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药物的治愈率非常慢。其次，增加了HIV和结核分枝杆菌的共同感染，目前的药物治疗会导致有害的HIV/TB药物相互作用。第三，是结核分枝杆菌对当前药物的耐药性的增加。在结核病药物开发计划中，我们针对结核分枝杆菌的细胞壁的形成，这是一个验证的药物靶标。 DTDP-rhamnose是TB细胞壁形成所需的生物合成前体。在人类中找不到DTDP-RHAMNOSE。依次作用的两种酶是DTDP-6-脱氧-D-xylo-4-己糖3,5- epimerase（RMLC）和DTDP-6-脱氧-L- lyxo-4- lyxo-4-羟基甲磺酸还原酶（RMLC）。这些酶已被证明对于史氏菌和结核分枝杆菌的生长至关重要。已经获得了来自非TB细菌来源的蛋白质的晶体结构，并且已经确定了结核分枝杆菌蛋白的RMLC。此外，已经使用粘结在活性位点的底物模拟物DTDP-Rhamnose获得了TB RMLC。基于DTDP-6-脱氧-D-Xylo-4-己糖的转化，RMLC和RMLD都以活性形式和基于微量盘板的测定过表达，并与NADPH同时氧化为NADP对NADP的氧化为DTDP-RHAMNOSE的转化。酶是平衡的，因此将检测到任何一种酶的抑制剂。该测定已被证明是稳健且可再现的。在此，我们要求MLSCN使用此测定法对RMLC和/或RMLD的抑制剂进行筛选，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的命中。该项目的目的最终是开发针对结核病的新药。由于需要减少治疗时间，并且由于结核病和HIV-AID的共同感染很难治疗，因此由于抗肺结核的抗菌菌株而迫切需要这些药物。该项目的直接目的是找到具有开发到新药的可能性的化合物，因为它们抑制了结核细菌细胞壁形成所需的酶。