Overcoming Proteolytic Instability in a β-Turn Antimicrobial Peptide via Cyclization and Stereochemical Inversion to Combat MDR Bacteria

The rational design of proteolytically stable antimicrobial peptides (AMPs) is crucial for combating antibiotic resistance. Although cyclization has been extensively applied to stabilize α-helical and random-coiled AMPs, its impact on the structure-activity relationship of β-turn AMPs remains underexplored. Here, we engineered cyclized derivatives of P-07─a linear β-turn AMP developed by our group─via disulfide/lactam bond cyclization and d-amino acid substitution. The lead candidate, PT-17, exhibited remarkably enhanced stability, retaining potent broad-spectrum activity against MDR pathogens and a high therapeutic index. Additionally, PT-17 exhibited rapid membrane disruption ability, low potential to induce bacterial resistance, and synergy with conventional antibiotics. In a murine infection model, PT-17 achieved a significant reduction of <i>Escherichia coli</i> (MDR) loads in the main organs with undetectable toxicity. This study provides comprehensive evidence that cyclization and d-amino acid substitution confer synergistic stability to β-turn AMPs without compromising activity, offering a viable strategy for developing novel AMPs with clinical potential.