Mechanistic details of key integral membrane enzymes for antimicrobial discovery

项目摘要 标题：发现抗微生物的关键整体膜酶的机制细节 越来越多的抗药性细菌菌株对人类健康构成了重大威胁， 这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 是作为提供基本结构支撑的独特网络的肽聚糖层；因此， 找出削弱这一层的方法是一个理想的抗生素策略。目前，许多治疗方法都是针对 肽聚糖的合成途径及其在医学上的应用已经非常成功。这些酶 除了膜组分的情况外，参与该途径的人已经被广泛地表征。 最值得注意的是mray和Murg，它们是催化肽聚糖的膜步骤的基本蛋白质。 生物合成。有一些已知的mray抑制剂，如衣霉素，显示了它作为一种 抗生素靶点；然而，它们都没有在临床上找到用处。我们的团队已经开发出高效的 两种最有前景的天然产物卡普拉霉素和穆拉霉素的全合成方案，以及在 在上一个资助期，我们利用这一点创造了具有更好治疗潜力的新化合物。 在这项提议中，我们描述了我们计划使用我们的功能mray同系物来解决脂质中的结构 用EM和X-射线结晶学研究了各种缓蚀剂和底物类似物在环境中的作用。我们有 为Murg开发了一种新的检测方法，使我们能够识别新的抑制剂。我们将进一步筛选更多的 化合物，并用Murg.我们将进一步探讨MURG与脂质的相互作用 双层的和灰色的。我们的新型抑制剂APPB和CPPB对细菌病原体和 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 复合库。有效性的广度引导我们追寻其他磷酸转移酶的结构， 细菌WECA和人DPAGT1，与我们的化合物形成复合体。这将允许更有针对性的小规模 分子发展。其目的是1)进行mray结构和力学研究。 缓蚀剂的开发，2)对Murg的机理和结构进行研究，3)开发新的和 改进的磷酸转移酶抑制剂。我们由结构生物学家和合成化学家组成的联合团队 为实现这些重要目标提供了一种创新方法。

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.