Chemical genetics of M. tuberculosis DosRST signaling and persistence

结核分枝杆菌是一种传染病导致死亡的主要原因--150万 每年死亡人数和新增活动性结核病病例1000万例。情况没有改善的一个主要原因是结核病 治疗漫长且具有挑战性，需要6个月或更长时间的多种抗生素治疗，副作用严重。 效果。这种养生法会导致广泛的不依从，导致复发并促进 耐多药结核病(MDR-TB)。 结核分枝杆菌非常成功，部分原因是它能够对宿主免疫作出反应而进入休眠状态 压力。MTB有一个双组分调节系统(TCS)，DosRST，当被缺氧诱导时，一氧化氮 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)。 核糖核酸菌被认为推动了结核病的长疗程治疗。因此，我们假设药物的抑制剂 依赖DosRST的适应将降低耐药的NRP Mtb的存活率，并可能缩短 疗程。由创新的基于记者的全细胞表型筛选的540,000个化合物 文库，我们发现了四种新的抑制剂，通过直接靶向DOSS和DosRST来抑制DosRST信号转导 DOST感应器激活酶。这些一流的化学探针HC101、HC102、HC103和HC106代表着一种 抑制结核分枝杆菌持久性的新策略。在低氧条件下，所有四种化合物都抑制mtb nrp相关基因。 生理，包括三酰甘油的合成和存活。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直接针对一个血红素基团 而HC102和HC103则抑制感受器激酶的自磷酸化。 研究TCS的一个关键障碍是缺乏在整个细胞中对细菌起作用的化学探针。 这项提议的目标是使用这些化学探针作为新的工具来剖析生化机制。 DOSS/T感受器激酶功能的研究以及条件感受器激酶抑制对结核分枝杆菌生理学的影响。目标 1将使用生化和构效关系(SAR)研究来定义 探测器。在目标2中，将使用遗传方法来识别与抗药性有关的氨基酸残基。 这些化合物是激活剂功能所必需的。AIM 3将结合使用CRISPR干扰(CRISPRi) 通过化学探针的处理，确定条件性DosRST抑制在 在体外和感染期间。此R01将为TCS功能定义新的机制，并生成 概念数据证实DosRST是开发新的结核病药物的目标。 总体影响：这些研究将克服长期阻碍结核病治疗的障碍 小分子在新靶点上的开发和对TCS功能的迫切需要的理解 在体外和感染期间。

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.