Mechanisms of Mycobacterium Tuberculosis pH-driven Adaptation

 描述（由申请人提供）：与每年夺去100多万人生命的持续结核病（TB）流行作斗争是全球卫生的主要挑战之一。结核分枝杆菌（Mtb）依赖于环境线索，以适应其生理和促进其在感染期间的生存。结核分枝杆菌定植的两个重要宿主小生境--巨噬细胞和坏死性肉芽肿是酸性环境。pH依赖性适应是至关重要的结核病发病机制，并可能指向新的治疗目标，但pH重塑结核病的生理机制仍然知之甚少。在初步研究中，我们试图发现pH诱导的双组分调节剂PhoPR或Mtb在酸性pH下存活的选择性抑制剂，这在此之前是不可用的。使用一个创新的，全细胞表型筛选> 273，000化合物库，我们成功地确定了抑制PhoPR调节子的化学探针，包括碳酸酐酶（CA）抑制剂乙氧唑胺（ETZ）。用ETZ处理Mtb复制phoPR突变表型并完全抑制整个细胞中的Mtb CA活性，支持我们的新假设，即CA生理学调节PhoPR信号传导。我们还发现了化学探针，功能独立的PhoPR和选择性抑制结核分枝杆菌生长在酸性pH值。我们假设，许多这些探针的目标生理，只有在酸性pH值的增长是必不可少的。经过一系列的优先级测定，我们已经确定了4种新的化学探针用于机械分析和优化。这些都是创新的化学遗传工具，将定义新的途径所需的结核分枝杆菌生存在酸性pH值的指导下，我们已经发现的新的化学探针，本申请的统一目标是定义的功能和治疗潜力的化学探针，调节pH值驱动的适应通过PhoPR依赖（目标1）和PhoPR独立（目标2）的机制。这些基础和应用研究将定义pH驱动的发病机制的新机制，并生成支持新的结核病治疗方法开发的概念验证数据。我们提出的研究的基本原理是phoPR和pH驱动的适应对于Mtb发病机制至关重要;因此，我们预测研究影响这些途径的化学物质和基因将发现新的致病机制和药物靶点。因为我们已经发现了在整个结核分枝杆菌细胞中发挥作用的新型化合物，我们有能力将我们的发现迅速转化为新药开发候选物。具体目标是：1）定义控制PhoPR依赖性发病机制的新的调节机制，并确定该途径作为治疗靶点的适用性;以及2）鉴定在酸性pH下选择性抑制Mtb生长的化学探针靶向的途径，并定义它们在Mtb发病机制中的作用。总体影响：确定结核分枝杆菌pH驱动的发病机制的新机制，开发针对这些pH依赖性适应途径的化学探针，并评估结核病治疗的新方法。

 DESCRIPTION (provided by applicant): Combating the ongoing tuberculosis (TB) epidemic, that claims over 1 million lives per year, is one of the major challenges in global health. Mycobacterium tuberculosis (Mtb) relies on environmental cues to adapt its physiology and promote its survival during infection. Two important host niches colonized by Mtb, the macrophage (M∅) and necrotic granuloma, are acidic environments. pH-dependent adaptations are critical to Mtb pathogenesis and may point to new therapeutic targets, but the mechanisms by which pH remodels Mtb physiology remain poorly understood. In preliminary studies we sought to discover selective inhibitors of the pH-inducible two component regulator PhoPR or Mtb survival at acidic pH, which have heretofore been unavailable. Using an innovative, whole-cell phenotypic screen of a >273,000 compound library, we successfully identified chemical probes that inhibit the PhoPR regulon, including the carbonic anhydrase (CA) inhibitor ethoxzolamide (ETZ). Treatment of Mtb with ETZ copies phoPR mutant phenotypes and fully inhibits Mtb CA activity in whole cells, supporting our novel hypothesis that CA physiology modulates PhoPR signaling. We also discovered chemical probes that function independently of PhoPR and selectively inhibit Mtb growth at acidic pH. We hypothesize that many of these probes are targeting physiology that is only essential for growth at acidic pH. Following a series of prioritization assays, we have identified 4 novel chemical probes to use for mechanistic analyses and optimizations. These are innovative chemical genetic tools that will define new pathways required for Mtb survival at acidic pH. Guided by the novel chemical probes that we have discovered, the unifying goal of this application is to define the function and therapeutic potential of chemical probes that modulate pH-driven adaptation via PhoPR-dependent (Aim 1) and PhoPR-independent (Aim 2) mechanisms. These basic and applied studies will define new mechanisms of pH-driven pathogenesis and generate proof-of-concept data supporting the development of new TB therapeutics. The rationale for our proposed studies is that phoPR and pH-driven adaptation are essential for Mtb pathogenesis; hence we predict that studying chemicals and genes impacting these pathways will uncover new pathogenic mechanisms and drug targets. Because we have already discovered novel compounds that function in whole Mtb cells, we are well-positioned to rapidly translate our findings into new drug development candidates. The Specific Aims are to: 1) Define new regulatory mechanisms controlling PhoPR-dependent pathogenesis and determine the suitability of the pathway as a therapeutic target; and, 2) Identify pathways targeted by chemical probes that selectively inhibit Mtb growth at acidic pH, and define their roles in Mtb pathogenesis. Overall Impact: Define new mechanisms of Mtb pH-driven pathogenesis, develop chemical probes targeting these pH-dependent adaptation pathways, and evaluate novel approaches to TB therapy.