HTS assays for the methylerythritol-4-phosphate pathway

描述(申请人提供)：这项建议是基于对甲基赤藓糖醇-4-磷酸(MEP)途径的研究，该途径合成异戊二烯基二磷酸(IPP)和二甲基烯丙基二磷酸(DMAPP)的通用前体，在该途径中，我们已经确定了结核分枝杆菌和其他细菌病原体的潜在药物靶点。这些靶标也存在于疟原虫和弓形虫物种中。最近，病原菌中参与MEP途径的酶作为新型抗微生物药物的靶点引起了极大的关注；MEP途径中的所有酶都被认为是新药的潜在靶点，因为MEP途径在革兰氏阳性和革兰氏阴性细菌中都是必不可少的，而且人类细胞不包含直系物。此外，抑制途径第二酶(IspC)的天然抗生素磷霉素也抑制革兰氏阴性细菌的生长，我们已经证明MEP途径第一酶(DXS)的抑制剂也抑制结核分枝杆菌的生长。然而，由于缺乏底物和适当的高温超导测试，很少有实验室能够详细研究这一主题。我们以前在这方面的工作是为了从细菌病原体中鉴定MEP途径的酶并为底物开发有效的合成方法；我们目前的目标是寻找和开发先前表征的酶的抑制剂，为开发新型抗菌化合物提供潜在的线索。 与公共卫生相关：这项提案的目标是开发高通量筛查，以确定结核分枝杆菌中甲基赤霉醇4-磷酸生物合成细菌的抑制剂，这可能会产生新的先导化合物，并最终产生治疗结核病的新药物。

DESCRIPTION (provided by applicant): This proposal is based on studies of the methylerythritol-4-phosphate (MEP) pathway that synthesizes the universal precursors of isoprenoid compounds, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), in which we have identified potential drug targets in Mycobacterium tuberculosis and other bacterial pathogens. These targets also exist in Plasmodium and Toxoplasma species. The enzymes involved in the MEP pathway in pathogenic bacteria have recently generated a great deal of attention as a source of targets for novel antimicrobials; all enzymes in the MEP pathway are thought to be potential targets for novel drugs as the MEP pathway has been demonstrated to be essential in both Gram-positive and Gram-negative bacteria and human cells do not contain orthologs. In addition, a natural antibiotic, fosmidomycin, which inhibits the second enzyme in the pathway (IspC) also inhibits the growth of Gram-negative bacteria and we have shown that inhibitors of the first enzyme in the MEP pathway (Dxs) also inhibits the growth of M. tuberculosis. However, few laboratories have been able to pursue this topic in detail due to a lack of substrates and appropriate HTS amenable assays. Our previous work in this area was aimed at characterizing MEP pathway enzymes from bacterial pathogens and developing efficient syntheses for the substrates; our present goal is to identify and develop inhibitors of previously characterized enzymes to provide potential leads for development of novel antimicrobial compounds. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to develop high throughput screens to identify inhibitors of the methylerythritol 4- phosphate biosynthetic bacteria in Mycobacterium tuberculosis, which will potentially generate new lead compounds and eventually new drugs for the treatment of tuberculosis.