Mechanistic details of key integral membrane enzymes for antimicrobial discovery

项目概要 标题：用于抗菌发现的关键整合膜酶的机制细节 越来越多的抗生素耐药细菌菌株对人类健康构成重大威胁， 使得开发新的治疗策略变得至关重要。细菌细胞壁的主要成分 肽聚糖层是一种独特的网状结构，提供必要的结构支持；因此， 找出削弱这一层的方法是一种理想的抗生素策略。目前，许多治疗方法都针对 肽聚糖合成途径及其应用在医学上非常成功。酶 除膜成分外，参与该途径的已被广泛表征。 最值得注意的是 MraY 和 MurG，它们是催化肽聚糖膜步骤的必需蛋白质 生物合成。有一些已知的 MraY 抑制剂，例如衣霉素，证明了其作为治疗药物的潜力。 抗生素靶标；然而，它们都没有在临床上发挥作用。我们集团已开发出高效 两种最有前途的天然产物Capuramycin和muraymycin的全合成方案，以及 在上一个资助期，我们利用这一点来创造具有改善治疗潜力的新型化合物。 在这个提案中，我们描述了我们的计划，即使用我们的功能性MraY同系物来解析脂质中的结构 通过 EM 和 X 射线晶体学分析具有各种抑制剂和底物类似物的环境。我们有 开发了一种新的 MurG 检测方法，使我们能够鉴定新型抑制剂。我们将进一步筛选更多 化合物并用 MurG 解析其结构。我们将进一步探讨 MurG 与脂质的相互作用 双层和MraY。我们的新型抑制剂 APPB 和 CPPB 对细菌病原体具有广泛的功效 显示出作为抗癌疗法的潜力。我们将利用结构工作来设计下一轮 化合物库。有效性的广度促使我们追求其他磷酸转移酶的结构， 细菌 WecA 和人类 DPAGT1，与我们的化合物复合。这将允许更有针对性的小 分子发育。目的是 1) 对 MraY 和 抑制剂的开发，2) 进行 MurG 的机制和结构研究，3) 开发新颖和 改进的磷酸转移酶抑制剂。我们的结构生物学家和合成化学家联合团队 提供了一种创新方法来实现这些重要目标。

Project Summary Title: Mechanistic details of key integral-membrane enzymes for antimicrobial discovery The increasing number of antibiotic resistant strains of bacteria represents a significant threat to human health, making the development of novel therapeutic strategies critical. The major component of the bacterial cell wall is the peptidoglycan layer that is a unique meshwork providing essential structural support; therefore, identifying ways to weaken this layer is an ideal antibiotic strategy. Currently, numerous therapeutics target the peptidoglycan synthesis pathway and their use has been extremely successful in medicine. The enzymes involved in the pathway have been extensively characterized except in the case of the membrane components. Most notable are MraY and MurG, essential proteins that catalyze the membrane steps of peptidoglycan biosynthesis. There are a few known inhibitors of MraY, such as tunicamycin, demonstrating its potential as an antibiotic target; however, none of them has found usefulness in the clinic. Our group has developed efficient total synthesis schemes for two of the most promising natural products, capuramycin and muraymycin, and in the last funding period we have leveraged this to create novel compounds with improved therapeutic potential. In this proposal, we describe our plans to use our functional MraY homologs to solve structures in a lipid environment with various inhibitors and substrate analogs by EM and X-ray crystallography. We have developed a new assay for MurG that allowed us to identify novel inhibitors. We will further screen additional compounds and solve their structures with MurG. We will further explore the MurG interaction with the lipid bilayer and MraY. Our novel inhibitors, APPB and CPPB, have broad efficacy against bacterial pathogens and show potential as anti-cancer therapeutics. We will leverage the structural work to design the next round of compound libraries. The breadth of effectiveness leads us to pursue structures of other phosphotransferases, bacterial WecA and human DPAGT1, in complex with our compounds. This will allow for more targeted small molecule development. The aims are to 1) perform structural and mechanistic studies of MraY and the development of inhibitors, 2) carry out mechanistic and structural studies of MurG, and 3) develop novel and improved phosphotransferase inhibitors. Our combined team of structural biologists and synthetic chemists provides an innovative approach to achieve these important goals.