All-hydrocarbon Stapling Modification and Optimization of Scorpion Toxin-Derived Peptide TtAP-2 Against Multidrug- Resistant Bacterial Infections

The widespread overuse of antibiotics has accelerated the global spread of multidrug‑resistant bacteria. Meanwhile, the clinical efficacy of conventional agents continues to decline, highlighting an urgent need for novel therapeutics. Antimicrobial peptides (AMPs) mainly exert their antimicrobial activity by disrupting bacterial cell membranes, a mechanism that impedes the development of resistance, making them an ideal candidate to replace traditional antibiotics. TtAP-2 is a cationic, linear antimicrobial peptide originally isolated from the venom of Tityus trinitatis , which displays modest activity against clinically resistant strains. Nevertheless, its high conformational flexibility and susceptibility to proteolytic degradation severely impede its advancement as a drug candidate. All-hydrocarbon stapling is achieved by introducing non-natural amino acids between the side chains of the peptide chain to form covalent bridges, thereby conferring a more stable α-helical conformation upon linear peptides. Consequently, this conformational stabilization not only reduces the protease recognition and degradation of the peptide chain by proteases but also enhances antimicrobial activity. In this study, the antimicrobial peptide TtAP-2 underwent structural modification using all-hydrocarbon stapling, in which the non-natural amino acids S ₅ and R ₈ were incorporated at positions i, i + 4 and i, i + 7. Subsequently, 15 stapled peptide derivatives were designed and synthesized, while their minimum inhibitory concentrations (MICs) against pathogenic bacteria, enzymatic stability, and hemolytic toxicity were evaluated. Among these, the stapled peptide derivative TtAP-2-4 exhibits good activity against multidrug-resistant Gram-positive bacteria and pronounced protease resistance, highlighting its potential as a drug candidate for treating drug-resistant infections.