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:
 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.   
     
     
         2 . The method of  claim 1 , wherein the polypeptide comprises an alpha helix and the alpha helix mediates the interaction with the protein. 
     
     
         3 . The method of  claim 1  or  2 , wherein the polypeptide comprises at least two non-natural amino acids with olefinic side chains. 
     
     
         4 . The method of any one of  claims 1  to  3 , wherein the polypeptide comprises a stabilized alpha helix. 
     
     
         5 . The method of any one of  claims 1  to  4 , wherein the sulfhydryl group of the cysteine residue in the protein is at a distance between 1 A 0  and 10 A 0  from the alpha carbon of the amino acid in the polypeptide. 
     
     
         6 . The method of any one of  claims 1  to  4 , wherein the sulfhydryl group of the cysteine residue in the protein is at a distance between 4 A 0  and 9 A 0  from the alpha carbon of the amino acid in the polypeptide. 
     
     
         7 . The method of any one of  claims 1  to  4 , wherein the sulfhydryl group of the cysteine residue in the protein is at a distance between 4 A 0  and 6 A 0  from the alpha carbon of the amino acid in the polypeptide. 
     
     
         8 . The method of any one of  claims 1  to  4 , wherein the sulfhydryl group of the cysteine residue in the protein is at a distance between 5 A 0  and 8 A 0  from the alpha carbon of the amino acid in the polypeptide. 
     
     
         9 . The method of any one of  claims 1  to  4 , wherein the sulfhydryl group of the cysteine residue in the protein is at a distance between 1 A 0  and 6 A 0  from the alpha carbon of the amino acid in the polypeptide. 
     
     
         10 . The method of any one of  claims 1  to  9 , further comprising substituting the amino acid in the polypeptide with a non-natural amino acid, or other chemical moiety linked to the stapled peptide, comprising an electrophilic group thereby creating a modified polypeptide that can covalently bind to the protein. 
     
     
         11 . The method of any one of  claims 1  to  9 , further comprising appending an electrophilic chemical moiety to the position in the polypeptide. 
     
     
         12 . The method of any one of  claim 11 , wherein the position in the polypeptide is the N-terminus or the C-terminus of the polypeptide. 
     
     
         13 . The method of  claim 11  or  12 , wherein the electrophilic moiety comprises an electrophilic acrylamide or an electrophilic substituted acrylamide. 
     
     
         14 . A method for identifying an amino acid or a position in a polypeptide for modification to facilitate covalent binding to a protein that the polypeptide binds to, the method comprising:
 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.   
     
     
         15 . The method of  claim 14 , wherein the polypeptide comprises an alpha helix and the alpha helix mediates the interaction with the interacting protein. 
     
     
         16 . The method of  claim 14  or  15 , wherein the polypeptide comprises at least two non-natural amino acids with olefinic side chains. 
     
     
         17 . The method of any one of  claims 14  to  16 , wherein the polypeptide comprises a stabilized alpha helix. 
     
     
         18 . The method of any one of  claims 14  to  17 , wherein the sulfhydryl group of the cysteine residue in the interacting protein is between 1 A 0  and 10 A 0  from the alpha carbon of an amino acid in the polypeptide. 
     
     
         19 . The method of any one of  claims 14  to  17 , wherein the sulfhydryl group of the cysteine residue in the interacting protein is between 4 A 0  and 9 A 0  from the alpha carbon of an amino acid in the polypeptide. 
     
     
         20 . The method of any one of  claims 14  to  17 , wherein the sulfhydryl group of the cysteine residue in the interacting protein is between 4 A 0  and 6 A 0  from the alpha carbon of an amino acid in the polypeptide. 
     
     
         21 . The method of any one of  claims 14  to  17 , wherein the sulfhydryl group of the cysteine residue in the interacting protein is between 5 A 0  and 8 A 0  from the alpha carbon of an amino acid in the polypeptide. 
     
     
         22 . The method of any one of  claims 14  to  17 , wherein the sulfhydryl to group of the cysteine residue in the interacting protein is between 1 A 0  and 6 A 0  from the alpha carbon of an amino acid in the polypeptide. 
     
     
         23 . The method of any one of  claims 14  to  22 , further comprising substituting the amino acid in the polypeptide with a non-natural amino acid, or other chemical moiety linked to the stapled peptide, comprising an electrophilic group thereby creating a modified polypeptide that can covalently bind to the protein. 
     
     
         24 . The method of any one of  claims 14  to  22 , further comprising appending an electrophilic chemical moiety to the position in the polypeptide. 
     
     
         25 . The method of any one of  claim 24 , wherein the position in the polypeptide is the N-terminus or the C-terminus of the polypeptide. 
     
     
         26 . The method of  claim 24  or  25 , wherein the electrophilic moiety comprises an electrophilic acrylamide or an electrophilic substituted acrylamide. 
     
     
         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 . The method of  claim 27 , wherein the polypeptide comprises an alpha helix and the alpha helix mediates the interaction with the target protein. 
     
     
         29 . The method of  claim 27  or  28 , wherein the polypeptide comprises at least two non-natural amino acids with olefinic side chains. 
     
     
         30 . The method of any one of  claims 27  to  29 , wherein the polypeptide comprises a stabilized alpha helix. 
     
     
         31 . The method of any one of  claims 27  to  30 , wherein the sulfhydryl group of the cysteine residue in the target protein is between 1 A 0  and 10 A 0  from the alpha carbon of the amino acid in the polypeptide. 
     
     
         32 . The method of any one of  claims 27  to  30 , wherein the sulfhydryl group of the cysteine residue in the target protein is between 1 A 0  and 6 A 0  from the alpha carbon of the amino acid in the polypeptide. 
     
     
         33 . The method of any one of  claims 27  to  30 , wherein the sulfhydryl group of the cysteine residue in the target protein is between 4 A 0  and 9 A 0  from the alpha carbon of the amino acid in the polypeptide. 
     
     
         34 . The method of any one of  claims 27  to  30 , wherein the sulfhydryl group of the cysteine residue in the target protein is between 4 A 0  and 6 A 0  from the alpha carbon of the amino acid in the polypeptide. 
     
     
         35 . The method of any one of  claims 27  to  30 , wherein the sulfhydryl group of the cysteine residue in the target protein is between 5 A 0  and 8 A 0  from the alpha carbon of the amino acid in the polypeptide. 
     
     
         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 . The method of  claim 36 , wherein the modified polypeptide comprises an alpha helix and the alpha helix mediates the interaction with the target protein. 
     
     
         38 . The method of  claim 36  or  37 , wherein the modified polypeptide comprises at least two non-natural amino acids with olefinic side chains. 
     
     
         39 . The method of any one of  claims 36  to  38 , wherein the modified polypeptide comprises a stabilized alpha helix. 
     
     
         40 . The method of any one of  claims 36  to  39 , wherein the sulfhydryl group of the cysteine residue in the target protein is between 1 A 0  and 10 A 0  from the alpha carbon of the amino acid in the polypeptide. 
     
     
         41 . The method of any one of  claims 36  to  39 , wherein the sulfhydryl to group of the cysteine residue in the target protein is between 1 A 0  and 6 A 0  from the alpha carbon of the amino acid in the polypeptide. 
     
     
         42 . The method of any one of  claims 36  to  39 , wherein the sulfhydryl group of the cysteine residue in the target protein is between 4 A 0  and 9 A 0  from the alpha carbon of the amino acid in the polypeptide. 
     
     
         43 . The method of any one of  claims 36  to  39 , wherein the sulfhydryl group of the cysteine residue in the target protein is between 4 A 0  and 6 A 0  from the alpha carbon of the amino acid in the polypeptide. 
     
     
         44 . The method of any one of  claims 36  to  39 , wherein the sulfhydryl group of the cysteine residue in the target protein is between 5 A 0  and 8 A 0  from the alpha carbon of the amino acid in the polypeptide. 
     
     
         45 . The method of any one of  claims 1  to  44 , wherein the polypeptide or the modified polypeptide is at least 5 amino acids in length but less than 100, less than 75, less than 60, less than 50, less than 40, less than 30, less than 29, less than 28, less than 27, less than 26, less than 25, less than 24, less than 23, less than 22, less than 21, less than 20, less than 19, or less than 18 amino acids in length. 
     
     
         46 . The method of any one of  claims 1  to  44 , wherein the polypeptide or the modified polypeptide is 5 to 40 amino acids in length. 
     
     
         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 . The polypeptide of  claim 47 , wherein the polypeptide is 5 to 40 amino acids in length. 
     
     
         49 . The polypeptide of  claim 47 , wherein the polypeptide is 5 to 30 amino acids in length. 
     
     
         50 . The polypeptide of  claim 47 , wherein the polypeptide is 8 to 30 amino acids in length. 
     
     
         51 . The polypeptide of any one of  claims 47  to  50 , wherein the polypeptide comprises a stabilized alpha helix. 
     
     
         52 . The polypeptide of any one of  claims 47  to  51 , wherein the non-natural amino acid comprising an electrophilic group is 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-acryloylpyyrolidine-2-carboxamide; (R)-1-acryloylpyrrolidine-2-carboxamide; (E)-4-(dimethylamino)but-2-enamide; and acrylamide. 
     
     
         53 . The polypeptide of any one of  claims 47  to  52 , wherein the at least two non-natural amino acids with olefinic side chains are both S-pentenyl alanine. 
     
     
         54 . The polypeptide of any one of  claims 47  to  52 , wherein the at least two non-natural amino acids with olefinic side chains are different non-natural amino acids. 
     
     
         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 . The stabilized polypeptide of  claim 55 , wherein the at least two non-natural amino acids with olefinic side chains are both S-pentenyl alanine. 
     
     
         57 . The stabilized polypeptide of  claim 55 , wherein the at least two non-natural amino acids with olefinic side chains are different non-natural amino acids. 
     
     
         58 . The stabilized polypeptide of  claim 55 , wherein the polypeptide is 5 to 30 amino acids in length. 
     
     
         59 . The stabilized polypeptide of  claim 55 , wherein the polypeptide is 8 to 30 amino acids in length. 
     
     
         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 variant of 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. 
       
     
     
         62 . The peptide variant of  claim 61 , wherein the electrophilic group has the structure: 
       
         
           
           
               
               
           
         
         wherein, n=1 to 9, R 1 =amine, ether, thioether, amide, or carbon, R 2  is a nitrogen-containing heterocycle (e.g., pyrrolidine, piperidine, or piperizine), substituted aniline, or amine and R 3 =acryloyl, beta-methyl acryloyl, beta-alkyl acryloyl, alpha-cyano acryloyl, vinylsulfonyl, alpha-fluoro acetyl, alpha-chloro acetyl, alpha-bromo acetyl or alpha-iodo acetyl linked to the N or the nitrogen-containing heterocycle, substituted aniline or amine. 
       
     
     
         63 . The peptide variant of  claim 61 , wherein one Xaa is replace by: a disubstituted aniline (aminobenzene), 3S-1-pyrrolidine-3-carboxylic acid; D-homoproline; L-homoproline; isonipecotic acid; D-nipecotic acid; L-nipecotic acid; D-proline; L-proline; trans-4-dimethylaminocrotonic acid; and acrylic acid or the peptide variant has an amino terminal group selected from: a substituted aniline (aminobenzene), 3S-1-pyrrolidine-3-carboxylic acid; D-homoproline; L-homoproline; isonipecotic acid; D-nipecotic acid; L-nipecotic acid; and D-proline; L-proline; 
     
     
         64 . The peptide variant of  claim 61 , wherein one Xaa is replaced by: lysine, ornithine, diaminobutanoic acid, and diaminopropionic acid. 
     
     
         65 . The peptide variant of  claim 61 , wherein R 3  is a C8 alkylene and x is 3. 
     
     
         66 . An internally cross-linked peptide comprising 15 contiguous amino acids
 wherein:   a) the side chains of at least one pair (e.g., one or two pairs) of amino acids separated by 2, 3 or 6 amino acids are replaced by the linking group, R3, which connects the alpha carbons of the pair of amino acids wherein:   each R3 is alkylene, alkenylene or alkynylene (e.g., a C6, C7, C8 or C11 alkenylene)   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 C1 to C10 alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl; and   b) the peptide includes at least one natural or non-natural amino acid having a side replaced by an electrophilic group.   
     
     
         67 . The internally cross-linked peptide of  claim 66 , wherein the electrophilic group has the structure: 
       
         
           
           
               
               
           
         
         wherein, n=1 to 9, R 1 =amine, ether, thioether, amide, or carbon, R 2  is a nitrogen-containing heterocycle (e.g., pyrrolidine, piperidine, or piperizine), substituted aniline, or amine and R 3 =acryloyl, beta-methyl acryloyl, beta-alkyl acryloyl, alpha-cyano acryloyl, vinylsulfonyl, alpha-fluoro acetyl, alpha-chloro acetyl, alpha-bromo acetyl or alpha-iodo acetyl linked to the N or the nitrogen-containing heterocycle, substituted aniline or amine. 
       
     
     
         68 . The internally cross-linked peptide of  claim 66 , wherein the amino acid having a side chain substituted by an electrophilic group is selected from the group consisting of: a disubstituted aniline (aminobenzene), 3S-1-pyrrolidine-3-carboxylic acid; D-homoproline; L-homoproline; isonipecotic acid; D-nipecotic acid; L-nipecotic acid; D-proline; L-proline; trans-4-dimethylaminocrotonic acid; and acrylic acid or the peptide variant has an amino terminal group selected from: a disubstituted aniline (aminobenzene), 3S-1-pyrrolidine-3-carboxylic acid; D-homoproline; L-homoproline; isonipecotic acid; D-nipecotic acid; L-nipecotic acid; and D-proline; L-proline; lysine, ornithine, diaminobutanoic acid, and diaminopropionic acid.