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）是一种传染病导致死亡的主要原因——150万人