Peptidomimetic macrocycles with improved properties

Abstract

The present invention provides biologically active peptidomimetic macrocycles with improved properties relative to their corresponding polypeptides. The invention additionally provides methods of preparing and using such macrocycles, for example in therapeutic applications.

1 - 71 . (canceled) 
     
     
         72 . 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 apparent affinity (K d *) of the parent polypeptide and the candidate polypeptide for human serum proteins;   d) determining that the candidate polypeptide that has a K d * greater than or equal to the K d * of the parent polypeptide;   e) measuring the alpha-helical content of the candidate polypeptide in the presence of human serum; and   f) determining that the candidate polypeptide that has an alpha-helical content of 25% or greater as the parent polypeptide.   
     
     
         73 . The method of  claim 72 , wherein the candidate polypeptide comprises an alpha-helical domain of a BCL-2 family member. 
     
     
         74 . The method of  claim 72 , wherein the candidate polypeptide comprises a BH3 domain. 
     
     
         75 . The method of  claim 72 , wherein the candidate polypeptide has a K d * of 1 to 700 micromolar. 
     
     
         76 . The method of  claim 72 , wherein the candidate polypeptide has a K d * of about 70 micromolar or lower. 
     
     
         77 . The method of  claim 72 , wherein the candidate polypeptide has a K d * of about 1 to 10 micromolar. 
     
     
         78 . The method of  claim 72 , wherein the candidate polypeptide has an alpha-helical content of 50% or greater. 
     
     
         79 . The method of  claim 72 , wherein the candidate polypeptide has an alpha-helical content of 75% or greater. 
     
     
         80 . The method of  claim 72 , wherein the candidate polypeptide possesses an estimated free fraction in human blood of about 0.1-50%, and the estimated free fraction is defined by the equation 
       
         
           
             
               FreeFraction 
               = 
               
                 
                   
                     K 
                     d 
                     * 
                   
                   
                     
                       K 
                       d 
                       * 
                     
                     + 
                     
                       
                         [ 
                         HSA 
                         ] 
                       
                       total 
                     
                   
                 
                  
                 
                     
                 
                  
                 
                   
                     and 
                      
                     
                       
 
                     
                     [ 
                     HSA 
                     ] 
                   
                   total 
                 
                  
                 
                     
                 
                  
                 is 
                  
                 
                     
                 
                  
                 700 
                  
                 
                     
                 
                  
                 
                   micromolar 
                   . 
                 
               
             
           
         
       
     
     
         81 . The method of  claim 72 , wherein the candidate polypeptide possesses an estimated free fraction in human blood of about 0.1-10% 
     
     
         82 . The method of  claim 72 , wherein the candidate polypeptide penetrates cell membranes by an energy-dependent process. 
     
     
         83 . The method of  claim 72 , wherein the crosslinker spans 1 or 2 turns of the alpha-helix. 
     
     
         84 . The method of  claim 72 , wherein the length of the crosslinker is about 5 Å to about 9 Å per turn of the alpha-helix. 
     
     
         85 . The method of  claim 72 , wherein the candidate polypeptide carries a net positive charge at pH 7.4. 
     
     
         86 . The method of  claim 72 , wherein the candidate polypeptide provides a therapeutic effect. 
     
     
         87 . The method of  claim 72 , wherein the candidate polypeptide possesses an apparent affinity to human serum proteins of about 1 micromolar or weaker. 
     
     
         88 . The method of  claim 72 , wherein the candidate polypeptide possesses an apparent affinity to human serum proteins of about 3 micromolar or weaker. 
     
     
         89 . The method of  claim 72 , wherein the candidate polypeptide possesses an apparent affinity to human serum proteins of about 10 micromolar or weaker.