High-Throughput Identification of RNA polymerase inhibitors in vivo

描述(申请人提供)：细菌RNA聚合酶既是基因调控机制研究的中心目标，也是新抗生素开发的极具吸引力的目标。通过识别和使用新的RNA聚合酶抑制剂，机制研究和抗生素开发都将成为可能。由于RNA聚合酶的表面结构和调节特性以及对特定抗生素的需求在不同的细菌谱系中是不同的，因此发现谱系特异的抑制剂是一个特别吸引人的目标。这些谱系特异的抑制剂将为研究RNA聚合酶的生物学功能提供强大的新分子探针，并为补充抗生素开发管道提供有价值的先导化合物。最近的研究将抑制剂-RNA聚合酶共晶结构与抑制剂对机制步骤的影响联系起来，充分证明了抑制剂在剖析RNA聚合酶复杂的酶机制中的作用。新的抑制剂，特别是影响容易结晶的水热菌RNA聚合酶的抑制剂，将对这些研究特别有价值。对新抗生素的需求是显而易见的。美国和世界现在都面临着抗生素开发的危机。医院已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传播，伊拉克战争的退伍军人带着无法治愈的鲍曼不动杆菌感染归来。RNA聚合酶是一种已被证实的药物靶点，必须在寻找新的抗生素时充分利用。谱系特异的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细菌，使用荧光信号原位检测重组细菌RNA聚合酶的活性。生物报告试验已经在发挥作用，非常适合高通量筛选，并且具有识别能够进入细菌细胞并在细菌细胞内发挥作用的抑制剂的重要优势。其具体目标是：(1)建立炭疽杆菌和水生芽孢杆菌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分析的健壮和可重复性；(3)使用密歇根大学麦迪逊分校提供的化合物文库进行初始筛选，然后将分析提供给NIH资助的高通量筛选设施；以及(4)将高通量分析扩展到抗生素开发的其他高优先级目标。

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.