Breaking barriers in peptide therapeutics: hydrocarbon stapling of Temporin-FL peptide for improved bacterial membrane disruption

Antibiotic-resistant bacteria are a serious global health concern that necessitates the use of alternate therapies. Particularly, alpha-helical antimicrobial peptides (AMPs) are promising candidates against resistant bacterial strains but face limitations due to instability and cytotoxicity. Hydrocarbon stapling presents a potential solution by stabilising the helical structure of AMPs, thereby improving their stability and efficacy. In this study, we employed computational methods to design and evaluate stapled variants of the AMP Temporin-FL. We designed 15 stapled variants, all of which demonstrated increased reactivity, as indicated by their HOMO–LUMO energy gap evaluations. Lyticity index analysis indicated that Temp_1_8 and Temp_2_9 are less toxic. These two stapled peptides, along with the native peptide as control, were subjected to membrane simulations which confirmed that the stapled variants retained their helical structure more effectively than the Native-Temporin-FL. Specifically, Temp_1_8 displayed enhanced stability, as shown by its free energy landscape and RMSF analysis. Additionally, the contact map for Temp_1_8 revealed stronger membrane interactions compared to the Native-Temporin-FL and Temp_2_9 peptides. Lipid disruption analysis further demonstrated that Temp_1_8 significantly destabilised the membrane, with marked increases in membrane thickness fluctuations and lipid lateral diffusion. These findings suggest that Temp_1_8 peptide can act as a membrane-targeted therapeutic agent.