Sequence-Activity Relationships and Integrated In Silico and In Vitro Mechanistic Study of the Antimicrobial Peptide PEP-4 against Staphylococcus aureus

Abstract The rise of antimicrobial resistance among ESKAPE pathogens demands novel therapeutic strategies. Here, we evaluate a panel of five synthetic peptides through integrated in vitro and in silico analyses, highlighting PEP-4 as a promising antibacterial lead peptide. Our findings demonstrate the key role of C-terminal hydrophobic patterning in antimicrobial efficacy. PEP-4 exhibits strong antibacterial activity (MIC 15.6–62.5 μg mL–1, corresponding to ∼10.5–41.9 μM) and consistently shows bactericidal effects against both Gram-positive and Gram-negative strains. Molecular dynamics simulations show sustained, charge-selective interaction of PEP-4 with an anionic S. aureus-like bilayer and only intermittent association with a zwitterionic mammalian model, supporting electrostatically driven membrane recognition as the initial step of its mechanism; no bilayer destabilization was observed in either system over the simulated time scale. Additionally, molecular docking suggests potential secondary intracellular protein targets. Notably, PEP-4 retains activity in the presence of lysyl-phospholipid-mediated resistance in Staphylococcus aureus, yet exhibits only transient interactions with zwitterionic mammalian membranes. This selective membrane binding is consistent with low cytotoxicity to humans, corroborated by other in silico predictions. It suggests a potential therapeutic window, pending experimental validation, supporting PEP-4 as a promising peptide scaffold for next-generation antimicrobial design.