Leucine/Isoleucine Substitution Scanning of Tryptophan Residues to Reduce the Toxicity of β-Hairpin Peptide N2W2

The unique bactericidal mechanism of antimicrobial peptides (AMPs) significantly reduces the likelihood of pathogenic bacteria developing resistance to them. However, AMPs exhibiting high antimicrobial activity are often associated with significant toxicity. In our prior studies, the peptide N2W2 (WKWKWWKWKW-NH2) exhibited potent antimicrobial activity but also high toxicity. In this study, the tryptophan of N2W2 was replaced with leucine or isoleucine, and the impact of amino acid substitutions at various positions on the SAR was examined. The analogs, especially those with terminal amino acid substitutions, maintained the potent antimicrobial activity of N2W2 while significantly lowering its toxicity, attributed to distinct changes in their secondary structure under varying membrane conditions. Peptide IL exhibited the highest therapeutic index, and its enantiomer D-IL demonstrated potent antibacterial activity, minimal cytotoxicity, and exceptional stability. In conclusion, this study offers a simple strategy to minimize the toxicity of AMPs, thereby facilitating their clinical translation as antibacterial agents.