Improve the Stability and Activity of Antimicrobial Peptides by the Proline-Based PXXP Hinge Structure

Developing a simple and effective strategy to enhance the stability of antimicrobial peptides (AMPs) is critical for successful AMP design. In this study, we leveraged the property of proline to form hinge-like structures and designed a series of repetitive symmetrical sequence AMPs with different proline-based hinge centers (PWWP, PKKP, and PWKP), proposing a template of (KW)_nPXXP(WK)_n-NH₂ (where XX = WW, n = 1-4 or XX = KK, WK, n = 2-4). The corresponding templates without hinge structures, (KW)_n(WK)_n-NH₂ (n = 1-4), were used as controls. Through comprehensive evaluations of activity, toxicity, and stability, we identified two promising AMP candidates, (KW)₃PK and (KW)₃PWK, which demonstrated excellent antibacterial activity, cell selectivity, and stability. Our findings indicate that incorporating a proline-containing PXXP hinge structure into AMP sequences could serve as an effective strategy to enhance AMP stability.