Data_Sheet_1_Backbone Cyclization and Dimerization of LL-37-Derived Peptides Enhance Antimicrobial Activity and Proteolytic Stability.docx

Can antimicrobial activity and peptide stability of alpha-helical peptides be increased by making them into dimers and macrocycles? Here, we explore that concept by using KR-12 as the starting point for peptide engineering. KR-12 has previously been determined as the minimalized antimicrobial fragment of the human host defense peptide LL-37. Backbone-cyclized KR-12 dimers, tethered by linkers of two to four amino acid residues, were synthesized and their antimicrobial activity, proteolytic stability and structures characterized. A modified KR-12 sequence, with substitutions at previously identified key residues, were also included in the screening panel. The backbone cyclized KR-12 dimers showed improved antimicrobial activity and increased stability compared to monomeric KR-12. The most active cyclic dimer displayed 16-fold higher antibacterial activity compared to KR-12 against Pseudomonas aeruginosa and Staphylococcus aureus, and 8-fold increased fungicidal activity against Candida albicans. It also showed increased hemolytic and cytotoxic activity. Enhanced antimicrobial activity coincided with increased membrane permeabilization of liposomes with one distinct discrepancy: monomeric KR-12 was much less disruptive of liposomes with bacterial lipid composition compared to liposomes from fungal lipid extract. Circular dichroism showed that the four-residue linked most active cyclic dimer had 65% helical content when bound to lyso-phosphatidylglycerol micelles, indicating that the helical propensity of the parent peptide is maintained in the new macrocyclic form. In conclusion, the current work on KR-12 suggests that dimerization together with backbone cyclization is an effective strategy for improving both potency and stability of linear antimicrobial peptides.

能否通过将α螺旋肽制备为二聚体与大环化合物，提升其抗菌活性与肽稳定性？本研究以KR-12作为肽工程化的起始模板，对上述构想展开探索。KR-12此前已被证实为人类宿主防御肽LL-37的最小活性抗菌片段。本研究合成了由2至4个氨基酸残基连接臂锚定的骨架环化KR-12二聚体，并对其抗菌活性、蛋白水解稳定性与结构进行了表征。同时，将在已确认的关键残基位点上进行取代修饰的KR-12突变序列纳入筛选体系。相较于单体KR-12，骨架环化KR-12二聚体展现出更优异的抗菌活性与更高的稳定性。活性最优的环化二聚体相较于KR-12，对铜绿假单胞菌（Pseudomonas aeruginosa）与金黄色葡萄球菌（Staphylococcus aureus）的抗菌活性提升了16倍，对白色念珠菌（Candida albicans）的杀菌活性提升了8倍，但其溶血活性与细胞毒性也有所增强。抗菌活性的提升与脂质体膜通透化能力的增强相契合，但存在一处显著差异：相较于真菌脂质提取物制备的脂质体，单体KR-12对细菌脂质组成的脂质体的破坏能力要弱得多。圆二色谱（Circular dichroism）分析显示，由4个氨基酸残基连接的活性最优环化二聚体与溶血磷脂酰甘油胶束结合时，螺旋含量达65%，表明亲本肽的螺旋倾向在新型大环结构中得以保留。综上，本项针对KR-12的研究表明，二聚化结合骨架环化是提升线性抗菌肽活性与稳定性的有效策略。