N-terminal acetylation of antimicrobial peptide L163 improves its stability against protease degradation

Antimicrobial peptide L163 was computationally designed by our laboratory; L163 is active against multidrug-resistant (MDR) bacteria but is easily degraded in the plasma and by trypsin. Amino acid substitution, cyclization, and amino-terminal (N-terminal) acetylation were performed to obtain L163 analogs with high stability in the plasma and in trypsin solutions. The stability, antimicrobial activity, and biosafety of L163 and its analogs were investigated. Comparison with unmodified L163 indicated that N-terminal acetylation enhanced the stability against pH, plasma, and trypsin degradation, and phenylalanine (Phe) substitution for leucine (Leu) and cysteine bridge (S-S) cyclization decreased the stability. N-terminal acetylation also enhanced antimicrobial activity against MDR Streptococcus Sc181, Listeria monocytogenes, and Enterococcus E1478F; did not change the activity against MDR Staphylococcus aureus 9, Staphylococcus sciuri P254, and Staphylococcus aureus RN4220; and decreased the activity against Candida tropicalis, Candida albicans, and Enterococcus faecalis Fbc35. Phe substitution for Leu and S-S cyclization decreased the antimicrobial activity. The negative effect of these modifications was detected against biofilm formation by the tested microbial strains. Comparison of Phe substitution for Leu and S-S cyclization indicated that N-terminally acetylated L163 (L163-Ac) is the best candidate. L163-Ac peptide had the highest antibacterial activity and enhanced tolerance to temperature, pH, plasma, and trypsin and low toxicity.