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: 10119676
• 负责人: Robert B Abramovitch
• 金额: $ 71.8万
• 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/10119676
• 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/antagonist; 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150万人死亡的主要传染病 死亡和1000万新的活动性结核病病例。情况没有改善的一个主要原因是结核病 治疗是漫长和具有挑战性的，需要6个月或更长时间的多种抗生素， 方面的影响.该方案引起广泛的不依从性，导致复发并促进 耐多药结核病。 结核分枝杆菌是非常成功的，部分原因是它能够进入休眠状态，以响应宿主的免疫反应。 压力结核分枝杆菌有一个双组分调节系统（TCS），Dossip，当缺氧诱导时， 氧化物（NO）或一氧化碳（CO）重塑Mtb生理机能以促进非复制持久性（NRP）。 NRP细菌被认为推动了结核病的长期治疗。因此，我们假设， DosRST依赖性适应将降低耐药NRP Mtb的存活率，并可能起到缩短耐药NRP Mtb的存活时间的作用。 治疗过程。通过一种创新的、基于荧光素酶的全细胞表型筛选， 文库中，我们发现了四种新的抑制剂，它们通过直接靶向DoS来抑制DoS信号传导， DOT传感器激酶。这些一流的化学探针，HC 101，HC 102，HC 103和HC 106，代表了 抑制结核分枝杆菌持久性的新策略。在缺氧条件下，所有四种化合物均抑制Mtb NRP相关的 生理学，包括三酰甘油的合成和生存。作用机制研究表明， 抑制DoS和DoS T激酶，但通过不同的机制; HC 101和HC 106直接靶向血红素基团 HC 102和HC 103抑制传感器激酶的自磷酸化。 研究TCS的一个关键障碍是缺乏针对全细胞中细菌的化学探针。 这项提议的目的是利用这些化学探针作为新的工具来剖析生物化学机制 的DoS/T传感器激酶功能和条件性传感器激酶抑制对Mtb生理的影响。目的 1将使用生物化学和结构-活性关系（SAR）研究来确定 probes.在目标2中，将使用遗传方法来鉴定与抗结核病相关的氨基酸残基。 激酶功能所需的化合物。目标3将使用CRISPR干扰（CRISPRi）， 用化学探针治疗，以确定条件性剂量抑制的生物学影响， 体外和感染期间。该R 01将定义TCS功能的新机制，并生成 概念数据验证Dosplatin作为新的结核病药物开发的目标。 总体影响：这些研究将克服长期阻碍结核病治疗的障碍， 新靶点的小分子开发，并将对TCS功能的迫切需要的理解引入 体外和感染期间。