High-Throughput Identification of RNA polymerase inhibitors in vivo

体内 RNA 聚合酶抑制剂的高通量鉴定

• 批准号: 7169444
• 负责人: Robert Landick
• 金额: $ 18.38万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7169444
• 关键词: Bacillus anthracis; NIH Roadmap Initiative tag; RNA directed DNA polymerase; anthrax; anthrax toxin; anthrax vaccines; antibiotics; bacteria infection mechanism; bacterial genetics; bioterrorism /chemical warfare; chemical registry /resource; drug discovery /isolation; fluorescent dye /probe; high throughput technology; inhibitor /antagonist

DESCRIPTION (provided by applicant): Bacterial RNA polymerase is both a central target in mechanistic studies of gene regulation and a highly attractive target for new antibiotic development. Both mechanistic studies and antibiotic development will be enabled by identification and use of new RNA polymerase inhibitors. Because the surface structure and the regulatory properties of RNA polymerase as well as the need for specific antibiotics vary among different bacterial lineages, discovery of lineage-specific inhibitors is a particularly attractive goal. These lineage- specific inhibitors will provide powerful new molecular probes for investigating the biological function of RNA polymerase and valuable lead compounds to replenish the antibiotic development pipeline. The utility of inhibitors for dissecting the complex enzymatic mechanism of RNA polymerase is well documented by recent studies that correlate inhibitor-RNA polymerase co-crystal structures with effects of inhibitors on steps in the mechanism. New inhibitors, especially inhibitors that affect easily crystallized Thermus aquaticus RNA polymerase, will be especially valuable for these studies. The need for new antibiotics is clear. The United States and the world now confront a crisis in antibiotic development. Hospitals have become breeding grounds for opportunistic pathogens, leading to more than 90,000 deaths/yr in the US alone. Methicillin-resistant Staphylococcus aureus (MRSA) is already spreading rapidly outside hospitals in the US and globally, and veterans of the Iraq war are returning with untreatable infections of Acinetobacter baumannii. RNA polymerase is a proven drug target and must be fully exploited in the quest for new antibiotics. Lineage-specific RNA polymerase inhibitors are excellent lead compounds for development of new antibiotics targeting the bacteria that pose the greatest threats to human health. We will develop a novel high-throughput screen for lineage-specific RNA polymerase inhibitors using specialized bioreporter bacteria that detect the activity of recombinant bacterial RNA polymerases in situ using a fluorescent signal. The bioreporter assay already is working, is ideally adaptable for high-throughput screening, and has the important advantage of identifying inhibitors that can enter and work within a bacterial cell. The specific aims are to (1) establish a high-throughput screen of inhibitors of B. anthracis and T. aquaticus RNA polymerase; (2) validate robust and reproducible behavior of the high-throughput assay using known RNA polymerase inhibitors; (3) perform an initial screen using compound libraries available at UW Madison and then supply the assay to NIH-funded high-throughput screening facilities; and (4) extend the high-throughput assay to other high priority targets for antibiotic development.

描述（由申请人提供）：细菌RNA聚合酶既是基因调节的机理研究的核心目标，又是新型抗生素发育的极具吸引力的靶标。机械研究和抗生素发展都将通过鉴定和使用新的RNA聚合酶抑制剂来启用。因为RNA聚合酶的表面结构和调节性能以及对不同细菌谱系的特定抗生素的需求各不相同，因此发现谱系特异性抑制剂的发现是一个特别有吸引力的目标。这些谱系 - 特异性抑制剂将为研究RNA聚合酶和有价值的铅化合物的生物学功能提供强大的新分子探针，以补充抗生素发育管道。最近的研究很好地证明了抑制剂解剖RNA聚合酶的复杂酶促机制的实用性，这些研究将抑制剂-RNA聚合酶共晶结构与抑制剂对机制步骤的影响相关联。对于这些研究，新的抑制剂，尤其是影响易于结晶的热水生RNA聚合酶的抑制剂，对于这些研究特别有价值。对新抗生素的需求很明显。现在，美国和世界面临着抗生素发展的危机。医院已经成为机会性病原体的繁殖地，仅美国就会导致90,000多人死亡。耐甲氧西林金黄色葡萄球菌（MRSA）已经在美国和全球的医院外迅速传播，伊拉克战争的退伍军人正在以不可治疗的baumannii感染返回。 RNA聚合酶是一个经过验证的药物靶标，必须在寻求新的抗生素中充分利用。谱系特异性的RNA聚合酶抑制剂是开发针对细菌的新抗生素的极佳铅化合物，该细菌对人类健康构成最大威胁。我们将使用专门的生物孢子菌细菌开发一个新型的高通量筛选，用于谱系特异性RNA聚合酶抑制剂，该细菌使用荧光信号检测重组细菌RNA聚合酶的活性。 Biorporter分析已经在起作用，理想适用于高通量筛选，并且具有鉴定可以在细菌细胞中输入和工作的抑制剂的重要优势。具体的目的是（1）建立炭疽芽孢杆菌和t. apecaties t. apepaticus RNA聚合酶抑制剂的高通量筛选； （2）使用已知的RNA聚合酶抑制剂验证高通量测定的鲁棒和可重复行为； （3）使用UW Madison上可用的复合库执行初始屏幕，然后将测定法提供给NIH资助的高通量筛选设施； （4）将高通量测定法扩展到其他高优先级目标，以进行抗生素开发。