Metabolome of non-replicating M. tuberculosis

描述(申请人提供)：现在普遍认为，需要新药才能使全球结核病控制成为现实。结核分枝杆菌基因组序列的完成为大大加快新药开发奠定了基础。虽然对结核转录组和蛋白质组的研究报道有限，但这些全球大分子分析仍然不能直接促进药物发现，除非基因/蛋白质功能已知或蛋白质晶体结构可用。阐明生物体中低分子化合物的全部组成--代谢组--正被公认为是全面了解任何病原体的补充，也可能是最容易处理的方法，包括理性药物发现所需的洞察力。这项R-21建议试图定义非复制持久性(NRP)阶段的结核杆菌的代谢组，这种生理状态被认为是结核病所需的长时间治疗的原因。该代谢物将通过高分辨色谱(CCC，LC)和光谱分析(MS，核磁共振)进行鉴定。最初的研究将利用卡介苗的高生物量来评估几种分离方案，以优化(次级)代谢物的拆分。然后，该方案将用于在NRP中定义结核分枝杆菌的代谢组。随后的实验将集中于了解在NRP下产生的独特代谢物的生长阶段特异性。获得的信息不仅将补充有关细胞壁化学的广泛知识，而且有望：1)确定可能与最近为其他分枝杆菌所描述的代谢相关的新的次生代谢物；2)在代谢向低氧环境的转变过程中帮助澄清事件和确定低分子量标志物，特别是考虑到在这种适应过程中上调的基因有70%是未知功能的；3)通过对哺乳动物细胞中未发现的结核病特异性化合物的类比，为药物开发提供线索；4)有助于理解新发现的抗结核药物的作用机制。这项研究的操作假设是，阐明结核分枝杆菌的代谢组将克服全球大分子分析在获得对休眠和药物发现的关键见解方面的固有限制。

DESCRIPTION (provided by applicant): It is now generally accepted that new drugs are needed to make global tuberculosis control a reality. The completion of the sequence of the M. tuberculosis genome has laid the foundation for greatly accelerating new drug discovery. While a limited number of studies of the TB transcriptome and proteome have been reported, these global macromolecular analyses are still not able to contribute directly to drug discovery except where gene/protein function are known or protein crystal structures are available. Elucidating the full complement of low molecular weight compounds in an organism -the metabolome - is becoming recognized as a complementary and perhaps the most tractable approach to a comprehensive understanding of any pathogen including the insight necessary for rational drug discovery. This R-21 proposal seeks to define the metabolome of the tubercle bacillus in the stage of non-replicating persistence (NRP), the physiological state considered to be responsible for the required long treatment duration for TB. The metabolome will be elucidated by high-resolution chromatography (CCC, LC) and -spectroscopy (MS, NMR). Initial studies will utilize a high biomass of BCG to evaluate several separation schemes in optimizing the resolution of (secondary) metabolites. This protocol will then be used to define the metabolome of M. tuberculosis in NRP. Subsequent experiments will focus on understanding the growth-phase specificity of unique metabolites produced under NRP. The information obtained will not only complement the extensive knowledge of the chemistry of the cell wall but is also expected to I) identify novel secondary metabolites possibly related to those recently described for other mycobacteria, 2) help to clarify events and identify low MW markers during the metabolic shift to a low oxygen environment especially considering that >70% of genes up-regulated during this adaptation are of unknown function, 3) provide leads for drug development through analoging around TB-specific compounds not known to occur in mammalian cells and 4) assist in the understanding of the mechanism of action of newly discovered anti-TB agents. The operating hypothesis for this study is that elucidating the metabolome of Mycobacterium tuberculosis will overcome limitations inherent in global macromolecular analyses with respect to gaining key insights into dormancy and drug discovery.