Department of Pharmacy, National University of Singapore, 117543, Singapore, Singapore.
Bioinformatics Institute, Agency of Science, Technology and Research (A*STAR), 138671, Singapore, Singapore; School of Chemistry, University of Southampton, SO17 1BJ, Southampton, United Kingdom.
Acta Biomater. 2021 Nov;135:214-224. doi: 10.1016/j.actbio.2021.09.004. Epub 2021 Sep 8.
Synthetic β-hairpin antimicrobial peptides (AMPs) offer a useful source for the development of novel antimicrobial agents. β-hairpin peptides generally consist of two side strands bridged by a reverse turn. In literature, most studies focused on the modifications of the side strands to manipulate the stability and activity of β-hairpin peptides, and much less is known about the impact of the turn region. By designing a series of de novo β-hairpin peptides with identical side strands but varied turns, we demonstrated that mutations of only 2 to 4 amino acids at the turn region could impart a wide range of antimicrobial profiles among synthetic β-hairpin AMPs. BTT2-4 and BTT6 displayed selective potency against Gram-negative bacteria, with minimum inhibitory concentrations (MICs) of 4-8 µM. In contrast, BTT1 exhibited broad-spectrum activity, with MICs of 4-8 µM against both Gram-positive and Gram-negative strains. Additionally, BTT1 was potent against methicillin-resistant Staphylococcus aureus (MRSA) and colistin-resistant Enterobacterales. The antimicrobial potency of BTT1 persisted after 14 days of serial passage. Mechanistic studies revealed that interactions between lipopolysaccharide (LPS) and the peptides were critical to their membranolytic activity against the bacterial inner membrane. Aside from folding stability, we observed that a degree of conformational flexibility was required for disruptive membrane interactions. STATEMENT OF SIGNIFICANCE: By examining the significance of the turn region of β-hairpin peptides, we present valuable knowledge to the design toolkit of novel antimicrobial peptides as alternative therapeutics to overcome antibiotic resistance. Our de novo designed synthetic peptides displayed selective activity against Gram-negative bacteria and potent activity against clinically relevant antibiotic-resistant strains (e.g. colistin-resistant Enterobacterales and methicillin-resistant Staphylococcus aureus). The bactericidal activity of our peptides was shown to be robust in the presence of proteolytic trypsin and saline, conditions that could suppress peptide activity. Our peptides were also determined to be non-cytotoxic against a human cell line.
合成 β-发夹抗菌肽（AMPs）为开发新型抗菌剂提供了有用的来源。β-发夹肽通常由两个侧链通过反向转弯桥接而成。在文献中，大多数研究都集中在侧链的修饰上，以操纵β-发夹肽的稳定性和活性，而对转弯区域的影响知之甚少。通过设计一系列具有相同侧链但不同转弯的新型 β-发夹肽，我们证明了转弯区域中仅 2 到 4 个氨基酸的突变可以赋予合成β-发夹 AMP 广泛的抗菌谱。BTT2-4 和 BTT6 对革兰氏阴性菌具有选择性效力，最小抑菌浓度（MIC）为 4-8 μM。相比之下，BTT1 表现出广谱活性，对革兰氏阳性和革兰氏阴性菌株的 MIC 均为 4-8 μM。此外，BTT1 对耐甲氧西林金黄色葡萄球菌（MRSA）和多粘菌素耐药肠杆菌科有效。BTT1 的抗菌效力在连续传代 14 天后仍然存在。机制研究表明，肽与脂多糖（LPS）之间的相互作用对其针对细菌内膜的膜溶解活性至关重要。除了折叠稳定性外，我们还观察到需要一定程度的构象灵活性来进行破坏性的膜相互作用。意义陈述：通过检查β-发夹肽转弯区域的意义，我们为新型抗菌肽的设计工具包提供了有价值的知识，作为克服抗生素耐药性的替代治疗方法。我们设计的新型合成肽对革兰氏阴性菌具有选择性活性，对临床相关的抗生素耐药菌株（如多粘菌素耐药肠杆菌科和耐甲氧西林金黄色葡萄球菌）具有强大的活性。在存在蛋白酶胰酶和生理盐水的情况下，我们的肽显示出强大的杀菌活性，这些条件可能会抑制肽的活性。我们的肽也被确定对人细胞系无细胞毒性。
