Mechanistic details of key integral membrane enzymes for antimicrobial discovery

项目摘要 标题：用于抗菌剂发现的关键整合膜酶的机制细节 越来越多的抗生素耐药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 是肽聚糖层，其是提供基本结构支撑的独特网络;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，许多疗法靶向于 肽聚糖合成途径及其在医学中的应用已经非常成功。的酶 除了膜组分的情况外，参与该途径的其他组分已经被广泛表征。 最值得注意的是MraY和MurG，它们是催化肽聚糖的膜步骤的必需蛋白质 生物合成有几种已知的MraY抑制剂，如衣霉素，证明了其作为抗肿瘤药物的潜力。 抗生素靶点;然而，它们中没有一个在临床上有用。我们的团队开发了高效的 两种最有前途的天然产物卡普霉素和穆雷霉素的全合成方案， 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。 在这个建议中，我们描述了我们的计划，使用我们的功能性MraY同源物来解决脂质结构 环境与各种抑制剂和底物类似物的EM和X射线晶体学。我们有 开发了一种新的MurG检测方法，使我们能够识别新的抑制剂。我们将进一步筛选额外的 化合物，并解决他们的结构与MurG。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疗效， 显示出作为抗癌治疗剂潜力。我们将利用结构工作来设计下一轮的 复合图书馆有效性的广度使我们追求其他磷酸转移酶的结构， 细菌WecA和人DPAGT 1，与我们的化合物复合。这将有助于更有针对性的小型 分子发展目的是1）进行MraY和MraY的结构和机制研究， 开发抑制剂，2）进行MurG的机制和结构研究，3）开发新的和 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 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.