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聚合酶既是基因调控机制研究的中心靶点，也是新抗生素开发的高度吸引人的靶点。通过鉴定和使用新的RNA聚合酶抑制剂，机制研究和抗生素开发将成为可能。由于RNA聚合酶的表面结构和调控特性以及对特定抗生素的需求在不同的细菌谱系中有所不同，因此发现谱系特异性抑制剂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聚合酶的生物学功能和有价值的先导化合物提供强有力的新分子探针，以补充抗生素的开发管道。抑制剂在分析RNA聚合酶复杂的酶促机制方面的效用已被最近的研究充分证明，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联系起来。新的抑制剂，特别是影响易结晶的水生热菌RNA聚合酶的抑制剂，将对这些研究特别有价值。对新型抗生素的需求是显而易见的。美国和全世界现在都面临着抗生素开发的危机。医院已经成为机会性病原体的滋生地，仅在美国每年就导致9万多人死亡。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蔓延，伊拉克战争退伍军人回国时感染了无法治愈的鲍曼不动杆菌。RNA聚合酶是一种已被证实的药物靶点，必须在寻求新的抗生素中充分利用。谱系特异性RNA聚合酶抑制剂是开发针对对人类健康构成最大威胁的细菌的新抗生素的优秀先导化合物。我们将开发一种新的高通量筛选谱系特异性RNA聚合酶抑制剂，使用专门的生物报告菌，利用荧光信号原位检测重组细菌RNA聚合酶的活性。生物报告者试验已经开始工作，非常适合于高通量筛选，并且具有识别可以进入细菌细胞并在细菌细胞内起作用的抑制剂的重要优势。具体目的是：(1)建立高通量筛选炭疽芽胞杆菌和水蛭RNA聚合酶抑制剂的方法；(2)利用已知的RNA聚合酶抑制剂验证高通量测定的稳健性和可重复性；(3)使用威斯康星大学麦迪逊分校提供的化合物文库进行初步筛选，然后向美国国立卫生研究院资助的高通量筛选设施提供检测；(4)将高通量分析扩展到抗生素开发的其他高优先目标。