Rational β-turn engineering of a disulfide-free β-hairpin-like antimicrobial peptide W2PG with enhanced stability, selectivity, and in vivo efficacy

Antimicrobial resistance (AMR) is an increasing global concern, while the development of new antibiotics remains limited. Antimicrobial peptides (AMPs) offer an alternative due to their broad-spectrum and membrane-targeting features, but their translation is restricted by instability and cytotoxicity. Here, we describe a β-turn engineering strategy that incorporates a Pro-Gly motif and aromatic residues to create a short, disulfide-free β-hairpin-like AMP (W2PG). W2PG showed broad-spectrum activity (MIC 4–8 μM) with low hemolysis and cytotoxicity, along with improved protease and serum stability. It caused concentration-dependent membrane permeabilization and depolarization, consistent with molecular dynamics simulations indicating aromatic-assisted anchoring and stable insertion. In murine infection models, W2PG achieved efficacy comparable to polymyxin B without observable organ toxicity. These results suggest that β-turn-guided design can balance stability, selectivity, and activity, providing a useful scaffold for peptide antibiotic development.