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

描述(由申请人提供)：出于三个主要原因，需要新药来治疗结核病。第一个原因是目前的药物治愈率很低。其次，艾滋病毒和结核分枝杆菌的混合感染正在增加，用目前的药物治疗会导致有害的艾滋病毒/结核病药物相互作用。第三，对现有药物产生抗药性的结核分枝杆菌的流行率越来越高。在结核病药物开发计划中，我们的目标是结核分枝杆菌细胞壁的形成，这是一个已被证实的药物靶点。DTDP-鼠李糖是结核细胞壁形成所必需的生物合成前体。DTDP-鼠李糖在人类中没有发现。顺次作用的两种酶是dTDP-6-脱氧-D-木糖-4-己糖3，5-差向异构酶(RMLC)和dTDP-6-脱氧-L-Lyxo-4-己六糖还原酶(RMLD)。这些酶已被证明对污垢分枝杆菌和结核分枝杆菌的生长至关重要。对于非结核细菌来源的蛋白质，已经获得了两者的晶体结构，对于结核分枝杆菌蛋白质，已经确定了RmlC。此外，还得到了Tb RmlC，底物模拟物dTDP-鼠李糖结合在活性中心。RmlC和RmlD都以活性形式过表达，建立了一种基于dTDP-6-脱氧-D-木糖-4-己糖转化为dTDP-鼠李糖并伴随NADPH氧化为NADP的微量平板分析方法。这些酶是平衡的，这样就可以检测到任何一种酶的抑制物。该分析已被证明是可靠的和可重复性的。在此，我们要求MLSCN使用此方法筛选RmlC和/或RmlD的抑制剂，并与我们的实验室以及我们的X射线结晶学家和药物化学家合作优化结果。该项目的最终目的是开发治疗结核病的新药。由于结核病的耐药菌株，因为治疗时间需要减少，以及结核病和艾滋病毒/艾滋病的混合感染很难治疗，因此迫切需要这些药物。该项目的直接目的是寻找有潜力开发成新药的化合物，因为它们抑制了结核病细菌细胞壁形成所需的酶。

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.