Novel β-hairpin amphiphiles utilizing tryptophan-interacting propensities exhibit multimodal antibacterial mechanism and promote wound healing in drug-resistant infections

INTRODUCTION: The emergence of multidrug-resistant bacteria constitutes a severe global health threat that requires effective countermeasures. In this context, antimicrobial peptides (AMPs) represent promising candidates for the development of new antibiotics. OBJECTIVES: In this study, the unique structural properties of tryptophan were leveraged to develop novel β-hairpin amphiphiles. Specifically, via tryptophan-mediated interstrand π-π and CH-π interactions, the β-hairpin conformation was stabilized without resorting to traditional disulfide bonds, thereby enabling the construction of the simplest β-sheet scaffold. METHODS: Peptides were synthesized via the Fmoc solid-phase method, and their secondary structures were determined by circular dichroism spectroscopy. In vitro experiments included the determination of minimum inhibitory concentration (MIC), evaluations of stability (salts, serum, and extreme pH), and assessments of toxicity (cytotoxicity and hemolysis). Mechanisms of action were explored by measuring inner and outer membrane permeability, combined with fluorescence microscopy, electron microscopy, flow cytometry, assays for respiratory chain dehydrogenase activity, adenosine triphosphate (ATP) alteration, and reactive oxygen species (ROS) generation, as well as transcriptomic analysis. In vivo efficacy was validated in mouse models of Escherichia coli-induced peritonitis-sepsis and methicillin-resistant Staphylococcus aureus (MRSA)-infected skin wounds. RESULTS: The lead peptide WIF exhibited broad-spectrum antibacterial activity against both Gram-negative and Gram-positive pathogens with a geometric mean MIC of 3.11 μM and demonstrated excellent biocompatibility, as indicated by a geometric mean selectivity index of 41.16. Mechanistic investigations revealed a multimodal antibacterial mechanism involving cell membrane disruption, inhibition of energy metabolism, and excessive reactive oxygen species accumulation. In vivo studies demonstrated WIF's efficacy in treating Escherichia coli-induced peritonitis-sepsis and MRSA-infected skin wounds, coupled with inflammation-modulating properties and minimal systemic toxicity. CONCLUSION: This study designed highly efficient, low-toxicity β-hairpin antimicrobial peptides with a multimodal mechanism of action, advancing β-hairpin-based biomaterials as promising alternatives to traditional antibiotics for combating drug-resistant infections.