Mechanistic details of key integral-membrane enzymes for antimicrobial discovery

摘要/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，一种催化肽聚糖第一步膜步骤的必不可少的蛋白质。 生物合成。Mray是一种完整的膜蛋白，它抵制机械论的理解。有一种 很少有已知的mray抑制剂，如核苷抗生素，显示其作为抗生素靶点的潜力； 然而，没有一种药物进入临床开发计划。我们的团队已经开发出高效的总 两种最有希望的天然产物，卡普拉霉素和穆拉霉素的合成方案，允许 改进化合物(如UT-324)的鉴定。在这个提案中，我们描述了mray的纯化样品 适用于抑制分子、酶底物模拟、病毒蛋白E或MURG的结构研究。 纯化的mray酶用于体外活性测定，以表征同系物和各种抑制剂。 我们扩展了我们的合成策略，以生成目标广泛的化学库，然后对它们进行测试 酶和细菌生长抑制活性。将这些努力结合到一个计划中，就会产生反馈 加强项目的结构和药物化学方面的循环。令人兴奋的是，我们目前的努力 已经产生了一种很有希望的共晶，它展示了一个模型 在低分辨率下抑制了络合物。这个应用程序描述了我们的目标是开发一种彻底的机械 这将使我们能够设计和识别作为药物发现先导化合物的新型抑制剂。 其目标是：1)扩展目标小分子文库，以确定新的mray抑制剂；2)开发一种 利用结构和生化研究全面理解mray在各种功能中的作用 相关州。我们由结构生物学家和合成化学家组成的联合团队提供了一种创新的 实现这些重要目标的方法。

Abstract/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 is MraY, an essential protein that catalyzes the first membrane step of peptidoglycan biosynthesis. MraY is an integral membrane protein that has resisted mechanistic understanding. There are a few known inhibitors of MraY, such as nucleoside antibiotics, demonstrating its potential as an antibiotic target; however, none has been advanced into clinical development programs. Our group has developed efficient total synthesis schemes for two of the most promising natural products, capuramycin and muraymycin, allowing for the identification of improved compounds (e.g. UT-324). In this proposal we describe purified samples of MraY suitable for structural studies with inhibitor molecules, enzymatic substrate mimics, the viral protein E, or MurG. Purified MraY enzymes are used in in vitro activity assays for characterizing homologs and various inhibitors. We expand our synthetic strategy to generate broadly targeted chemical libraries and then test these for enzyme and bacterial growth inhibitory activities. Combining these efforts in one program creates a feedback loop that strengthens structural and medicinal chemistry aspects of the projects. Excitingly, our current efforts toward MraY structural characterization have yielded a promising co-crystal that demonstrates a model of an inhibited complex at low resolution. This application describes our goal of developing a thorough mechanistic picture of MraY that will allow us to design and identify novel inhibitors as lead compounds for drug discovery. The aims are to 1) expand on targeted small molecule libraries to identify new MraY inhibitors and 2) develop a full mechanistic understanding using structural and biochemical studies of MraY in a variety of functionally relevant states. Our combined team of structural biologists and synthetic chemists provides an innovative approach to achieve these important goals.