Chemical genetics of M. tuberculosis DosRST signaling and persistence

结核分枝杆菌 DosRST 信号传导和持久性的化学遗传学

• 批准号: 10470823
• 负责人: Robert B Abramovitch
• 金额: $ 73.04万
• 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: 2020-09-22 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10470823
• 关键词: Air; American; Amino Acids; Animal Model; Anti-Inflammatory Agents; Antibiotics; Bacteria; Biochemical; Biological; CRISPR interference; Carbon Monoxide; Cause of Death; Cavia; Cells; Cessation of life; Chemicals; Communicable Diseases; Data; Development; Drug Tolerance; Drug resistance; Evolution; Genes; Genetic Screening; Genetic study; Goals; Granuloma; HIV Infections; Health; Heme Group; Hypoxia; Immune; Immune system; Immunity; In Vitro; Infection; Libraries; Mission; Multidrug-Resistant Tuberculosis; Mus; Mycobacterium tuberculosis; National Institute of Allergy and Infectious Disease; Nitric Oxide; Oryctolagus cuniculus; Pharmaceutical Preparations; Phenotype; Phosphotransferases; Physiology; Population; Predisposition; Regimen; Relapse; Reporter; Resistance; Signal Transduction; Structure-Activity Relationship; System; Triglycerides; Tuberculosis; Virulence; aging population; base; chemical genetics; disease transmission; genetic approach; genetic selection; global health; heme a; in vivo; inhibitor; innovation; mutant; non-compliance; non-tuberculosis mycobacteria; nonhuman primate; pressure; resistant strain; response; sensor; side effect; small molecule; tool; tuberculosis drugs; tuberculosis treatment

Mycobacterium tuberculosis (Mtb) is the leading cause of death by an infectious disease — 1.5 million deaths and 10 million new active TB cases each year. A major reason the situation is not improving is that TB treatment is lengthy and challenging, requiring 6 months or more of multiple antibiotics with serious side effects. This regimen causes widespread non-compliance leading to relapse and promoting the evolution of multidrug-resistant TB (MDR-TB). Mtb is remarkably successful, in part, due to its ability to become dormant in response to host immune pressures. Mtb has a two-component regulatory system (TCS), DosRST, that when induced by hypoxia, nitric oxide (NO) or carbon monoxide (CO) remodels Mtb physiology to promote non-replicating persistence (NRP). NRP bacteria are thought to drive the long course of TB treatment. Therefore, we hypothesize that inhibitors of DosRST-dependent adaptation will reduce survival of drug-tolerant NRP Mtb and could function to shorten the course of therapy. By an innovative, reporter-based whole-cell phenotypic screen of a >540,000 compound library, we have discovered four new inhibitors that inhibit DosRST signaling by directly targeting the DosS and DosT sensor kinases. These first-in-class chemical probes, HC101, HC102, HC103 and HC106, represent a new strategy to inhibit Mtb persistence. Under hypoxia, all four compounds inhibit Mtb NRP-associated physiologies, including triacylglycerol synthesis and survival. Mechanism of action studies show they directly inhibit DosS and DosT kinases, but by distinct mechanisms; HC101 and HC106 directly target a heme group embedded in the kinases, while HC102 and HC103 inhibit sensor kinase autophosphorylation. A critical barrier to studying TCS is the lack of chemical probes that function against bacteria in whole cells. The goal of this proposal is to use these chemical probes as new tools to dissect the biochemical mechanisms of DosS/T sensor kinase function and the impact of conditional sensor kinase inhibition on Mtb physiology. Aim 1 will use biochemical and structure-activity relationship (SAR) studies to define mechanisms of action of the probes. In Aim 2, genetic approaches will be used to identify amino acid residues associated with resistance to the compounds and required for kinase function. Aim 3 will use CRISPR interference (CRISPRi), combined with treatment with the chemical probes, to define the biological impact of conditional DosRST inhibition both in vitro and during infection. This R01 will define new mechanisms for TCS function and generate proof-of- concept data validating DosRST as a target for the development of new TB drugs. OVERALL IMPACT: These studies will surmount obstacles that have long stymied TB therapy by focusing small molecule development on new targets and bringing critically needed understanding of TCS function in vitro and during infection.

结核分枝杆菌 (Mtb) 是导致 150 万人死亡的传染病的主要原因 每年新增 1000 万活动性结核病病例，情况没有改善的一个主要原因是结核病。 治疗时间长且具有挑战性，需要 6 个月或更长时间使用多种抗生素，且副作用严重 这种治疗方案会导致广泛的不依从性，从而导致复发并促进疾病的发展。 耐多药结核病（MDR-TB）。 Mtb 非常成功，部分原因在于它能够响应宿主免疫而进入休眠状态 结核分枝杆菌有一个两部分调节系统（TCS），即 DosRST，当缺氧、硝酸盐诱导时，该系统会产生压力。 一氧化氮 (NO) 或一氧化碳 (CO) 重塑 Mtb 生理机能，促进非复制持久性 (NRP)。 NRP 细菌被认为能够驱动结核病的长期​​治疗，因此，我们捕获了该抑制剂。 DosRST 依赖性适应会降低耐药 NRP Mtb 的存活率，并可能缩短 通过基于报告基因的创新全细胞表型筛选超过 540,000 种化合物。 在库中，我们发现了四种新的抑制剂，它们通过直接靶向 DosS 来抑制 DosRST 信号传导 这些一流的化学探针 HC101、HC102、HC103 和 HC106 代表了 DosT 传感器激酶。 抑制 Mtb 持久性的新策略 在缺氧条件下，所有四种抑制 Mtb NRP 相关的化合物。 生理学，包括三酰甘油合成和生存的作用机制研究表明它们直接。 抑制 DosS 和 DosT 激酶，但通过不同的机制 HC101 和 HC106 直接靶向血红素基团； 嵌入激酶中，而 HC102 和 HC103 抑制传感器激酶自磷酸化。 研究 TCS 的一个关键障碍是缺乏针对全细胞细菌的化学探针。 该提案的目标是使用这些化学探针作为剖析生化机制的新工具 DosS/T 传感器激酶功能的研究以及条件传感器激酶抑制对 Mtb 生理学的影响。 1 将使用生化和结构-活性关系（SAR）研究来定义作用机制 在目标 2 中，将使用遗传方法来鉴定与抗性相关的氨基酸残基。 Aim 3 将使用 CRISPR 干扰 (CRISPRi) 来组合化合物和激酶功能所需的化合物。 通过化学探针处理，以确定条件 DosRST 抑制的生物学影响 该 R01 将定义 TCS 功能的新机制并生成证据。 概念数据验证了 DosRST 作为开发新结核病药物的目标。 总体影响：这些研究将通过重点关注克服长期阻碍结核病治疗的障碍 新靶标的小分子开发，并带来对 TCS 功能的迫切需要的理解 体外和感染期间。