Metabolome of non-replicating M. tuberculosis

描述（由申请人提供）：现在普遍认为，需要新的药物来实现全球结核病控制。完成了M.结核病基因组为大大加快新药发现奠定了基础。虽然已经报道了有限数量的TB转录组和蛋白质组的研究，但这些全球大分子分析仍然不能直接有助于药物发现，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量化合物的完整补充-代谢组-正被认为是全面了解任何病原体的补充，也许是最容易处理的方法，包括合理药物发现所需的洞察力。该R-21提案旨在定义处于非复制持久性（NRP）阶段的结核杆菌的代谢组，该生理状态被认为是结核病所需的长治疗时间的原因。将通过高分辨率色谱法（CCC、LC）和质谱法（MS、NMR）阐明代谢物组。最初的研究将利用高生物量的卡介苗，以评估优化（次级）代谢产物的分辨率的几种分离方案。该方案将用于确定M的代谢组。NRP中的结核病随后的实验将侧重于了解NRP下产生的独特代谢物的生长阶段特异性。所获得的信息不仅将补充细胞壁化学的广泛知识，而且还有望I）鉴定可能与最近描述的其他分枝杆菌的次级代谢物相关的新次级代谢物，2）在代谢向低氧环境转变的过程中，有助于澄清事件并鉴定低MW标记物，特别是考虑到>70%的基因在低氧环境中表达，在这种适应过程中调节的蛋白质具有未知的功能，3）通过模拟哺乳动物细胞中未知的TB特异性化合物为药物开发提供线索，以及4）帮助理解新发现的抗TB剂的作用机制。本研究的操作假设是，阐明结核分枝杆菌的代谢组将克服全球大分子分析中固有的局限性，从而获得对休眠和药物发现的关键见解。

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.