Ligation of stapled polypeptides

Abstract

The present invention provides technology for making large (e.g., greater than 50 amino acids), semi-synthetic, stapled or stitched proteins. The method essentially involves ligating a synthetically produced stapled or stitched peptide to a larger protein. Modified version of IL-13 and MYC are provided as illustrative examples.

1 . A method of preparing a stitched or stapled protein, the method comprising steps of:
 providing a stitched or stapled peptide;   providing a second protein or peptide to which the stitched or stapled peptide is to be ligated; and   ligating the stitched or stapled peptide to the second protein or peptide.   
     
     
         2 . The method of  claim 1 , wherein the stitched or stapled peptide comprises an alpha-helix. 
     
     
         3 . The method of  claim 1 , wherein the stitched or stapled peptide is less than 30 amino acids in length. 
     
     
         4 . (canceled) 
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 1 , wherein the stapled peptide is of formula: 
       
         
           
           
               
               
           
         
       
       wherein
 each instance of L 1  and L 2  is, independently, a bond, cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkenylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkynylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkenylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkynylene; substituted or unsubstituted arylene; substituted or unsubstituted heteroarylene; or substituted or unsubstituted acylene; 
 each instance of R a  is, independently, hydrogen; cyclic or acyclic, branched or unbranched, substituted or unsubstituted aliphatic; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; cyclic or acyclic, substituted or unsubstituted acyl; or R a  is a suitable amino protecting group; 
 each instance of R b  is, independently, a suitable amino acid side chain; hydrogen; cyclic or acyclic, branched or unbranched, substituted or unsubstituted aliphatic; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; cyclic or acyclic, substituted or unsubstituted acyl; substituted or unsubstituted hydroxyl; substituted or unsubstituted thiol; substituted or unsubstituted amino; cyano; isocyano; halo; or nitro; 
 each instance of R e  is, independently, —R E , —OR E , —N(R E ) 2 , or —SR E , wherein each instance of R E  is, independently, hydrogen, cyclic or acyclic, branched or unbranched, substituted or unsubstituted aliphatic; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; substituted or unsubstituted acyl; a resin; a suitable hydroxyl, amino, or thiol protecting group; or two R E  groups together form a substituted or unsubstituted 5- to 6-membered heterocyclic or heteroaromatic ring; 
 each instance of R f  is, independently, hydrogen; cyclic or acyclic, branched or unbranched, substituted or unsubstituted aliphatic; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; substituted or unsubstituted acyl; a resin; a suitable amino protecting group; a label optionally joined by a linker, wherein the linker is selected from cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkenylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkynylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkenylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkynylene; substituted or unsubstituted arylene; substituted or unsubstituted heteroarylene; or substituted or unsubstituted acylene; or R f  and R a  together form a substituted or unsubstituted 5- to 6-membered heterocyclic or heteroaromatic ring; 
 each instance of R LL  is, independently, hydrogen; cyclic or acyclic, branched or unbranched, substituted or unsubstituted aliphatic; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; substituted or unsubstituted acyl; substituted or unsubstituted hydroxyl; substituted or unsubstituted thiol; substituted or unsubstituted amino; azido; cyano; isocyano; halo; nitro; 
 or two adjacent R LL  groups are joined to form a substituted or unsubstituted 5- to 8-membered cycloaliphatic ring; substituted or unsubstituted 5- to 8-membered cycloheteroaliphatic ring; substituted or unsubstituted aryl ring; or substituted or unsubstituted heteroaryl ring; 
 each instance of X AA  is, independently, a natural or unnatural amino acid; 
 each instance of z is, independently, an integer between 2 to 6; 
 each instance of j is, independently, an integer between 1 to 10; 
 each instance of s and t is, independently, an integer between 0 and 100; 
 each instance of q is, independently, an integer between 0 to 2; 
 
       and wherein
    corresponds to a single or double bond. 
 
     
     
         7 - 8 . (canceled) 
     
     
         9 . The method of  claim 1 , wherein the stitched peptide is of formula: 
       
         
           
           
               
               
           
         
       
       wherein
 each instance of K, L 1 , L 2 , and M, is, independently, a bond, cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkenylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkynylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkenylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkynylene; substituted or unsubstituted arylene; substituted or unsubstituted heteroarylene; or substituted or unsubstituted acylene; 
 each instance of R a  is, independently, hydrogen; cyclic or acyclic, branched or unbranched, substituted or unsubstituted aliphatic; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; cyclic or acyclic, substituted or unsubstituted acyl; or R a  is a suitable amino protecting group; 
 each instance of R b  is, independently, a suitable amino acid side chain; hydrogen; cyclic or acyclic, branched or unbranched, substituted or unsubstituted aliphatic; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; cyclic or acyclic, substituted or unsubstituted acyl; substituted or unsubstituted hydroxyl; substituted or unsubstituted thiol; substituted or unsubstituted amino; cyano; isocyano; halo; or nitro; 
 each instance of R e  is, independently, —R E , —OR E , N(R E ) 2 , or —SR E , wherein each instance of R E  is, independently, hydrogen, cyclic or acyclic, branched or unbranched, substituted or unsubstituted aliphatic; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; substituted or unsubstituted acyl; a resin; a suitable hydroxyl, amino, or thiol protecting group; or two R E  groups together form a substituted or unsubstituted 5- to 6-membered heterocyclic or heteroaromatic ring; 
 each instance of R f  is, independently, hydrogen; cyclic or acyclic, branched or unbranched, substituted or unsubstituted aliphatic; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; substituted or unsubstituted acyl; a resin; a suitable amino protecting group; a label optionally joined by a linker, wherein the linker is selected from cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkenylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted alkynylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkenylene; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroalkynylene; substituted or unsubstituted arylene; substituted or unsubstituted heteroarylene; or substituted or unsubstituted acylene; or R f  and R a  together form a substituted or unsubstituted 5- to 6-membered heterocyclic or heteroaromatic ring; 
 each instance of R KL , R LL , and R LM , is, independently, hydrogen; cyclic or acyclic, branched or unbranched, substituted or unsubstituted aliphatic; cyclic or acyclic, branched or unbranched, substituted or unsubstituted heteroaliphatic; substituted or unsubstituted aryl; substituted or unsubstituted heteroaryl; substituted or unsubstituted acyl; substituted or unsubstituted hydroxyl; substituted or unsubstituted thiol; substituted or unsubstituted amino; azido; cyano; isocyano; halo; nitro; 
 or two adjacent R KL  are joined to form a substituted or unsubstituted 5- to 8-membered cycloaliphatic ring; substituted or unsubstituted 5- to 8-membered cycloheteroaliphatic ring; substituted or unsubstituted aryl ring; or substituted or unsubstituted heteroaryl ring; two adjacent R KL  are joined to form a substituted or unsubstituted 5- to 8-membered cycloaliphatic ring; substituted or unsubstituted 5- to 8-membered cycloheteroaliphatic ring; substituted or unsubstituted aryl ring; or substituted or unsubstituted heteroaryl ring; or two adjacent R LM  groups are joined to form a substituted or unsubstituted 5- to 8-membered cycloaliphatic ring; substituted or unsubstituted 5- to 8-membered cycloheteroaliphatic ring; substituted or unsubstituted aryl ring; or substituted or unsubstituted heteroaryl ring; 
 each instance of X AA  is, independently, a natural or unnatural amino acid; 
 each instance of y and z is, independently, an integer between 2 to 6; 
 each instance of j is, independently, an integer between 1 to 10; 
 each instance of p is, independently, an integer between 0 to 10; 
 each instance of s and t is, independently, an integer between 0 and 100; 
 each instance of u, v, and q, is, independently, an integer between 0 to 2; 
 
       and wherein
    corresponds to a single or double bond. 
 
     
     
         10 - 11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein the step of providing a stitched or stapled peptide comprises steps of:
 synthetically producing an unstitched or unstapled peptide, wherein the peptide comprises unnatural amino acid residues suitable for stitching or stapling; and   stitching or stapling the unstitched or unstapled peptide.   
     
     
         13 . The method of  claim 1 , wherein the step of providing a stapled peptide comprises steps of:
 providing a peptide with at least two α-methyl,α-alkenylglycine or α-hydro,α-alkenylglycine residues; and   stapling the peptide using a Grubbs catalyst to form a macrocyclic hydrocarbon staple.   
     
     
         14 . The method of  claim 1 , wherein the step of providing a stitched peptide comprises steps of:
 providing a peptide with at least two α-methyl,α-alkenylglycine or α-hydro,α-alkenylglycine residues and at least one dialkenylglycine residue; and   stitching the peptide using a Grubbs catalyst to form at least two macrocyclic hydrocarbon staples.   
     
     
         15 . The method of  claim 13 , wherein the two α-methyl,α-alkenylglycine or α-hydro,α-alkenylglycine residues are at positions i and i+3, at positions i and i+4, and/or at positions i and i+7. 
     
     
         16 - 17 . (canceled) 
     
     
         18 . The method of  claim 13 , wherein the α-methyl,α-alkenylglycine or α-hydro,α-alkenylglycine residue is of the formula: 
       
         
           
           
               
               
           
         
       
       wherein n is an integer between 1 and 10, inclusive; and R b  is H or methyl. 
     
     
         19 . (canceled) 
     
     
         20 . The method of  claim 1 , wherein the second protein or peptide is prepared recombinantly. 
     
     
         21 . The method of  claim 1 , wherein the second protein or peptide is prepared synthetically. 
     
     
         22 . The method of  claim 1 , wherein the second protein or peptide is treated with a protease to yield an N-terminal cysteine. 
     
     
         23 - 26 . (canceled) 
     
     
         27 . The method of  claim 1 , wherein the second protein is produced by having cysteine as the second residue in an expression construct following an N-terminal methionine; wherein the methionine is processed during protein expression to yield an N-terminal cysteine. 
     
     
         28 . The method of  claim 1 , wherein the step of ligating comprises ligating using Expressed Protein Ligation (EPL). 
     
     
         29 . The method of  claim 1 , wherein the step of ligating produces a scarless protein. 
     
     
         30 - 36 . (canceled) 
     
     
         37 . A method of preparing a stitched or stapled protein, the method comprising steps of:
 providing an unstitched or unstapled peptide, wherein the peptide comprises unnatural amino acid residues suitable for stitching or stapling;   providing a second protein or peptide to which the unstitched or unstapled peptide is to be ligated;   ligating the unstitched or unstapled peptide to the second protein or peptide; and   stitching or stapling the unstitched or unstapled peptide.   
     
     
         38 - 39 . (canceled) 
     
     
         40 . A stapled IL-13, wherein Helix A or Helix D includes a hydrocarbon staple. 
     
     
         41 - 50 . (canceled) 
     
     
         51 . A stapled cMyc, wherein the leucine zipper includes a hydrocarbon staple. 
     
     
         52 . A stapled Max, wherein the DNA binding basic region of Max includes a hydrocarbon staple. 
     
     
         53 - 55 . (canceled)