Discovery and Optimisation of Novel Bombinin-Derived Peptides from Bombina variegata against Staphylococcus aureus

Amphibian skin-secreted antimicrobial peptides (AMPs) have garnered significant attention for their excellent biological activity and low propensity for drug resistance over the past 40 years. Bombinins and bombinin H, two classes of AMPs isolated from the skin secretions of Bombina species, demonstrate strong antimicrobial activity against broad-spectrum microorganisms. In this study, two novel peptides, bombinin-like peptide 7S and bombinin-H2L, were identified from the toad, Bombina variegata. While both peptides exhibited broad-spectrum antimicrobial activity, they also showed relatively high cytotoxicity. To explore the structure-activity relationship and enhance therapeutic potential, bombinin-H2L, which displayed stronger average antimicrobial activity, was used as a template. With the aid of bioinformatics analysis, a series of bombinin-H2L analogues were designed by increasing the net positive charges and/or adjusting the amphiphilicity of the parent peptide. Among these analogues, [Arg^{8, 15}]BH2L and [Lys^{7, 8}]BH2L demonstrated high therapeutic efficacy and specificity toward clinically isolated, drug-resistant Staphylococcus aureus strains in both in vitro and ex vivo tests. Their notable biosafety profiles, sensitivity to diverse environments, and ability to disrupt biofilms highlight their potential for further development. Additionally, studies on the mechanism of [Arg^{8, 15}]BH2L and [Lys^{7, 8}]BH2L revealed a membrane-targeted antimicrobial mechanism, with its antibacterial function exerted by disrupting the integrity of bacterial membranes. These findings provide valuable insights into structural modifications of bombinin H peptides for enhanced activity, and [Arg^{8, 15}]BH2L and [Lys^{7, 8}]BH2L have the potential as promising candidates for novel antibacterial agents in treated bacterial skin infections.