Bellavita Rosa, Palladino Sara, Rangel Karyne, Lechuga Guilherme Curty, Imbò Lorenzo Emiliano, Falcigno Lucia, D'Auria Gabriella, da Silva Lara Leonardo, de Souza Pereira Mirian Cláudia, De-Simone Salvatore Giovanni, Galdiero Stefania, Falanga Annarita
Department of Pharmacy, School of Medicine, University of Naples Federico II, Via Domenico Montesano 49, 80131 Napoli, Italy.
Center for Technological Development in Health (CDTS), National Institute of Science and Technology for Innovation in Neglected Population Diseases (INCT-IDPN), Oswaldo Cruz Foundation (FIOCRUZ), Rio de Janeiro 21040-900, Brazil.
Antibiotics (Basel). 2025 Sep 10;14(9):913. doi: 10.3390/antibiotics14090913.
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.
替莫瑞辛 -1 是一种由天蚕素 A、短杆菌肽和聚亮氨酸序列组合而成的杂合抗菌肽，对锥虫具有强大的杀锥虫活性，对哺乳动物细胞具有中等细胞毒性。在本研究中，我们研究了该肽与原生动物和真核细胞膜相互作用时的作用方式。为此，我们使用脂质体作为仿生模型进行了一系列生物物理测定，并进行了基于荧光的实验，如脂质混合、膜泄漏以及涉及硫黄素和劳丹的测定。替莫瑞辛 -1 对原生动物和真核细胞膜表现出强大的膜溶解活性，导致显著的膜融合和泄漏，进而形成孔道。此外，我们还对脂质体相互作用进行了结构研究，观察到在膜相互作用过程中保守的螺旋结构。核磁共振研究证实了所有获得的数据，提供了溶液中游离替莫瑞辛 -1 的结构及其与胶束相互作用的方式。此外，替莫瑞辛 -1 对细胞内形式的[此处原文缺失相关内容]表现出高选择性，与苯硝唑联合使用时表现出相加作用，突出了其有前景的治疗活性。总之，阐明替莫瑞辛 -1 的作用机制对于优化其结构、提高靶点选择性以及通过应用化学策略和递送系统偶联推动下一代抗菌肽的合理设计至关重要。
