HTS Screen of TB RmlC & RmlD dTDP-Rhamnose Formation Enzymes

TB RmlC 的 HTS 筛查

• 批准号: 7363783
• 负责人: Michael R McNeil
• 金额: $ 2.5万
• 依托单位: COLORADO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7363783
• 关键词: Active Sites; Bacteria; Binding; Biological Assay; Cell Wall; Chemicals; Drug Delivery Systems; Drug Interactions; Drug Kinetics; Enzyme Inhibitor Drugs; Enzyme Inhibitors; Enzymes; Equilibrium; Growth; HIV Infections; High Pressure Liquid Chromatography; Human; Infection; Inhibitory Concentration 50; Lead; Modification; Mus; Mycobacterium tuberculosis; NADP; Oxidoreductase; Pathway interactions; Pharmaceutical Preparations; Prevalence; Proteins; Purpose; Rate; Research; Resistance; Rhamnose; Roentgen Rays; Serum; Source; Structure; Testing; Time; Toxic effect; Tuberculosis; Vero Cells; Work; base; cell preparation; dTDP-4-dehydrorhamnose 3,5-epimerase; dTDP-6-deoxy-D-xylo-4-hexulose; drug development; inhibitor/antagonist; mouse model; oxidation; programs; tuberculosis drugs

DESCRIPTION (provided by applicant): New drugs are needed against tuberculosis (TB) for three major reasons. The first reason is because the rate of cure with the present drugs is very slow. Secondly, increasing co-infection of HIV and Mycobacterium tuberculosis is occurring and treatment with present drugs results in harmful HIV/TB drug interactions. Thirdly, is the increasing prevalence of M. tuberculosis resistant to the present drugs. In a TB drug development program, we are targeting the formation of the cell wall of M. tuberculosis, a proven drug target. dTDP-rhamnose is a required biosynthetic precursor for TB cell wall formation. dTDP-rhamnose is not found in humans. Two of the enzymes which act sequentially for its formation are dTDP-6-deoxy-D-xylo-4-hexulose 3,5-epimerase (RmlC) and dTDP-6-deoxy-L- lyxo-4-hexulose reductase (RmlD). These enzymes have been shown to be essential for the growth of M. smegmatis and M. tuberculosis. The crystal structures of both have been obtained for proteins from non-TB bacterial sources and RmlC has been determined for the M. tuberculosis protein. Moreover TB RmlC has been obtained with the substrate mimic, dTDP-rhamnose, bound in the active site. Both RmlC and RmlD have been over-expressed in active form and a microtiter plate based assay based on the conversion of dTDP-6-deoxy-D-xylo-4-hexulose to dTDP-rhamnose with the concomitant oxidation of NADPH to NADP has been developed. The enzymes are balanced so that an inhibitor of either enzyme will be detected. The assay has been shown to be robust and reproducible. Herein we request that this assay be used to screen for inhibitors of RmlC and/or RmlD by the MLSCN and the resulting hits be optimized in concert with our lab and with our X-ray crystallographer and medicinal chemist collaborators. The purpose of this project is ultimately to develop new drugs against tuberculosis. These drugs are badly needed because of resistant strains of tuberculosis, because the treatment time needs to be decreased, and because co-infection of TB and HIV-AIDS is difficult to treat. The immediate purpose of the project is to find compounds that have the potential to be developed into new drugs because they inhibit enzymes required for the formation of the cell wall of the tuberculosis bacterium.

描述（由申请人提供）：由于三个主要原因，需要针对结核病（TB）的新药。第一个原因是因为目前的药物治愈率非常慢。其次，艾滋病毒和结核分枝杆菌的合并感染正在增加，用现有药物治疗会导致有害的艾滋病毒/结核药物相互作用。第三，是M.结核病对目前的药物有抗药性。在结核病药物开发项目中，我们的目标是M.结核病，一个被证实的药物靶点。dTDP-鼠李糖是TB细胞壁形成所需的生物合成前体。dTDP-鼠李糖不存在于人体中。对于其形成顺序起作用的两种酶是dTDP-6-脱氧-D-木-4-己酮糖3，5-差向异构酶（RmlC）和dTDP-6-脱氧-L-来苏-4-己酮糖还原酶（RmlD）。这些酶对M.耻垢分枝杆菌和M.结核已经获得了来自非TB细菌来源的蛋白质的晶体结构，并且已经确定了M.结核蛋白此外，TB RmlC已获得与底物模拟物，dTDP-鼠李糖，结合在活性位点。RmlC和RmlD都以活性形式过表达，并且已经开发了基于dTDP-6-脱氧-D-木-4-己酮糖转化为dTDP-鼠李糖并伴随NADPH氧化为NADP的基于微量滴定板的测定。酶是平衡的，以便检测到任一酶的抑制剂。已证明该试验具有耐用性和重现性。在此，我们要求将该测定用于通过MLSCN筛选RmlC和/或RmlD的抑制剂，并与我们的实验室以及我们的X射线晶体学家和药物化学家合作者一起优化所得命中。该项目的最终目的是开发治疗结核病的新药。由于结核病耐药菌株的存在、治疗时间的缩短以及结核病和艾滋病毒/艾滋病的合并感染难以治疗，迫切需要这些药物。该项目的直接目的是寻找具有开发新药潜力的化合物，因为它们抑制结核菌细胞壁形成所需的酶。