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）治疗需要至少6个月的治疗与四种药物：异烟肼，利福平，吡嗪酰胺，乙胺丁醇。然而，随着耐药结核分枝杆菌（Mtb）的进化和传播，目前可用的疗法已经不足以控制多药耐药（MDR）TB。因此，发现新的抗生素对于结核病的长期控制至关重要。我们的实验室已经进行了两个屏幕的抑制剂的Dossip双组分信号通路使用低氧诱导的GFP报告菌株。为了找到DosRST抑制剂，我们筛选了抑制报告GFP荧光（I类抑制剂）但不抑制生长的化合物，因为在筛选条件下生长不需要DosRST。第一次筛选是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），鉴定了3种经验证的多柔比星抑制剂。我们使用340，000个化合物NIH分子图书馆计划（MLP）集合重复筛选，并鉴定了3种新的抑制剂。值得注意的是，这两种筛选还鉴定了许多抑制Mtb生长的化合物，但不依赖于我们称为II类抑制剂的Dosplatin。这些化合物可能靶向结核分枝杆菌生长所必需的途径，并可能包括新的结核病治疗方法的线索。我们已经建立了一系列二级检测、化学信息学管道和突变体筛选平台，以确认、分类、优先排序和定义II类抑制剂的作用机制。该管道用于表征ICCB收集的II类抑制剂，然而，我们尚未表征MLP收集的1250个II类命中。本R 03的目标是确认、分类和优先考虑MLP集合中的Mtb生长抑制剂，并启动表征研究。这项工作将定义和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。NIH为我们提供了1250个樱桃挑选化合物，排列在384孔板中，适合于10个高通量二级检测。使用已建立的高通量试验，我们将确认表型特性（目标1A）的命中，包括：体外生长抑制以确认其活性;半数最大有效浓度;真核细胞毒性;以及巨噬细胞中Mtb生长的抑制。还将测试樱桃采摘物的机械特性（Aim 1B），包括使用已知抗性基因（包括mmpL 3、katG、folC、mmpR 5）中的一组突变体的抗性;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学管道的支持下，化合物将根据生物学和化学特性进行优先排序（目标1C）。将从100个优先命中的新鲜粉末中购买并确认测试的活性和与TB药物的协同相互作用（目标1D）。总体影响：该R 03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。