Performance optimization of ultrashort antimicrobial peptides through backbone amide cyclization

Antimicrobial resistance demands therapeutics that combine potent activity with low resistance potential. Ultrashort antimicrobial peptides (AMPs) are attractive due to their manufacturability and tunability, but activity is often lost upon sequence minimization because of conformational instability. Here, we establish a conformational engineering strategy based on backbone amide cyclization to restore ultrashort AMP function. Using a snake venom-derived cathelicidin template ZY4, we identified a minimal Trp/Lys/Arg-rich motif and generated the ultrashort cyclic peptide WKR-cyl, which exhibits enhanced antibacterial potency, membrane selectivity, and proteolytic stability compared with linear and disulfide-cyclized analogs. Mechanistically, amide cyclization stabilizes membrane-active conformations and increases hydrophobic driving forces, reducing the energetic barrier for membrane insertion. WKR-cyl preferentially targets peptidoglycan-rich bacterial surfaces, enabling rapid membrane permeabilization and bactericidal activity. WKR-cyl shows potent activity against MRSA, strong anti-biofilm activity, high plasma stability, and minimal resistance induction, and demonstrates therapeutic efficacy in murine MRSA pneumonia and skin infection models, supporting development of clinically translatable ultrashort anti-infective peptides.