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: 10267727
• 负责人: Robert B Abramovitch
• 金额: $ 72.59万
• 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/10267727
• 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）是传染病的主要死亡原因 - 150万 每年死亡和1000万个新的活跃结核病病例。情况之所以没有改善的主要原因是结核病 治疗是漫长的挑战，需要6个月或更长时间的多种抗生素 效果。这种方案导致宽度不合规，从而释放并促进 多药耐药结核病（MDR-TB）。 MTB非常成功，部分原因是它能够响应宿主免疫而处于休眠状态 压力。 MTB具有两个组件调节系统（TCS），DOSRST，当时是由缺氧诱导的 氧化物（NO）或一氧化碳（CO）重塑MTB生理学，以促进非复制持久性（NRP）。 NRP细菌被认为可以推动结核病治疗的漫长过程。因此，我们假设 DOSR的依赖性适应将降低耐药NRP MTB的存活率，并可以缩短 治疗过程。通过创新的，基于记者的全细胞表型屏幕的> 540,000化合物 图书馆，我们发现了四个新的抑制剂，这些抑制剂通过直接针对Doss和 DOST传感器激酶。这些第一类化学问题HC101，HC102，HC103和HC106代表 抑制MTB持久性的新战略。在缺氧下，所有四种化合物都抑制了MTB NRP相关的 生理学，包括三酰基甘油合成和存活。行动研究机制直接显示 抑制DOSS和DOST激酶，但通过不同的机制； HC101和HC106直接针对血红素组 嵌入激酶中，而HC102和HC103抑制传感器激酶自磷酸化。 研究TCS的关键障碍是缺乏针对全细胞中细菌作用的化学问题。 该建议的目的是将这些化学问题用作新工具来剖析生化机制 DOSS/T传感器激酶功能以及条件传感器激酶抑制对MTB生理学的影响。目的 1将使用生化和结构活性关系（SAR）研究来定义 问题。在AIM 2中，遗传方法将用于鉴定氨基酸与抗性有关 激酶功能所需的化合物。 AIM 3将使用CRISPR干扰（CRISPRI），合并 使用化学问题处理，以定义有条件的DOSRST抑制的生物学影响 体外和感染期间。该R01将定义用于TCS功能的新机制，并生成证明 概念数据将DOSRST验证为开发新结核病药物的目标。 总体影响：这些研究将通过关注长期阻碍结核病治疗的障碍 在新靶标上开发小分子，并对TCS功能产生迫切需要理解 体外和感染期间。