Dual-Mechanism Antimicrobial Peptides from a Nature-Inspired Scaffold.

Antimicrobial peptides are promising alternatives to conventional antibiotics, yet systematic strategies to enhance their potency and elucidate their mechanisms of action remain limited. Here, we generated and evaluated a focused library of 20 peptides derived from the lead peptide L3. Across clinically relevant pathogens, including Escherichia coli, Klebsiella pneumoniae, Staphylococcus aureus, and Candida albicans, several variants showed enhanced antibacterial activity, reducing MIC values to as low as 32 μg/mL (G2-4). Additional candidates (G1-8, G2-1, G2-2, G2-10) achieved MICs of 64 μg/mL against E. coli. Studies in environmental Escherichia isolates revealed species-specific susceptibility patterns. Mechanistic investigations demonstrated minimal membrane-lytic activity at concentrations exceeding their MICs, indicating that membrane disruption is not their primary mode of action. In contrast, in vitro transcription/translation assays demonstrated potent inhibition of protein expression. These results demonstrate how targeted sequence refinement can substantially enhance antimicrobial potency while modulating interactions with bacterial membranes and the transcription/translation machinery.