Design, antimicrobial activity and mechanism of action of Arg-rich ultra-short cationic lipopeptides

The increasing emergence of multidrug-resistant microorganisms represents one of the greatest challenges in the clinical management of infectious diseases, and requires the development of novel antimicrobial agents. To this aim, we de novo designed a library of Arg-rich ultra-short cationic antimicrobial lipopeptides (USCLs), based on the Arg-X-Trp-Arg-NH2 peptide moiety conjugated with a fatty acid, and investigated their antibacterial potential. USCLs exhibited an excellent antimicrobial activity against clinically pathogenic microorganisms, in particular Gram-positive bacteria, including multidrug resistant strains, with MIC values ranging between 1.56 and 6.25 μg/mL. The capability of the two most active molecules, Lau-RIWR-NH2 and Lau-RRIWRR-NH2, to interact with the bacterial membranes has been predicted by molecular dynamics and verified on liposomes by surface plasmon resonance. Both compounds inhibited the growth of S. aureus even at sub MIC concentrations and induced cell membranes permeabilization by producing visible cell surface alterations leading to a significant decrease in bacterial viability. Interestingly, no cytotoxic effects were evidenced for these lipopeptides up to 50–100 μg/mL in hemolysis assay, in human epidermal model and HaCaT cells, thus highlighting a good cell selectivity. These results, together with the simple composition of USCLs, make them promising lead compounds as new antimicrobials.

多重耐药微生物的不断涌现，已成为感染性疾病临床诊疗领域面临的重大挑战之一，亟需开发新型抗菌制剂。为此，本研究基于与脂肪酸缀合的精氨酸-X-色氨酸-精氨酸-NH2肽片段，从头设计了一类富含精氨酸的超短阳离子抗菌脂肽（ultra-short cationic antimicrobial lipopeptides, USCLs）文库，并对其抗菌潜能展开了探究。此类USCLs对临床致病微生物展现出优异的抗菌活性，尤其针对包括多重耐药菌株在内的革兰氏阳性菌，其最低抑菌浓度（minimum inhibitory concentration, MIC）范围为1.56~6.25 μg/mL。本研究通过分子动力学模拟，预测了两种活性最强的分子——Lau-RIWR-NH2与Lau-RRIWRR-NH2与细菌细胞膜的结合能力，并通过表面等离子体共振技术在脂质体上验证了该结合特性。即便在亚抑菌浓度下，这两种化合物仍可抑制金黄色葡萄球菌（Staphylococcus aureus, S. aureus）的生长，通过引发可见的细胞表面形态改变诱导细胞膜通透性增加，进而显著降低细菌存活率。值得注意的是，在溶血试验、人体表皮模型及HaCaT细胞实验中，浓度高达50~100 μg/mL的此类脂肽未表现出任何细胞毒性，由此凸显出其良好的细胞选择性。上述研究结果结合USCLs简洁的分子构成，使其有望成为新型抗菌制剂的先导化合物。