Discovery and validation of drug targets in vulnerable pathways of Mtb

描述(申请人提供)：耐药结核分枝杆菌(结核分枝杆菌)对全球健康的威胁越来越大。许多创新的遗传学研究已经确定了对结核分枝杆菌生存至关重要的基因和途径；然而，这些发现几乎没有产生新的有效药物靶标和靶标抑制物。同样，最近的研究已经确定了数百种使用全细胞筛选的有效抗结核分枝杆菌的新药物；然而，这项工作产生的有前途的候选药物很少。问题一直是在可行的药物靶点和已知具有全细胞活性的化合物之间找到良好的匹配。越来越明显的是，并不是所有的必要代谢过程都代表着良好的药物靶点。然而，多年的药物开发努力已经确立了细胞壁生物合成、蛋白质合成和DNA超螺旋作为关键的细胞过程，这些过程确实包含了极好的抗菌药靶点。事实上，这些途径的一些抑制剂正在临床上用作抗B细胞药物。呼吸抑制是结核分枝杆菌的第四个可用药途径，新的呼吸抑制剂贝达奎兰证明了这一点。我们建议发现和开发针对这些可药物过程的抑制剂。我们已经开发了一种广泛检测细胞壁生物合成抑制剂的屏幕，并证明了它在专门识别具有抑制细胞壁的新类化合物方面的有效性。我们的筛选/发现方法也可以用来识别其他可药物细胞过程特有的抑制剂。在这里，我们 建议充分描述我们细胞壁抑制剂筛选中剩余的HITs，扩展我们的筛选方法，以发现蛋白质合成、DNA超螺旋和呼吸等可药物过程中的新抑制剂和新靶点，验证每个靶点，并将选定的HITS开发为优化的药物先导。对于R21阶段，我们将：1)鉴定和验证我们细胞壁生物合成抑制剂筛选中已经确定的有前景的新HIT化合物的靶标。2)使用我们新颖的全细胞启动子-报告筛选方法，发现新的热门化学类别，这些类别可以抑制MTB中高度脆弱的蛋白质合成、DNA超螺旋和呼吸过程。然后，将使用耐药突变的全基因组测序来确定每一次有希望的命中的细胞靶点。对于R33阶段，我们将从一系列目标中选择至少10个最活跃的目标，并：3)通过生化和遗传学研究验证每个目标的假定目标。然后，使用尖端的代谢组学分析，通过研究每一次HIT治疗时Mtb内的代谢结果，进一步研究每一次HIT的作用模式。5)最后，我们将对至少六个化合物进行Hit-to-Lead优化。

DESCRIPTION (provided by applicant): Drug-resistant Mycobacterium tuberculosis (Mtb) is an increasing threat to global health. A number of innovative genetic studies have identified genes and pathways that are essential to Mtb survival; however, these discoveries have produced few new validated drug targets and target-inhibitors. Similarly, recent studies have identified hundreds of novel hits active against Mtb using whole-cell screening; yet, very few promising drug-candidates have resulted from this work. The problem has been to find good matches between viable drug targets and compounds with known whole-cell activity. It is becoming increasingly apparent that not all essential metabolic processes represent good drug targets. However, years of drug development efforts have established cell wall biosynthesis, protein synthesis, and DNA supercoiling as critical cellular processes that do contain excellent targets for antibacterials. In fact, some inhibitors of these pathways are in clinical use as anti-B drugs. Inhibition of respiration comprises a fourth druggable pathway in Mtb, as demonstrated by the new respiration inhibitor bedaquiline. We propose to discover and develop inhibitors that target these druggable processes. We have already developed a screen that broadly detects cell wall biosynthesis inhibitors, and proved its effectiveness for specifically identifying new classes of compounds with that inhibit the cell wall. Our screening/discovery approach can also be adapted to identify inhibitors that are specific to other druggable cellular processes. Here, we propose to fully characterize the remaining hits from our cell wall inhibitor screen, expand our screening approach to uncover new inhibitors and novel targets in the druggable processes of protein synthesis, DNA supercoiling and respiration, validate each target, and develop selected hits into optimized drug leads. For the R21 phase we will: 1) Identify and validate the targets of promising new hit compounds already identified in our cell wall biosynthesis inhibitor screen. 2) Discover new hit chemical classes that inhibit the highly vulnerable processes of protein synthesis, DNA supercoiling and respiration in Mtb, using our novel whole cell promoter-reporter screening approach. The cellular targets of each promising hit will then be determined using whole-genome sequencing of resistant mutants. For the R33 phase we will select at least 10 of the most active hits across a range of targets and: 3) Validate the putative target of each hit wit biochemical and genetic studies. Then, further investigate the mode of action of each hit by studying the metabolic consequences within Mtb upon treatment with each hit, using a cutting edge metabolomic assay. 5) Finally, we will perform hit-to-lead optimization of at least six compounds.