Mechanistic details of key integral-membrane enzymes for antimicrobial discovery

用于抗菌发现的关键整合膜酶的机制细节

• 批准号: 9324275
• 负责人: William M. Clemons
• 金额: $ 44.88万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9324275
• 关键词: Anabolism; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Architecture; Bacteria; Bacterial Infections; Biochemical; Biochemistry; Biological Assay; Biological Process; Cell Wall; Complex; Coupled; Crystallization; Cytolysis; Development; Drug Design; Drug Targeting; E protein; Encapsulated; Enzyme Tests; Enzymes; Escherichia coli; Evolution; Feedback; Generations; Genus Mycobacterium; Goals; Growth; Health; Human; Integral Membrane Protein; Investigational Drugs; Knowledge; Lead; Libraries; Life; Lipid Bilayers; Lipids; Medical; Medicine; Membrane; Methods; Modeling; Modernization; Molecular Target; Multienzyme Complexes; Natural Products; Nature; Nucleosides; Pathway interactions; Penicillin-Binding Proteins; Peptidoglycan; Peptidyltransferase; Pharmaceutical Chemistry; Pharmaceutical Preparations; Pharmacology; Program Development; Proteins; Reaction; Resistance; Resolution; Route; Sampling; Scheme; Series; Site-Directed Mutagenesis; Source; Structural Biologist; Structural Chemistry; Structure; Synthesis Chemistry; Testing; Viral Proteins; Work; analog; antimicrobial; base; biochemical tools; clinical development; design; drug development; drug discovery; enzyme biosynthesis; feeding; improved; in vitro activity; inhibitor/antagonist; innovation; insight; interest; multi-drug resistant pathogen; new therapeutic target; next generation; novel; novel therapeutics; pathogen; programs; protein E; resistant strain; small molecule inhibitor; small molecule libraries; structural biology; success; therapeutic target; translocase

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.

摘要/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，一种催化肽聚糖的第一个膜步骤的必需蛋白质 生物合成MraY是一种不可或缺的膜蛋白，它抵抗了机械的理解。有 一些已知的MraY抑制剂，如核苷类抗生素，证明其作为抗生素靶标的潜力; 然而，还没有一种被推进临床开发计划。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合成方案， 改进化合物的鉴定（例如UT-324）。在本提案中，我们描述了纯化的MraY样品 适用于抑制剂分子、酶底物模拟物、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，用于表征同系物和各种抑制剂。 我们扩展了我们的合成策略，以产生广泛靶向的化学文库，然后测试这些化合物的活性。 酶和细菌生长抑制活性。将这些努力结合在一个程序中， 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 MraY结构表征已经产生了一种有前途的共晶，展示了一种模型 在低分辨率下抑制复合物。这个应用程序描述了我们的目标，开发一个彻底的机械 这将使我们能够设计和识别新的抑制剂作为药物发现的先导化合物。 目的是1）扩展靶向小分子文库以鉴定新的MraY抑制剂，2）开发一种新的MraY抑制剂。 利用MraY的结构和生物化学研究，在各种功能上全面了解 相关国家。我们的结构生物学家和合成化学家的联合团队提供了一个创新的 为实现这些重要目标而努力。