Improved proteolytic stability of chicken cathelicidin-2 derived peptides by D-amino acid substitutions and cyclization.

A truncated version of host defense peptide chicken cathelicidin-2, C1-15, possesses potent, broad spectrum antibacterial activity. A variant of this peptide, F(2,5,12)W, which contains 3 phenylalanine to tryptophan substitutions, possesses improved antibacterial activity and lipopolysaccharide (LPS) neutralizing activity compared to C1-15. In order to improve the proteolytic resistance of both peptides we engineered novel chicken cathelicidin-2 analogs by substitution of l- with D-amino acids and head-to-tail cyclization. Both cyclic and D-amino acid variants showed enhanced stability in human serum compared to C1-15 and F(2,5,12)W. The D-amino acid variants were fully resistant to proteolysis by trypsin and bacterial proteases. Head-to-tail cyclization of peptide F(2,5,12)W resulted in a 3.5-fold lower cytotoxicity toward peripheral blood mononuclear cells. In general, these modifications did not influence antibacterial and LPS neutralization activities. It is concluded that for the development of novel therapeutic compounds based on chicken cathelicidin-2 D-amino acid substitutions and cyclization must be considered. These modifications increase the stability and lower cytotoxicity of the peptides without altering their antimicrobial potency.