Temporizin-1 Meets the Membranes: Probing Membrane Inser-Tion and Disruption Mechanisms.

Temporizin-1, a hybrid antimicrobial peptide derived from the combination of Temporin A, Gramicidin peptide, and a poly-leu sequence, has strong trypanocide activity against and moderate cytotoxicity towards mammalian cells. In this study, we investigated the mode of action of the peptide upon interaction with protozoan and eukaryotic membranes. To this end, we conducted a series of biophysical assays using liposomes as biomimetic models, along with fluorescence-based experiments such as lipid mixing, membrane leakage, and assays involving Thioflavin and Laurdan. Temporizin-1 displayed potent membranolytic activity on protozoan and eukaryotic membranes, causing significant membrane fusion and leakage with consequent pore formation. In addition, we also performed structural studies on liposome interaction, where we observed a helical structure that is conserved during membrane interaction. The NMR study confirms all the data obtained, providing both the structure of free Temporizin-1 in solution and the way it interacts with micelles. Moreover, Temporizin-1 demonstrated high selectivity against intracellular forms of and exhibited an additive effect when combined with benznidazole, highlighting its promising therapeutic activity. In conclusion, elucidating the mechanism of action of Temporizin-1 is essential for optimizing its structure and improving target selectivity, and driving the rational design of next-generation antimicrobial peptides by applying chemical strategies and delivery system's conjugation.