Design of Potent, Non-Toxic Antimicrobial Agents Based upon the Structure of the Frog Skin Peptide, Temporin-1CEb from Chinese Brown Frog, Rana chensinensis

摘要

Temporin-1CEb shows antimicrobial activity against Gram-positive bacteria, but its therapeutic potential is limited by its haemolysis. In this study, eight temporin- 1CEb analogues with altered cationicities and hydrophobicities were synthesized. Increasing cationicity and amphipathicity by substituting neutral and non-polar amino acid residues on the hydrophilic face of the α-helix by five or six lysines increased antimicrobial potency approximately 10- fold to 40-fold, although when the number of positive charges was increased from +6 to +7, the antimicrobial potency was not additionally enhanced. The substitution of an L-lysine with a D-lysine, meanwhile maintaining the net charge and the mean hydrophobicity values, had only a minor effect on its antimicrobial activity, whereas significantly led a decrease in its haemolytic activity. Of all the peptides, L-K6 has the best potential as an antimicrobial agent because its antimicrobial activity against both Gram-positive and Gram-negative bacteria is substantial, and its haemolytic activity is negligible. L-K6 adopts an α-helix in 50% trifluoroethanol ?water and 30 mM SDS solutions. L-K6 killed 99.9% of E. coli and S. aureus at 4× MIC in 60 min, and its postantibiotic effect was >5 h. L-K6 affects the integrity of E. coli and S. aureus plasma membranes by rapidly inducing membrane depolarization.