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

表征分子库小分子存储库中发现的新型结核分枝杆菌抑制剂

• 批准号: 10029703
• 负责人: Robert B Abramovitch
• 金额: $ 7.45万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10029703
• 关键词: Air; American; Anti-Inflammatory Agents; Antibiotics; Biological; Biological Assay; Cellular biology; Cessation of life; Chemicals; Chemistry; Collection; Drug Targeting; Drug resistance; Drug resistance in tuberculosis; Ethambutol; Evolution; Fluorescence; Goals; Growth; Growth Inhibitors; HIV Infections; Health; Hypoxia; Immune system; Immunity; In Vitro; Institutes; Mission; Molecular Bank; Multidrug-Resistant Tuberculosis; Mycobacterium tuberculosis; National Institute of Allergy and Infectious Disease; Oxidation-Reduction; Pathway interactions; Pharmaceutical Preparations; Phenotype; Population; Powder dose form; Predisposition; Property; Pyrazinamide; Reporter; Resistance; Rifampin; Series; Signal Pathway; Testing; Therapeutic; Tuberculosis; United States National Institutes of Health; Work; aging population; base; chemical property; cytotoxicity; disease transmission; global health; high throughput screening; in vitro activity; inhibitor/antagonist; isoniazid; macrophage; medical schools; mutant; pH Homeostasis; preclinical study; programs; repository; resistance gene; resistant strain; scaffold; screening; small molecule; small molecule libraries; success; tuberculosis drugs; tuberculosis treatment

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.

结核病（TB）疗法需要至少6个月的四种药物治疗：异烟肼，利福平，吡嗪酰胺和乙酰胺醇。然而，随着耐药性分枝杆菌结核病（MTB）的进化和扩散，目前可用的疗法已经不足以控制多药耐药（MDR）TB。因此，新抗生素的发现对于TB的长期控制至关重要。我们的实验室已经使用缺氧诱导的GFP报告菌株进行了两个筛选，以用于DOSRST两个组分信号通路的抑制剂。为了找到DOSR的抑制剂，我们筛选了抑制报告基因GFP荧光（I类抑制剂）的化合物，但这并没有抑制生长，因为在筛查条件下并不需要DOSRST。第一个屏幕是哈佛医学院化学与细胞生物学研究所（ICCB）收集（约220,000个小分子），该分子鉴定出3个经过验证的DOSRST抑制剂。我们使用340,000种化合物NIH NIH分子文库计划（MLP）收集重复了屏幕，并确定了3种新的抑制剂。值得注意的是，两个筛选还确定了抑制MTB生长的许多化合物，但与DOSRST无关，我们称为II类抑制剂。这些化合物可能是针对MTB生长必不可少的途径，并且可能包括新的结核病疗法的铅。我们已经建立了一系列的次要测定，化学信息性管道和突变筛选平台，以确认，对，优先级和定义II类抑制剂的作用机理。该管道用于表征ICCB收集中II类抑制剂的表征，但是，我们尚未表征MLP集合中的1250级II级命中。该R03的目的是确认，对MLP收集的MTB增长抑制剂进行确认和优先级，并启动表征研究。这项工作将定义并确定抑制已知和新的MTB药物靶标的新的化学支架。 NIH为我们提供了1250个樱桃挑选化合物，在384孔板中排列，适用于10个高吞吐量辅助测定法。使用已建立的高通量测定法，我们将确认表型特性的命中（AIM 1A），包括：体外抑制生长以确认其活性；半最大的有效浓度；真核细胞毒性；并抑制巨噬细胞中MTB生长。樱桃小摘要还将测试机械性能（AIM 1B），包括使用已知抗性基因的面板突变体（包括MMPL3，KATG，KATG，FOLC，MMPR5）的耐药性；以及氧化还原和pH稳态的调节。在药物化学家和定义的化学信息疗法管道的支持下，这些化合物将根据生物学和化学性质（AIM 1C）优先考虑。将购买并确认来自100个优先命中的新鲜粉末，以进行测试活动以及与结核病药物的协同相互作用（AIM 1D）。总体影响：这个R03项目将提供新的100种优先级化合物的收藏，这些化合物适合作为新的结核病疗法持续研究。