Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen 9747 AG, The Netherlands.
ACS Synth Biol. 2021 Aug 20;10(8):1980-1991. doi: 10.1021/acssynbio.1c00161. Epub 2021 Aug 4.
A large number of antimicrobial peptides depend on intramolecular disulfide bonds for their biological activity. However, the relative instability of disulfide bonds has limited the potential of some of these peptides to be developed into therapeutics. Conversely, peptides containing intramolecular (methyl)lanthionine-based bonds, lanthipeptides, are highly stable under a broader range of biological and physical conditions. Here, the class-II lanthipeptide synthetase CinM, from the cinnamycin gene cluster, was employed to create methyllanthionine stabilized analogues of disulfide-bond-containing antimicrobial peptides. The resulting analogues were subsequently modified by adding lipid tails of variable lengths through chemical addition. Finally, the created compounds were characterized by MIC tests against several relevant pathogens, killing assays, membrane permeability assays, and hemolysis assays. It was found that CinM could successfully install methyllanthionine bonds at the intended positions of the analogues and that the lipidated macrocyclic core peptides have bactericidal activity against tested Gram-positive and Gram-negative pathogenic bacteria. Additionally, fluorescence microscopy assays revealed that the lipidated compounds disrupt the bacterial membrane and lyse bacterial cells, hinting toward a potential mode of action. Notably, the semisynthesized macrocyclic lipo-lanthipeptides show low hemolytic activity. These results show that the methods developed here extend the toolbox for novel antimicrobial development and might enable the further development of novel compounds with killing activity against relevant pathogenic bacteria.
大量的抗菌肽依赖于分子内二硫键来发挥其生物活性。然而，二硫键的相对不稳定性限制了其中一些肽类发展为治疗药物的潜力。相反，含有分子内（甲基）硫醚键的肽类，即硫肽，在更广泛的生物和物理条件下具有高度的稳定性。在这里，我们采用来自肉桂霉素基因簇的 II 类硫肽合成酶 CinM，来构建含有二硫键的抗菌肽的甲基硫醚稳定类似物。然后，通过化学添加的方式，在这些类似物中添加了不同长度的脂质尾巴来对其进行进一步修饰。最后，通过 MIC 测试对几种相关病原体、杀伤实验、膜通透性实验和溶血实验对所合成的化合物进行了表征。结果发现，CinM 可以成功地在类似物的预期位置上安装甲基硫醚键，并且脂质化的大环核心肽对测试的革兰氏阳性和革兰氏阴性致病菌具有杀菌活性。此外，荧光显微镜实验表明，脂质化化合物破坏了细菌膜并裂解了细菌细胞，提示了一种潜在的作用模式。值得注意的是，半合成的大环脂硫肽显示出较低的溶血活性。这些结果表明，这里开发的方法扩展了新型抗菌药物开发的工具包，并可能使具有针对相关致病菌的杀伤活性的新型化合物的进一步开发成为可能。
