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.