Stapled anoplin with perfluoroaryl-cysteine enhances antibacterial activity

Owing to their unique mechanism of action and broad-spectrum antimicrobial activity, antimicrobial peptides (AMPs) have gained considerable attention as promising antimicrobial alternatives. Anoplin is a natural antimicrobial peptide from the venom sac of the solitary wasp Anoplius samariensis featuring with broad-spectrum antibacterial activity, low hemolytic activity, and a low propensity to induce resistance. However, its relatively moderate potency and poor stability limited its further development and application. In this work, a peptide stapling strategy utilizing perfluoroaryl-cysteine or decafluorobiphenyl-cysteine was first applied to Anoplin, enabling the preparation of a series of conformationally constrained peptide analogues. The results indicated that these stapled peptides not only retained their antibacterial activity in the presence of physiological salt concentrations but also exhibited potent efficacy in serum, along with improved resistance to enzymatic degradation. In particular, Ano-(3-10) exhibited enhanced broad-spectrum antibacterial activity, low hemolytic toxicity, and efficacy under physiological salt and proteolytic conditions. Mechanistic studies indicated that these derivatives exert rapid antibacterial effects by disrupting outer bacterial membrane integrity, ultimately causing membrane collapse. Additionally, these analogs effectively inhibited biofilm formation. In a serial of passage experiments, E. coli and S. aureus failed to develop resistance to Ano-(3-10) over 15 passages. In summary, this study successfully developed a novel class of stapled antimicrobial peptide analogs with potent antimicrobial activity and high proteolytic stability, providing a new strategy for the optimization and application of AMPs.