Metabolomics approaches to TB drug development

描述（由申请人提供）：抗生素开发是未满足医疗需求的主要领域。结核病是世界范围内导致死亡的主要细菌原因，这方面的需求最大。控制结核病大流行的一个主要障碍是缺乏对其病原体结核分枝杆菌（Mtb）的复制型和非复制型都有效的药物。因此，了解针对复制性和非复制性结核分枝杆菌的新靶点是一个尚未满足科学需求的主要领域。一类对复制型和非复制型结核分枝杆菌都有活性的化合物是活性氮中间体（RNI），但研究不足。在体外，RNI杀死结核分枝杆菌的摩尔效力大于许多目前使用的结核病药物。因此，对RNI抗细菌机制的了解为复制和非复制结核分枝杆菌的潜在靶标提供了一个概念性蓝图。在之前的工作中，我们确定了Mtb中rni介导的损伤的蛋白质组靶点。这项工作的动机是，RNI介导的损伤靶点代表了潜在的药物靶点，这些靶点被与RNI无关的化合物抑制，将模仿RNI的杀灭分枝杆菌活性，并抵抗Mtb的RNI防御。令人惊讶的是，这项工作选择性地确定了结核分枝杆菌中间代谢酶作为一类潜在的药物靶点，而不是现有结核病药物的靶点。在这里，我们提出应用一种新的基于质谱的代谢组学工具，这不仅有助于确定RNI对复制和非复制结核分枝杆菌的抗细菌作用，而且更广泛地扩展了在生物化学全球代谢物水平上对结核分枝杆菌代谢网络的了解。为了做到这一点，我们将通过无偏倚的rni介导的Mtb代谢组杀伤的比较来推断药物化合物的MOA或靶标（类似于基因组学中已经证明的“纲要”方法）。作为概念验证，我们提出了初步数据，确认了代谢物分析（使用结核分枝杆菌的游离氨基酸池）的概念优点，以及结核分枝杆菌全球代谢物分析的技术可行性。我们特别寻求确定RNI的“通用”代谢组学特征，以解释它们对复制和非复制结核杆菌的精美效力。这些知识将为可能增强或协同天然免疫的新药物靶点提供新的视角。从更广泛的角度来看，这项工作不仅将推动在复制型和非复制型结核分枝杆菌中发现新的潜在药物靶点的努力，而且还将为未来研究具有不明MOA的抗结核化合物奠定基础。公共卫生相关性：抗生素开发是未满足医疗需求的主要领域。结核病是世界范围内导致死亡的主要细菌原因，这方面的需求最大。控制结核病大流行的一个主要障碍是缺乏对复制型和非复制型结核分枝杆菌（Mtb）都有效的药物，结核分枝杆菌是结核病的病原体。因此，了解针对可复制性和不可复制性结核分枝杆菌的新靶点，解决了当前大流行病尚未满足的一项重大科学需求。

DESCRIPTION (provided by applicant): Antibiotic development is a major area of unmet medical need. Nowhere is this need greater than in the case of tuberculosis (TB), the leading bacterial cause of deaths worldwide. A major roadblock to control of the TB pandemic is the lack of drugs active against both replicating and non-replicating forms of its causative agent, Mycobacterium tuberculosis (Mtb). Knowledge of new targets against both replicating and non-replicating Mtb is thus a major area of unmet scientific need. One potent, but understudied, class of compounds active against both replicating and non-replicating Mtb is the reactive nitrogen intermediates (RNI). In vitro, RNI kill Mtb with a molar potency greater than that of many currently used TB drugs. Knowledge of the antimycobacterial mechanism(s) of RNI thus represents a conceptual blueprint to potential targets in replicating and non-replicating Mtb alike. In previous work, we identified proteomic targets of RNI-mediated damage in Mtb. This work was motivated by the view that targets of RNI-mediated damage represented potential drug targets whose inhibition by compounds unrelated to RNI would mimic RNI's mycobactericidal activity and resist Mtb's RNI defenses. Surprisingly, this work selectively identified enzymes of Mtb's intermediary metabolism as a major class of potential drug targets, not targeted by existing TB drugs. Here, we propose to apply a novel mass spectrometry-based metabolomics tool that will not only help define the antimycobacterial actions of RNI against both replicating and non-replicating Mtb but more broadly expand knowledge of Mtb's metabolic network at the biochemically global level of metabolites. To do so, we will deduce the MOA or targets of pharmacologic compounds using unbiased, comparisons of RNI-mediated killing on the Mtb metabolome across and between a variety of settings (analogous to the already proven "compendium" approach used in genomics). As proof-of-concept validation, we present Preliminary Data that affirm the conceptual merit of metabolite profiling (using Mtb's free amino acid pools) as well as the technical feasibility of global metabolite profiling of Mtb. We specifically seek to identify a "universal" metabolomic signature of RNI that would explain their exquisite potency against replicating and non-replicating Mtb alike. Such knowledge would provide a fresh lens on new drug targets that might augment or synergize with natural immunity. From a broader point of view, this work will not only advance efforts to identify new potential drug targets in replicating and non- replicating Mtb but also lay the groundwork for future studies of antimycobacterial compounds with undefined MOA. PUBLIC HEALTH RELEVANCE: Antibiotic development is a major area of unmet medical need. Nowhere is this need greater than in the case of tuberculosis (TB), the leading bacterial cause of deaths worldwide. A major roadblock to control of the TB pandemic is the lack of drugs active against both replicating and non-replicating forms Mycobacterium tuberculosis (Mtb), the causative agent of TB. Knowledge of new targets against both replicating and non-replicating Mtb thus addresses a major unmet scientific need of the current pandemic.