Patent ID: 8399405

Claim:
A method of screening for an enhanced alpha-helical polypeptide with enhanced cellular efficacy in human whole blood, the enhanced alpha-helical polypeptide comprising a cross-linker connecting a first amino acid and a second amino acid of the polypeptide, the method comprising: (a) providing a sequence of a parent alpha-helical 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 mediated by binding the alpha-helical polypeptide to an intracellular target, in the presence and/or 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 alpha-helical polypeptide has a higher K d * and an equal or lower EC 50 than the parent alpha-helical polypeptide.