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 . A method of screening for an alpha-helical polypeptide, the method comprising:
 a) providing a parent polypeptide comprising a crosslinker connecting a first amino acid and a second amino acid of the parent polypeptide, wherein the parent polypeptide is at least 60% identical to any of the sequences in Tables 1, 2, 3 and 4;   b) replacing an acidic side chain of the parent polypeptide with a neutral side chain, wherein the acidic side chain is adjacent to a large hydrophobic side chain to produce a candidate polypeptide;   c) determining an in vivo efficacy (EC 50 ) in a whole cell assay of the parent polypeptide and the candidate polypeptide for human serum, wherein the in vivo efficacy is mediated by binding to a target in the presence and absence of human serum proteins;   d) determining that the candidate polypeptide that has a EC 50  greater than or equal to the EC 50  of the parent polypeptide;   e) measuring the alpha-helical content of the candidate polypeptide in the presence of human serum; and   f) selecting the candidate polypeptide that has an alpha-helical content of 25% or greater as the alpha-helical polypeptide.   
     
     
         38 . The method of  claim 37 , wherein the measuring of the EC 50  comprises using a cellular assay. 
     
     
         39 . The method of  claim 37 , further comprising calculating the apparent affinity (K d *) of the selected candidate polypeptide to human serum proteins. 
     
     
         40 . The method of  claim 39 , 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 human serum, EC' 50  is an in vitro efficacy measured in a whole cell assay in N% human serum, and P equals (N/100)×(700) micromolar. 
     
     
         41 . The method of  claim 37 , wherein the alpha-helical polypeptide has a K d * of 1 to 700 micromolar. 
     
     
         42 . The method of  claim 37 , wherein the alpha-helical polypeptide has a K d * of about 70 micromolar or lower. 
     
     
         43 . The method of  claim 37 , wherein the alpha-helical polypeptide has a K d * of about 1 to 10 micromolar. 
     
     
         44 . The method of  claim 37 , wherein the alpha-helical polypeptide comprises an alpha-helical domain of a BCL-2 family member. 
     
     
         45 . The method of  claim 37 , wherein the alpha-helical polypeptide comprises a BH3 domain. 
     
     
         46 . The method of  claim 37 , wherein the alpha-helical polypeptide has an alpha-helical content of 50% or greater. 
     
     
         47 . The method of claim  1 , wherein the alpha-helical polypeptide has an alpha-helical content of 75% or greater. 
     
     
         48 . The method of  claim 37 , wherein alpha-helical polypeptide penetrates cell membranes by an energy-dependent process. 
     
     
         49 . The method of  claim 48 , wherein the energy-dependent process is endocytosis. 
     
     
         50 . The method of  claim 37 , wherein both the first and second amino acids of the of the parent polypeptide are α,α-disubstituted. 
     
     
         51 . The method of  claim 37 , wherein the crosslinker spans 1 or 2 turns of the alpha-helix. 
     
     
         52 . The method of  claim 37 , wherein the length of the crosslinker is about 5 A to about 9 A per turn of the alpha-helix. 
     
     
         53 . The method of  claim 37 , wherein the crosslinker comprises between 8 and 16 consecutive bonds. 
     
     
         54 . The method of  claim 37 , wherein the crosslinker comprises between 10 and 13 consecutive bonds. 
     
     
         55 . The method of  claim 37 , wherein alpha-helical polypeptide carries a net positive charge at pH 7.4. 
     
     
         56 . The method of  claim 37 , wherein alpha-helical polypeptide provides a therapeutic effect.