the transfer to a number of other human E2 enzymes. (Example 4) Identification of Key Binding Residues in SAH-UBE2A-11

[0294] Alanine scanning mutagenesis (Figure 6) and charge reversal (amino acid substitution from R to E) mutagenesis (Figure 7) were performed on the SAH-UBE2A-11 (SEQ ID NO: 132) peptide to identify the key amino acid residues contributing to the activity of the functional E2 hA stapled peptide (Figures 6 and 7). In In vitro enzyme inhibition assays revealed that amino acids R7, L9, B10, F13, and L16 (at positions A4, A6, A7, A 10 , A 13 ) are particularly important for the function of the peptide (Figures 6 and 7). (Example 5) Direct binding of SAH-UBE2A to UBA1

[0295] The direct binding of SAH-UBE2A to UBA1 was evaluated by streptavidin capture (Figure 8), fluorescence polarization assay (Figure 9), and hydrogen-deuterium exchange mass spectrometry (Figure 10). Streptavidin capture of biotinylated peptide mixed with recombinant protein, followed by washing, elution, and detection by protein gel electrophoresis demonstrated the direct binding of SAH-UBE2A to UBA1 (Figure 8). Fluorescence polarization assays performed both in the presence and absence of ubiquitin and ATP demonstrated that SAH-UBE2A binds to UBA1 independently of ubiquitin and ATP (Figure 9). Hydrogen-deuterium exchange mass spectrometry showed that incubation with recombinant UBA1 protected SAH-UBE2A from deuterium incorporation in a dose-dependent manner compared to incubation with peptide alone. In contrast, incubation of the peptide with recombinant E2 did not result in significant protection (Figure 10). (Example 6) SAH-UBE2A binds to native UBA1 and inhibits the ubiquitin cascade in cell lysates

[0296] The direct binding of SAH-UBE2A to native UBA1 was demonstrated by streptavidin capture of biotinylated peptides mixed with HeLa cell lysates, followed by washing, elution, protein gel electrophoresis, and Western blotting for UBA1 (Figure 11). Inhibition of the ubiquitin cascade in cancer cell lysates was demonstrated by ubiquitination assays. The cytoplasmic fraction of HeLa cells was incubated with SAH-UBE2A or vehicle control in the presence of excess ubiquitin and an ATP regeneration solution, and then subjected to non-reducing gel electrophoresis and Western blotting for ubiquitin. SAH-UBE2A inhibited the formation of polyubiquitin chains in a dose-dependent manner compared to the vehicle control (Figure 12). (Example 7) Generation of additional E2 hA staple peptides

[0297] Additional stapled E2 hA peptides were designed and generated. Table 14 provides the sequences of the additional generated E2 hA staple peptides, each of which inhibited E1-mediated thioester transfer to E2. In other words, each of the E2 hA staple peptides in Table 14 was functional. Table 15 provides the sequences of the additional generated E2 hA staple peptides, each of which was found to have a compromised ability to inhibit E1-mediated thioester transfer to E2. In other words, the E2 hA staple peptides in Table 15 exhibited significantly impaired function, highlighting the importance of design, synthesis, testing, and iteration to achieve optimally functional E2 hA staple peptides for preclinical and clinical development.

[0298] Table 14. Generated functional E2 hA staple peptides. "B" is norleucine, "X1" is R-octenylalanine, and "X2" is S-pentenylalanine.

Table 14

[0299] Table 15. Generated non-functional E2 hA staple peptides. "B" is norleucine, "X1" is R-octenylalanine, and "X2" is S-pentenylalanine. [Table 15] Methods used in Examples 1-7

[0300] Synthesis and purification of staple peptides: Amino acids were successively added to rink amide AM resin, and staple peptides were generated using solid-phase Fmoc chemistry on a Symphony X peptide synthesizer. Two S-pentenylalanines or one S-pentenylalanine and one R-octenylalanine non-natural amino acids replaced two native amino acids at the i, i+4 or i, i+7 positions, respectively. All hydrocarbon staples were formed by olefin metathesis using the Grubbs first-generation ruthenium catalyst, followed by deprotection of the peptide and cleavage from the resin. As shown, the peptide was derivatized at the N-terminus with acetyl and the C-terminus was amidated or derivatized with FITC. Peptides were purified by reverse-phase high-performance liquid chromatography and mass spectrometry (LC / MS) and quantified by amino acid analysis.

[0301] In vitro binding assay: To evaluate the binding and affinity of a ligand for an acceptor protein, for example, a fluorescence polarization assay (FPA) can be used. The FPA technique measures molecular orientation and motion using polarization and a fluorescent tracer. When excited by polarization, a fluorescent tracer (e.g., FITC) bound to a molecule with a high apparent molecular weight (e.g., a FITC-labeled peptide bound to a large protein) emits a higher level of polarized fluorescence due to their slower rotational rates compared to a fluorescent tracer bound to a small molecule (e.g., a FITC-labeled peptide free in solution). As shown, the FP assay was performed in binding buffer containing or not containing ubiquitin (10 μM) and ATP (20 μM).

[0302] Streptavidin-biotin pull-down assay: For the pull-down of recombinant proteins, 10 pmol of recombinant human UBA1 was incubated with 5 nmol of C-terminally biotinylated SAH-UBE2A or DMSO vehicle control in a total volume of 1 mL of binding buffer (50 mM NaCl, 20 mM HEPES, pH 7.4, 5 mM DTT) at 4 °C for 1.5 h. Then, 30 μL of pre-equilibrated streptavidin agarose beads was added and incubated at 4 °C for an additional 1.5 h. The beads were pelleted by centrifugation and washed three times with NP-40 buffer (20 mM HEPES, pH 7.4, 50 mM NaCl, 5 mM DTT, 0.5% [v / v] NP-40), and then the protein samples were eluted from the beads by heating at 70 °C for 10 min in 3× SDS loading buffer. The samples were subjected to electrophoresis and silver staining (Pierce 24612). For the pull-down from cell lysates, 0.75 mg of HeLa cell lysate was incubated with 5 nmol of C-terminally biotinylated SAH-UBE2A or DMSO vehicle control in a total volume of 1 mL of binding buffer (50 mM NaCl, 20 mM HEPES, pH 7.4, 5 mM DTT) containing a protease-phosphatase inhibitor cocktail at 4 °C for 2 h. Then, 30 μL of pre-equilibrated streptavidin agarose beads (ThermoFisher 20357) was added and incubated at 4 °C for an additional 1.5 h. The beads were pelleted by centrifugation and washed three times with NP-40 buffer (20 mM HEPES, pH 7.4, 50 mM NaCl, 5 mM DTT, 0.5% [v / v] NP-40), and then the protein samples were eluted from the beads by heating at 70 °C for 10 min in 3× SDS loading buffer. The proteins were separated by SDS-PAGE gel electrophoresis and detected by Western blot against UBE1 (Abcam ab34711).

[0303] In vitro enzyme inhibition assay: Recombinant human E1 activating enzyme UBA1 (10 nM), E2 enzyme (150 nM), ubiquitin (1 μM), and Mg-ATP (20 μM) are combined in a buffer containing 50 mM NaCl and 50 mM HEPES pH 7.5 for 45 minutes at room temperature in the presence of a peptide or vehicle control (1% DMSO). The control reaction does not contain the E1 enzyme. The reaction is quenched by the addition of a loading dye containing SDS, and the samples are separated on a 4 - 12% Bis-Tris protein gel under non-reducing conditions. Proteins are visualized by silver staining. The conjugation of ubiquitin to E2 by E1 is monitored by the conversion of free E2 (17 kDa) to the E2-ubiquitin conjugate (approx. 26 kDa). The percent inhibition is calculated from densitometry of the silver-stained images. Error bars represent ±1 SEM calculated from three independent experiments.

[0304] HXMS analysis: Hydrogen-deuterium exchange mass spectrometry (HXMS) experiments were performed as described (Barclay, L. A. et al. Inhibition of Pro-apoptotic BAX by a noncanonical interaction mechanism. Molecular Cell 57, 873-886, doi: 10.1016 / j.molcel.2015.01.014 (2015)). SAH-UBE2A was used in 50 mM NaCl, 5m In a buffer containing MgCl2 of M, 500 nM of ubiquitin, 20 mM of ATP, 5 mM of DTT, 20 mM of HEPES, pH 7.4, incubated alone or with recombinant human UBA1, UBE2D2, or vehicle control at the indicated molar ratios for 10 minutes on ice. Deuterium labeling was initiated by an 18-fold dilution into D2O buffer (20 mM of HEPES, 50 mM of NaCl, pD 8). After 10 seconds of labeling, an equal volume of quenching buffer (0.8 M of guanidinium chloride, 0.8% [v / v] formic acid) was added to quench the labeling reaction. The sample was then injected, the peptide was captured intact, desalted on a VanGuard Pre-Column trap (2.1 mm × 5 mm, ACQUITY UPLC BEH C18, 1.7 μm) for 3 minutes, eluted from the trap using a 5% - 35% acetonitrile gradient over 6 minutes at a flow rate of 65 μL / min, and then separated using an ACQUITY UPLC HSS T3, 1.8 μm, 1.0 mm × 50 mm column on a Waters nanoACQUITY LC. A Waters Synapt G2Si mass spectrometer was operated in ion mobility mode and the data were analyzed. All mass spectra were processed using DynamX 3.0 (Waters Corporation). Deuterium levels were not corrected for back-exchange and were thus reported as relative values. All described changes were consistent in at least n = 2 biological replicates.

[0305] Cell Lysate Enzyme Inhibition Assay: The cell lysate assay was performed in assay buffer (pH 7.4) containing 1×ATP regeneration solution, 20 mM HEPES, 50 mM NaCl, 5 mM MgCl2, 2 mM DTT, 100 nM ubiquitin aldehyde, and a protease - phosphatase inhibitor cocktail, using the HeLa S100 lysate fraction at a final concentration of 1 mg / mL and the indicated concentrations of peptide or DMSO vehicle control. The reaction was initiated by adding ubiquitin to a final concentration of 100 μM and allowed to proceed for 30 minutes at room temperature, after which it was quenched with non - reducing LDS loading buffer. Proteins were separated by SDS - PAGE gel electrophoresis and detected by Western blot against polyubiquitin (Cell Signaling Technology CST3936). (Example 8) Modification of the SAH - E2 Peptide for Incorporation of an Electrophilic Warhead

[0306] To generate variants of additional E2 hA stapled peptides with the potential to form a covalent bond with Cys1039 of UBA1, several variants were generated in which position A8 or A 11 was replaced with a "warhead". The variants were based on the E2 hA sequences of Ubc15 (SEQ ID NO: 2), UBE2D2 (SEQ ID NO: 10), and UBE2A (SEQ ID NO: 4). Tables 16 and 17 provide the sequences of the warhead variants of the various E2 hA stapled peptides generated. The position of the warhead was determined based on the docking of the peptide to the crystal structure of human E1 (PDF 6dc6). Positions on or adjacent to the E1 - binding face of the peptide and in the vicinity of Cys1039 of UBA1 (SEQ ID NO: 845) were selected for replacement by the warhead. The alpha - carbon of amino acid consensus position A7 is estimated to be 5.4 angstroms from the sulfur of Cys1039 of UBA1 (SEQ ID NO: 845). The alpha - carbon of amino acid consensus position A8 is estimated to be 6.6 angstroms from the sulfur of Cys1039 of UBA1 (SEQ ID NO: 845). Amino acid consensus position A 11The alpha carbon is estimated to be 4.8 angstroms from the sulfur of Cys1039 of UBA1 (SEQ ID NO: 845).

[0307] Table 16. Staple peptides having the warheads of the generated E2 hA. "J" is the version with acrylamide of diaminobutanoic acid ("Dab"), "B" is norleucine, "X1" is R-octenylalanine, and "X2" is S-pentenylalanine [Table 16]

[0308] Table 17. Staple peptides having the warheads of the generated E2 hA. "J" is the version with acrylamide of diaminobutanoic acid ("Dab"), "B" is norleucine, "X1" is R-octenylalanine, and "X2" is S-pentenylalanine. [Table 17]

[0309] The E2 hA staple peptides having the warheads of SEQ ID NOs: 727, 841, 842, and 844 inhibited E1-mediated th...

Claims

【Claim 1】 The invention described in the specification.

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