Characterizing new M. tuberculosis inhibitors discovered in the Molecular Libraries Small Molecule Repository

结核病的治疗需要四种药物至少6个月的治疗：异烟肼、利福平、吡津酰胺和乙胺丁醇。然而，随着耐药结核分枝杆菌(Mtb)的进化和传播，现有的治疗方法已不足以控制多药耐药(MDR)结核病。因此，新抗生素的发现对结核病的长期控制至关重要。我们的实验室已经使用低氧诱导的GFP报告菌株对DosRST双组分信号通路的抑制剂进行了两次筛选。为了寻找DosRST抑制剂，我们筛选了抑制报告GFP荧光的化合物(I类抑制剂)，但不抑制生长，因为在筛选条件下DosRST不是生长所必需的。第一次筛查来自哈佛医学院化学和细胞生物学研究所(ICCB)收集的(约220,000个小分子)，鉴定出3种已验证的DosRST抑制剂。我们使用340,000个化合物NIH分子图书馆计划(MLP)收集重复筛选，并确定了3个新的抑制剂。值得注意的是，两种筛查都发现了许多抑制结核分枝杆菌生长的化合物，但不依赖于DosRST，我们称之为II类抑制剂。这些化合物可能针对结核分枝杆菌生长所必需的途径，并可能包括新的结核病疗法的先导。我们已经建立了一系列二级检测、化学信息管道和突变筛选平台来确认、分类、优先排序和定义II类抑制剂的作用机制。这条流水线被用来表征ICCB收集的II类抑制剂，然而，我们还没有表征MLP收集的1250个II类命中。R03的目标是对MLP收集中的Mtb生长抑制药进行确认、分类和优先排序，并启动表征研究。这项工作将定义并优先考虑抑制已知和新的结核分枝杆菌药物靶点的新化学支架。美国国立卫生研究院为我们提供了1250种樱桃精选化合物，排列在384孔板上，适用于10种高通量二次分析。使用已建立的高通量试验，我们将确认HITS的表型特性(目标1A)，包括：体外生长抑制以确认其活性；半最大有效浓度；真核细胞毒性；以及抑制巨噬细胞中的结核分枝杆菌生长。这些精选的樱桃还将接受机械性能(目标1B)的测试，包括使用一组已知抗性基因(包括MmpL3、katG、folC、MmpR5)中的突变进行抗性测试；以及氧化还原和pH动态平衡的调节。在药物化学家和明确的化学信息学渠道的支持下，将根据生物和化学性质(目标1C)对化合物进行优先排序。将购买来自100个优先命中的新鲜粉末，并确认其用于测试的活动以及与结核病药物的协同作用(AIM 1D)。总体影响：R03项目将提供100种优先化合物的新集合，这些化合物适合作为新的结核病治疗药物继续进行临床前研究。

Tuberculosis (TB) therapy requires at least 6 months of treatment with four drugs: isoniazid, rifampin, pyrazinamide, and ethambutol. However, with the evolution and spread of drug resistant Mycobacterium tuberculosis (Mtb), currently available therapies have become inadequate to control multi-drug resistant (MDR) TB. Therefore, the discovery of new antibiotics is critical for the long-term control of TB. Our lab has conducted two screens for inhibitors of the DosRST two-component signaling pathway using a hypoxia-inducible GFP reporter strain. To find DosRST-inhibitors, we screened for compounds that inhibited reporter GFP fluorescence (Class I inhibitors) but that did not inhibit growth, because DosRST is not required for growth under the screening conditions. The first screen was of the Harvard Medical School Institute of Chemistry and Cell Biology (ICCB) collection (~220,000 small molecules), which identified 3 verified DosRST inhibitors. We repeated the screen using the 340,000 compound NIH Molecular Libraries Program (MLP) collection, and identified 3 new inhibitors. Notably, both screens also identified numerous compounds that inhibit Mtb growth, but independent of DosRST, which we refer to as Class II inhibitors. These compounds are likely targeting pathways essential for Mtb growth and may include leads for new TB therapeutics. We have established a series of secondary assays, chemoinformatic pipelines and mutant screening platforms to confirm, classify, prioritize and define the mechanism of action of the Class II inhibitors. This pipeline was used to characterize Class II inhibitors from ICCB collection, however, we have not yet characterized the 1250 Class II hits from the MLP collection. The goal of this R03 is to confirm, classify and prioritize Mtb growth inhibitors from the MLP collection and initiate characterization studies. This work will define and prioritize new chemical scaffolds that inhibit known and new Mtb drug targets. The NIH provided us with 1250 cherry-pick compounds, arrayed in 384-well plates, suitable for 10 high throughput secondary assays. Using established, high-throughput assays, we will confirm the hits for phenotypic properties (Aim 1A), including: growth inhibition in vitro to confirm their activity; half-maximal effective concentration; eukaryotic cytotoxicity; and inhibition of Mtb growth in macrophages. The cherry-picks will also be tested for mechanistic properties (Aim 1B), including for resistance using a panel mutants in known resistance genes including mmpL3, katG, folC, mmpR5; and modulation of redox and pH homeostasis. With the support of a medicinal chemist and a defined chemoinformatics pipeline, the compounds will be prioritized based on biological and chemical properties (Aim 1C). Fresh powders from 100 prioritized hits will be purchased and confirmed for the tested activities and for synergistic interactions with TB drugs (Aim 1D). OVERALL IMPACT: This R03 project will deliver a new collection of 100 prioritized compounds that are suitable for continued preclinical studies as new TB therapeutics.