Bifunctional stapled polypeptides and uses thereof

Abstract

The invention relates to bifunctional stapled or stiched peptides comprising a targeting domain, a linker moiety, and an effector domain, that can be used to tether, or to bring into close proximity, at least two cellular entities (e.g., proteins). Certain aspects relate to bifunctional stapled or stiched peptides that bind to an effector biomolecule through the effector domain and bind to a target biomolecule through the targeting domain. Polypeptides and/or polypeptide complexes that are tethered by the bifunctional stapled or stiched peptides of the invention, where the effector polypeptide bound to the effector domain of the bifunctional stapled or stiched peptide modifies or alters the target polypeptide bound to the targeting domain of the bifunctional peptide. Usesses of the inventive bifunctional stapled or stiched peptides including methods for treatment of disease (e.g., cancer, inflammatory diseases) are also provided.

1 . A bifunctional peptide comprising:
 a targeting domain;   a linker moiety; and   an effector domain, wherein   the linker moiety links the targeting domain to the effector domain,   and at least one domain is stapled or stitched.   
     
     
         2 - 5 . (canceled) 
     
     
         6 . The peptide of  claim 1 , wherein at least one domain comprises the 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 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, a bond to the linker moiety, —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 of —N(R E ) 2  together form a substituted or unsubstituted 5- to 6-membered heterocyclic or heteroaromatic ring; 
 each instance of R f  is, independently, a bond to the linker moiety; 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 tether, wherein the tether 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  of a terminal amino acid 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 
    corresponds to a single or double bond. 
 
     
     
         7 - 11 . (canceled) 
     
     
         12 . The peptide of  claim 1 , wherein at least one domain comprises the 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 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, a bond to the linker moiety, —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 of —N(R E ) 2 together form a substituted or unsubstituted 5- to 6-membered heterocyclic or heteroaromatic ring; 
 each instance of R f  is, independently, a bond to the linker moiety; 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 tether, wherein the tether 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  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; two adjacent R KL  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; 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 
    corresponds to a single or double bond. 
 
     
     
         13 - 17 . (canceled) 
     
     
         18 . The peptide of  claim 1 , wherein the targeting domain is a stapled or stitched protein. 
     
     
         19 . The peptide of  claim 1 , wherein the effector domain is a stapled or stitched protein. 
     
     
         20 - 25 . (canceled) 
     
     
         26 . The peptide of  claim 1 , wherein the targeting domain binds a protein, a nucleic acid, or a phospholipid. 
     
     
         27 . The peptide of  claim 26 , wherein the targeting domain binds β-catenin, Myc, Ras, or hypoxia-inducible factor. 
     
     
         28 . (canceled) 
     
     
         29 . The peptide of  claim 1 , wherein the targeting domain is a ligand for β-catenin, Myc, Ras, hypoxia-inducible factor, or Sin3. 
     
     
         30 . The peptide of  claim 29 , wherein the targeting domain is a stapled version of Bcl9 or Tcf-4. 
     
     
         31 - 34 . (canceled) 
     
     
         35 . The peptide of  claim 29 , wherein the targeting domain is a stapled version of Max, Mad, or Mxi. 
     
     
         36 - 42 . (canceled) 
     
     
         43 . The peptide of  claim 1 , wherein the effector domain modulates the activity of an enzyme. 
     
     
         44 . The peptide of  claim 43 , wherein the enzyme is a ubiquitinating enzyme, a glycosylating enzyme, a histone deacetylase, a histone acetyl transferase, a kinase, a protease, a farnesyl transferase, an acetylase, a phosphatase, or a phosphorylating enzyme. 
     
     
         45 . (canceled) 
     
     
         46 . The peptide of  claim 44 , wherein the effector domain is a ligand for an E3 ubiquitin ligase. 
     
     
         47 . The peptide of  claim 46 , wherein the E3 ubiquitin ligase is hDM2, mDM2, ubiquitin protein ligase E3A, a RING finger domain, or an SCF E3 ligase complex. 
     
     
         48 . The peptide of  claim 47 , wherein the effector domain is a stapled version of p53. 
     
     
         49 - 91 . (canceled) 
     
     
         92 . A pharmaceutical composition comprising a stapled peptide of  claim 1 . 
     
     
         93 . A method of treating a disease, disorder, or condition in a subject, said method comprising administering a therapeutically effective amount of a stapled peptide of  claim 1  to a subject in need thereof. 
     
     
         94 . The method of  claim 93 , wherein the disease is cancer. 
     
     
         95 - 102 . (canceled) 
     
     
         103 . The peptide of  claim 92 , wherein the targeting domain is a stapled version of the Sin3 interacting domain (SID) of Mad1. 
     
     
         104 - 106 . (canceled) 
     
     
         107 . The peptide of  claim 1 , wherein the effector domain is a stapled version of MLL or cMyb. 
     
     
         108 - 112 . (canceled)