Discovery and validation of drug targets in vulnerable pathways of Mtb

描述（由申请人提供）：耐药结核分枝杆菌（Mtb）是对全球健康日益严重的威胁。一些创新的遗传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，这些发现并没有产生新的有效的药物靶点和靶点抑制剂。同样，最近的研究已经利用全细胞筛选确定了数百种对结核分枝杆菌有活性的新打击；然而，从这项工作中产生的有希望的候选药物很少。问题是如何在可行的药物靶点和已知的全细胞活性化合物之间找到良好的匹配。越来越明显的是，并非所有必需的代谢过程都代表良好的药物靶点。然而，多年的药物开发努力已经建立了细胞壁生物合成，蛋白质合成和DNA超卷曲作为关键的细胞过程，它们确实含有抗菌药物的优秀靶点。事实上，这些途径的一些抑制剂在临床中被用作抗b药物。呼吸抑制包括结核分枝杆菌的第四个可药物途径，如新的呼吸抑制剂贝达喹啉所证明的那样。我们建议发现和开发针对这些可药物过程的抑制剂。我们已经开发出一种广泛检测细胞壁生物合成抑制剂的筛选方法，并证明了其在特异性识别抑制细胞壁的新型化合物方面的有效性。我们的筛选/发现方法也可以用于识别对其他可药物细胞过程特异性的抑制剂。在这里,我们

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.