Department of Molecular Medicine, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Department of Molecular Biology, University of Wyoming, 1000 East University Avenue, Laramie, WY 82071, USA.
Cell Chem Biol. 2018 Mar 15;25(3):301-308.e12. doi: 10.1016/j.chembiol.2017.12.011. Epub 2018 Jan 11.
As resistance to antibiotics increases, the exploration of new targets and strategies to combat pathogenic bacteria becomes more urgent. Ideal protein targets are required for viability across many species, are unique to prokaryotes to limit effects on the host, and have robust assays to quantitate activity and identify inhibitors. Lipoprotein signal peptidase (Lsp) is a transmembrane aspartyl protease required for lipoprotein maturation and comprehensively fits these criteria. Here, we have developed the first in vitro high-throughput assay to monitor proteolysis by Lsp. We employed our high-throughput screen assay against 646,275 compounds to discover inhibitors of Lsp and synthesized a range of analogs to generate molecules with nanomolar half maximal inhibitory concentration values. Importantly, our inhibitors are effective in preventing the growth of E. coli cultures in the presence of outer-membrane permeabilizer PMBN and should facilitate development of antibacterial agents with a novel mechanism of action to treat antibiotic-resistant bacteria.
随着抗生素耐药性的增加，寻找新的靶点和策略来对抗致病菌变得更加紧迫。理想的蛋白靶标需要在许多物种中具有生存能力，并且是原核生物所特有的，以限制对宿主的影响，并且具有强大的测定法来定量活性和鉴定抑制剂。脂蛋白信号肽酶（Lsp）是一种跨膜天冬氨酸蛋白酶，对于脂蛋白成熟是必需的，并且完全符合这些标准。在这里，我们开发了第一个体外高通量测定法来监测 Lsp 的蛋白水解。我们针对 646,275 种化合物进行了高通量筛选，以发现 Lsp 的抑制剂，并合成了一系列类似物，以产生具有纳摩尔半数最大抑制浓度值的分子。重要的是，我们的抑制剂在存在外膜通透剂 PMBN 的情况下能够有效阻止大肠杆菌培养物的生长，并且应该有助于开发具有新型作用机制的抗菌剂来治疗抗药性细菌。
