Stabilized peptides for covalent binding to target protein

Abstract

Provided herein is a platform technology for designing stabilized peptides that covalently bind their target protein and thereby inhibit the activity of the target protein. Also provided are exemplary stabilized peptides that can be used for covalent modification of their target proteins.

1 . A method for identifying an amino acid or a position for modification in a polypeptide to facilitate covalent binding to a protein that the polypeptide binds to, the method comprising:
 (a) providing a protein comprising a cysteine residue, wherein the cysteine residue is in a region of the protein that binds a polypeptide;   providing the polypeptide that binds the target protein;   determining that the sulfhydryl group of the cysteine residue in the target protein is at a distance between 0.1 A 0  and 15 A 0  from the alpha carbon of an amino acid in the polypeptide, wherein the alpha carbon is on the first, the last or any internal amino acid of the polypeptide; and   identifying the amino acid as the amino acid to substitute with a non-natural amino acid or other chemical moiety linked to the stapled peptide comprising an electrophilic group; or   identifying the position of the amino acid in the polypeptide as the position to append an electrophilic moiety,   thereby facilitating covalent binding to the protein; or   (b) identifying an amino acid or a position in the polypeptide as the amino acid to modify by substitution with a non-natural amino acid or other chemical moiety linked to the peptide comprising an electrophilic group, or identifying the position of the amino acid in the polypeptide as the position to append an electrophilic moiety in order to facilitate covalent binding to an interacting protein, the alpha carbon of the amino acid having been previously determined to be between 0.1 A 0  and 15 A 0  from the sulfhydryl group of a cysteine residue in the interacting protein, wherein the cysteine residue is in a region of the interacting protein that binds the polypeptide, and wherein the alpha carbon of the amino acid is on the first, the last or any internal amino acid of the polypeptide.   
     
     
         2 .- 26 . (canceled) 
     
     
         27 . A method for covalently modifying a target protein with a stabilized polypeptide, the method comprising:
 providing a target protein comprising a cysteine residue, wherein the cysteine residue is in a region of the target protein that binds a polypeptide;   providing the polypeptide that binds the target protein;   determining that the sulfhydryl group of the cysteine residue in the target protein is between 0.1 A 0  and 15 A 0  from the alpha carbon of an amino acid in the polypeptide, wherein the alpha carbon is on the first, last, or any internal amino acid of the polypeptide;   substituting the amino acid in the polypeptide with a non-natural amino acid, or other chemical moiety linked to the polypeptide, comprising an electrophilic group thereby creating a modified polypeptide, or appending an electrophilic chemical moiety to the position of the amino acid in the polypeptide thereby creating a modified polypeptide; and   contacting the modified polypeptide with the target protein, thereby covalently modifying the target protein.   
     
     
         28 .- 35 . (canceled) 
     
     
         36 . A method comprising:
 contacting a target protein comprising a cysteine residue with: a modified polypeptide comprising a non-natural amino acid, or other chemical moiety linked to the polypeptide, comprising an electrophilic group; or a modified polypeptide comprising an electrophilic group appended to a position of the polypeptide, under conditions that allow the electrophilic group to react with the cysteine, thereby covalently modifying the target protein, wherein the cysteine residue is in a region of the interacting protein that binds the polypeptide, and   wherein the modified polypeptide comprising the non-natural amino acid, or other chemical moiety linked to the polypeptide, comprising an electrophilic group was modified by substitution of a polypeptide comprising an amino acid that was previously identified as having an alpha carbon between 0.1 A 0  and 12 A 0  from a nucleophilic sulfhydryl group of a cysteine residue in the target protein; or   wherein the modified polypeptide comprising an electrophilic group appended to a position of the polypeptide was modified by appending the electrophilic group to a position of the polypeptide that was previously identified as being between 0.1 A 0  and 12 A 0  from a nucleophilic sulfhydryl group of a cysteine residue in the target protein.   
     
     
         37 .- 46 . (canceled) 
     
     
         47 . A polypeptide comprising an amino acid sequence that is at least 5 amino acids in length but less than 40 amino acids in length that can covalently modify a protein the polypeptide it interacts with, wherein the protein the polypeptide interacts with comprises a cysteine in a region that interacts with the polypeptide, and wherein the polypeptide comprises an alpha helix that comprises an interacting region with the protein, and the alpha helix of the polypeptide comprises at least two non-natural amino acids with olefinic side chains and a non-natural amino acid, or other chemical moiety linked to the peptide, comprising an electrophilic group, and wherein the non-natural amino acid, or other chemical moiety linked to the peptide, comprising the electrophilic group can covalently bond with the cysteine in the protein. 
     
     
         48 .- 54 . (canceled) 
     
     
         55 . A stabilized alpha-helical polypeptide of 5 to 40 amino acids in length that specifically binds a target protein, the stabilized polypeptide comprising at least two non-natural amino acids with olefinic side chains, wherein the stabilized polypeptide's backbone is linked to an electrophilic acrylamide or substituted acrylamide, and wherein the linker is a nitrogen containing heterocycle, nitrogen containing heterocyclic amino acid, or amino-functionalized benzene ring, carbocycle, polycycle or heterocycle. 
     
     
         56 .- 59 . (canceled) 
     
     
         60 . An electrophilic acrylamide or substituted acrylamide linked to a stabilized helical polypeptide backbone via a linker, wherein the linker is selected from the group consisting of a nitrogen containing heterocycle, a nitrogen containing heterocyclic amino acid, an amino-functionalized benzene ring, an amino-functionalized carbocycle, an amino-functionalized polycycle, and an amino-functionalized heterocycle. 
     
     
         61 . An internally cross-linked peptide, wherein:
 (a) the internally cross-linked peptide comprises Formula I or a pharmaceutically acceptable salt thereof,   
       
         
           
           
               
               
           
         
         wherein; 
         each Xaa is independently a natural amino acid or a non-natural amino acid; 
         R 1  and R 2  are independently H or a C 1  to C 10  alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl; 
         R 3  is alkylene, alkenylene or alkynylene (e.g., a C 6 , C 7 , or C 11  alkenylene) substituted with 1-6 R 4 ; 
         x is 2, 3 or 6; 
         w and y are independently an integer from 5-20; 
         provided that: when x is 2, R 3  is C 4-13  alkylene, alkenylene or alkynylene; when x is 3, R 3  is C 4-13  alkylene, alkenylene or alkynylene; and when x is 6, R 3  is C 4-13  alkylene, alkenylene or alkynylene and provided that the sum of x, w and y is at least 12; 
         wherein the side chain of at least one Xaa replaced by an electrophilic group; or 
         (b) the internally cross-linked peptide comprises 15 amino acids, 
         wherein: 
         i) the side chains of at least one pair of amino acids separated by 2, 3 or 6 amino acids are replaced by the linking group, R 3 , which connects the alpha carbons of the pair of amino acids wherein: 
         each R3 is alkylene, alkenylene or alkynylene; 
         and the H of the alpha carbon of each pair of amino acids having their side chains replaced by linking group R3 is optionally, independently replaced by a C 1  to C 10  alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl; and 
         ii) the internally cross-linked peptide includes at least one natural or non-natural amino acid having a side replaced by an electrophilic group. 
       
     
     
         62 .- 68 . (canceled) 
     
     
         69 . A stabilized peptide comprising:
 (a) the amino acid sequence set forth in any one of SEQ ID NOs: 13-21, except for zero to three amino acid substitutions relative to the amino acid sequence set forth in any one of SEQ ID NOs: 13-21, wherein the stabilized peptide binds to BAX;   (b) the amino acid sequence set forth in any one of SEQ ID NOs: 24-33, except for zero to three amino acid substitutions relative to the amino acid sequence set forth in any one of SEQ ID NOs: 24-33, wherein the stabilized peptide binds to mediator of RNA polymerase II transcription subunit 22;   (c) the amino acid sequence set forth in any one of SEQ ID NOs: 35-45, except for zero to three amino acid substitutions relative to the amino acid sequence set forth in any one of SEQ ID NOs: 35-45, wherein the stabilized peptide binds to FADD;   (d) the amino acid sequence set forth in any one of SEQ ID NOs: 47-49, except for zero to three amino acid substitutions relative to the amino acid sequence set forth in any one of SEQ ID NOs: 47-49, wherein the stabilized peptide binds to myosin-X;   (e) the amino acid sequence set forth in any one of SEQ ID NOs: 51 and 53-56, except for zero to three amino acid substitutions relative to the amino acid sequence set forth in any one of SEQ ID NOs: 51 and 53-56, wherein the stabilized peptide binds to PDCD4;   (f) the amino acid sequence set forth in SEQ ID NO: 59 or 60, except for zero to three amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 59 or 60, wherein the stabilized peptide binds to DAXX;   (g) the amino acid sequence set forth in any one of SEQ ID NOs: 64-67, except for zero to three amino acid substitutions relative to the amino acid sequence set forth in any one of SEQ ID NOs: 64-67, wherein the stabilized peptide binds to p300;   (h) the amino acid sequence set forth in any one of SEQ ID NOs: 70-75, except for zero to three amino acid substitutions relative to the amino acid sequence set forth in any one of SEQ ID NOs: 70-75, wherein the stabilized peptide binds to tryptophanyl-tRNA synthetase; or   (i) the amino acid sequence set forth in any one of SEQ ID NOs: 76-96 and 98-116, except for zero to three amino acid substitutions relative to the amino acid sequence set forth in any one of SEQ ID NOs: 76-96 and 98-116, wherein the stabilized peptide binds to RAS; and   wherein the non-natural electrophile containing amino acid in the amino acid sequence set forth in any one of SEQ ID NOs: 13-21, 24-33, 35-45, 47-49, 51, 53-56, 59, 60, 64-67, 70-75, 76-96, and 98-116 is present.   
     
     
         70 . The stapled peptide of  claim 69 , wherein the amino acid substitutions are conservative amino acid substitutions. 
     
     
         71 . The stapled peptide of  claim 69 , wherein the stapled peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 13-21, 24-33, 35-45, 47-49, 51, 53-56, 59, 60, 64-67, 70-75, 76-96, and 98-116. 
     
     
         72 . The stapled peptide of  claim 69 , wherein the non-natural electrophile containing amino acid is selected from the group consisting of (S)-1-acryloylpyrrolidine-3-carboxamide; 1-acryloylpiperidine-4-carboxamide, (R)-1 acryloylpiperidine-3-carboxamide; (S)-1-acryloylpiperidine-3-carboxamide; (S)-1-acryloylpyrrolidine-2-carboxamide; (R)-1-acryloylpyrrolidine-2-carboxamide; (E)-4-(dimethylamino)but-2-enamide; and acrylamide. 
     
     
         73 . The peptide of  claim 69 , which is no more than 30 amino acids in length. 
     
     
         74 . A peptide comprising:
 (a) the amino acid sequence set forth in any one of SEQ ID NOs: 10-12, except for zero to three amino acid substitutions relative to the amino acid sequence set forth in any one of SEQ ID NOs: 10-12, wherein the peptide binds to BAX;   (b) the amino acid sequence set forth in SEQ ID NO: 23, except for zero to three amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 23, wherein the peptide binds to eiF4E;   (c) the amino acid sequence set forth in SEQ ID NO: 52, except for zero to three amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 52, wherein the peptide binds to PDCD4;   (d) the amino acid sequence set forth in SEQ ID NO: 58 or 61, except for zero to three amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 58 or 61, wherein the peptide binds to DAXX;   (e) the amino acid sequence set forth in SEQ ID NO: 63, except for zero to three amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 63, wherein the peptide binds to p300;   (f) the amino acid sequence set forth in SEQ ID NO: 69, except for zero to three amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 69, wherein the peptide binds to tryptophanyl-tRNA synthetase; or   (g) the amino acid sequence set forth in SEQ ID NO: 97 or 117, except for zero to three amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO: 97 or 117, wherein the peptide binds to RAS; and   wherein the non-natural electrophile containing amino acid in the amino acid sequence set forth in any one of SEQ ID NOs: 10-12, 23, 52, 58, 61, 63, 69, 97, and 117 is present.   
     
     
         75 . The stapled peptide of  claim 74 , wherein the amino acid substitutions are conservative amino acid substitutions. 
     
     
         76 . The stapled peptide of  claim 74 , wherein the stapled peptide comprises the amino acid sequence set forth in any one of SEQ ID NOs: 10-12, 23, 52, 58, 61, 63, 69, 97, and 117. 
     
     
         77 . The stapled peptide of  claim 74 , wherein the non-natural electrophile containing amino acid is selected from the group consisting of (S)-1-acryloylpyrrolidine-3-carboxamide; 1-acryloylpiperidine-4-carboxamide, (R)-1 acryloylpiperidine-3-carboxamide; (S)-1-acryloylpiperidine-3-carboxamide; (S)-1-acryloylpyrrolidine-2-carboxamide; (R)-1-acryloylpyrrolidine-2-carboxamide; (E)-4-(dimethylamino)but-2-enamide; and acrylamide. 
     
     
         78 . The peptide of  claim 74 , which is no more than 30 amino acids in length. 
     
     
         79 . A pharmaceutical composition comprising the stabilized peptide of  claim 69 . 
     
     
         80 . A pharmaceutical composition comprising the peptide of  claim 74 . 
     
     
         81 . A method of treating a disease in a subject in need thereof, the method comprising administering to the subject the polypeptide of  claim 47 . 
     
     
         82 . A method of treating a disease in a subject in need thereof, the method comprising administering to the subject the stabilized alpha-helical polypeptide of  claim 55 . 
     
     
         83 . A method of treating a disease in a subject in need thereof, the method comprising administering to the subject the internally cross-linked peptide of  claim 61 . 
     
     
         84 . A method of treating a disease in a subject in need thereof, the method comprising administering to the subject the stabilized peptide of  claim 69 , wherein the disease is a caner, autoimmunity, or an infection. 
     
     
         85 . A method of treating a disease in a subject in need thereof, the method comprising administering to the subject the peptide of  claim 74 , wherein the disease is a caner, autoimmunity, or an infection.