Peptidomimetic macrocycles

Abstract

The present invention provides biologically active crosslinked polypeptides with improved properties relative to their corresponding precursor polypeptides, having good cell penetration properties and reduced binding to human proteins. The invention additionally provides methods of identifying and making such improved polypeptides.

1 .- 36 . (canceled) 
     
     
         37 . An enhanced polypeptide comprising a cross-linker connecting a first amino acid and a second amino acid of the enhanced polypeptide, wherein the enhanced polypeptide is selected by a screening method comprising:
 (a) providing a sequence of a parent polypeptide comprising a cross-linker connecting a first amino acid and a second amino acid of the parent polypeptide sequence,   (b) producing a modified alpha-helical polypeptide having the same sequence as the parent polypeptide sequence except that at least one amino acid side chain that is not essential for target binding is different in comparison to the alpha-helical parent polypeptide sequence, wherein the modified alpha-helical polypeptide comprises a cross-linker connecting a first amino acid and a second amino acid of the modified alpha-helical polypeptide;   (c) measuring an in vitro activity (EC 50 ) of the modified alpha-helical polypeptide in a whole cell assay wherein activity is mediated by binding to a target, in the presence and absence of human serum;   (d) determining an apparent affinity (K d *) of the modified alpha-helical polypeptide for human serum proteins; and   (e) selecting the modified alpha-helical polypeptide as an enhanced alpha-helical polypeptide if the modified alpha-helical polypeptide has a K d * between about 1 and 700 micromolar.   
     
     
         38 . The enhanced alpha-helical polypeptide of  claim 37 , wherein the enhanced alpha-helical polypeptide has a K d * between about 1 and 70 micromolar. 
     
     
         39 . The enhanced alpha-helical polypeptide of  claim 37 , wherein the enhanced alpha-helical polypeptide has a K d * between about 1 and about 10 micromolar. 
     
     
         40 . The enhanced alpha-helical polypeptide of  claim 37 , wherein the enhanced alpha-helical polypeptide possesses an estimated free fraction in human blood of about 0.1-50%, wherein the estimated free fraction is defined by the equation 
       
         
           
             
               FreeFraction 
               = 
               
                 
                   K 
                   d 
                   * 
                 
                 
                   
                     K 
                     d 
                     * 
                   
                   + 
                   
                     
                       [ 
                       HSA 
                       ] 
                     
                     total 
                   
                 
               
             
           
         
       
       and [HSA] total  is 700 micromolar. 
     
     
         41 . The enhanced alpha-helical polypeptide of  claim 37 , wherein the enhanced alpha-helical polypeptide possesses an estimated free fraction in human blood of about 0.5-10%. 
     
     
         42 . The enhanced alpha-helical polypeptide of  claim 37 , wherein the first amino acid and the second amino acid are separated by three amino acids. 
     
     
         43 . The enhanced alpha-helical polypeptide of  claim 37 , wherein the cross-linker comprises between 6 and 14 consecutive bonds. 
     
     
         44 . The enhanced alpha-helical polypeptide of  claim 37 , wherein the cross-linker comprises between 8 and 12 consecutive bonds. 
     
     
         45 . The enhanced alpha-helical polypeptide of  claim 37 , wherein the enhanced alpha-helical polypeptide comprises a ring of about 18 atoms to 26 atoms. 
     
     
         46 . The enhanced alpha-helical polypeptide of  claim 37 , wherein the first amino acid and the second amino acid are separated by six amino acids. 
     
     
         47 . The enhanced alpha-helical polypeptide of  claim 37 , wherein the cross-linker comprises between 8 and 16 consecutive bonds. 
     
     
         48 . The enhanced alpha-helical polypeptide of  claim 37 , wherein the cross-linker comprises between 10 and 13 consecutive bonds. 
     
     
         49 . The enhanced alpha-helical polypeptide of  claim 37 , wherein the enhanced alpha-helical polypeptide comprises a ring of about 29 atoms to 37 atoms. 
     
     
         50 . The enhanced alpha-helical polypeptide of  claim 37 , wherein the length of the cross-linker is about 5 Å to about 9 Å per turn of an alpha-helix. 
     
     
         51 . The enhanced alpha-helical polypeptide of  claim 37 , wherein the enhanced alpha-helical polypeptide carries a net positive charge at pH 7.4. 
     
     
         52 . The enhanced alpha-helical polypeptide of  claim 37 , wherein the enhanced alpha-helical polypeptide comprises one or more of a halogen, an alkyl group, a fluorescent moiety, an affinity label, a targeting moiety, or a radioisotope. 
     
     
         53 . The enhanced alpha-helical polypeptide of  claim 37 , wherein the enhanced alpha-helical polypeptide provides a therapeutic effect. 
     
     
         54 . The enhanced alpha-helical polypeptide of  claim 37 , wherein the enhanced alpha-helical polypeptide possesses an apparent affinity to human serum proteins of about 1 micromolar or weaker. 
     
     
         55 . The enhanced alpha-helical polypeptide of  claim 37 , wherein the enhanced alpha-helical polypeptide possesses an apparent affinity to human serum proteins of about 3 micromolar or weaker. 
     
     
         56 . The enhanced alpha-helical polypeptide of  claim 37 , wherein the enhanced alpha-helical polypeptide possesses an apparent affinity to human serum proteins of about 10 micromolar or weaker. 
     
     
         57 . The enhanced alpha-helical polypeptide of  claim 37 , wherein K d * is defined by the equation 
       
         
           
             
               
                 EC 
                 50 
                 ′ 
               
               = 
               
                 
                   EC 
                   50 
                 
                 + 
                 
                   P 
                   ( 
                   
                     n 
                     
                       1 
                       + 
                       
                         
                           K 
                           d 
                           * 
                         
                         
                           EC 
                           50 
                         
                       
                     
                   
                   ) 
                 
               
             
           
         
       
       wherein n is 1, EC 50  is an in vitro efficacy measured in a whole cell assay in the absence of any human serum, and EC′ 50  is an in vitro efficacy measured in a whole cell assay in N % human serum wherein P equals (N/100)×(700) micromolar. 
     
     
         58 . The enhanced alpha-helical polypeptide of  claim 37 , wherein the enhanced alpha-helical polypeptide has a Formula (I): 
       
         
           
           
               
               
           
         
         wherein: 
         each A, C, D, and E is independently a natural or non-natural amino acid; 
         B is a natural or non-natural amino acid, amino acid analog, 
       
       
         
           
           
               
               
           
         
       
       [—NH-L 3 -CO—], [—NH-L 3 -SO 2 —], or [—NH-L 3 -];
 each R 1  and R 2  are independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, cycloalkylalkyl, heteroalkyl, heterocycloalkyl, or an additional cross-link L, unsubstituted or substituted with halo-; 
 R 3  is hydrogen, alkyl, alkenyl, alkynyl, arylalkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R 5 ; 
 L is a macrocycle-forming linker of formula -L 1 -L 2 -; 
 each L 1 , L 2  and L 3  are independently alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, cycloarylene, heterocycloarylene, or [—R 4 —K—R 4 -] n , each being optionally substituted with R 5 ; 
 each R 4  is alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene; 
 each K is O, S, SO, SO 2 , CO, CO 2 , or CONR 3 ; 
 each R 5  is independently halogen, alkyl, —OR 6 , —N(R 6 ) 2 , —SR 6 , —SOR 6 , —SO 2 R 6 , —CO 2 R 6 , a fluorescent moiety, a radioisotope or a therapeutic agent; 
 each R 6  is independently —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkylalkyl, heterocycloalkyl, a fluorescent moiety, a radioisotope or a therapeutic agent; 
 R 7  is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R 5 , or part of a cyclic structure with a D residue; 
 R 8  is —H, alkyl, alkenyl, alkynyl, arylalkyl, cycloalkyl, heteroalkyl, cycloalkylalkyl, heterocycloalkyl, cycloaryl, or heterocycloaryl, optionally substituted with R 5 , or part of a cyclic structure with an E residue; 
 each of v and w is independently an integer from 1-1000; 
 each of x, y, and z is independently an integer from 0-10; 
 u is an integer of value 1 to 10; and 
 n is an integer from 1-5.