Dap-modified antimicrobial peptides exhibit enhanced antimicrobial activity and potential for bacterial infection...

Antimicrobial peptides (AMPs) are promising candidates for addressing the antimicrobial resistance crisis due to their multiple antibacterial mechanisms and low resistance induction. However, the antibacterial activity, cytotoxicity and protease stability of natural AMPs are usually insufficient to be used as drugs. This study aimed to enhance the activity and stability of the arginine-rich AMP W3R6 through the incorporation of non-natural amino acids, such as 2,3-diaminopropionic acid (Dap) and 2,3-diaminobutanoic acid (Dab). Six analogues were synthesized by replacing arginine or tryptophan residues. Their antibacterial activity, hemolytic activity, cytotoxicity, serum stability, and mechanism of action were systematically evaluated. The lead analogue, W3R6-A1, exhibited significantly improved antibacterial potency, particularly against methicillin-resistant Staphylococcus aureus, with the MIC reduced from 6.25 μM to 0.78 μM. The residual amount of W3R6-A2 and -A3 in the serum after incubation for 3 h were higher than that of W3R6. All analogues showed reduced cytotoxicity against mammalian cells (>80% viability at 100 μM). Dap-modified analogues demonstrated the ability to disrupt bacterial cell membranes and biofilms, and exhibited a lower tendency to induce drug resistance. Two mouse models, burn infection and peritonitis-sepsis, were used to verify the therapeutic effects. W3R6-A1 effectively reduced bacterial load in tissues, attenuated pathological damage, and decreased the levels of pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) in serum, without showing in vivo toxicity. These results demonstrate that incorporation of non-proteinogenic amino acid Dap is a viable strategy for optimizing the antibacterial activity and cytotoxicity of arginine-rich cationic amphipathic AMPs.