Systems-based screen of compounds that target nitrogen metabolism of Mycobacterium tuberculosis.

结核分枝杆菌(Mtb)是结核病的病原体，目前是世界范围内最具破坏性的致死传染病，每年造成约800万结核病病例，导致100多万人死亡。与人类免疫缺陷病毒(HIV)的混合感染以及多重耐药和广泛耐药(MDR和XDR)结核菌株的出现，使结核分枝杆菌再次成为世界各地的主要公共卫生威胁。对结核分枝杆菌具有活性的现有药物数量有限，以及根除这种感染所需的长期多药方案，是结核病治疗的根本问题。迫切需要有效对抗结核分枝杆菌的新药。结核分枝杆菌的细胞内代谢是开发新型抗结核药物的一个有吸引力的靶点；然而，大多数研究都集中在碳代谢上。氮也是结核分枝杆菌的基本营养，但很少有研究试图阐明基本问题，如病原体在宿主内生长时氮源的性质。我们之前的研究已经确定，当结核杆菌在宿主细胞内生长时，主要氨基酸是结核杆菌的氮源。在这项研究中，我们的目标是推进这项研究，以检验氮代谢是结核病药物开发的一个未被探索和潜在有效的药物靶点的假设。我们将使用丝氨酸代谢作为这一假说的测试案例。在我们之前的研究中，我们证明了执行丝氨酸生物合成最后一步的酶SERC对于MTB的细胞内复制是必不可少的。这表明它对病原体在宿主内的生长也是必不可少的，因此是开发新药的良好靶点。为了证实这一假设，我们将首先在小鼠感染模型中测量结核分枝杆菌SERC突变体的毒力。下一步将是筛选化合物库‘库’，以识别任何目标SERC。传统的药物筛选识别对特定酶有活性的化合物，如SERC。然而，由于药物在宿主细胞中的渗透性差或失活，已鉴定的化合物往往无法在自然宿主中发挥作用。为了克服这个问题，我们建议筛选抗结核分枝杆菌整个活细胞的化合物。这是更困难的，因为Mtb会产生数百种不同的酶，每一种都可能被测试的化合物抑制。为了鉴定专门针对SERC的化合物，我们将搜索那些对野生型Mtb有活性的化合物，但不是补充丝氨酸的SERC突变菌株，这样它就不再需要SERC生长。然后，我们将把研究扩展到与氮代谢和从宿主细胞吸收氨基酸有关的其他关键基因。最后，我们将在MTB中开发一个氮和碳代谢的计算机模型，该模型可以用于设计针对氮代谢的药物组合，这些药物将有效地协同工作，并通过实验测试该模型的预测。

M. tuberculosis (Mtb), the etiological agent of TB, is presently the most devastating infectious agent of mortality worldwide, responsible for about 8 million cases of TB each year resulting in more than one million deaths. Co-infection with the human immunodeficiency virus (HIV), along with the emergence of multi- and extensively- drug resistant (MDR and XDR) strains of TB, has reaffirmed Mtb as a primary public health threat throughout the world. The limited number of drugs available that have activity against Mtb, and the prolonged multi-drug regimen needed to eradicate the infection, are the fundamental problems of TB treatment. New drugs active against Mtb are urgently needed. Intracellular metabolism of Mtb is an attractive target for development of novel anti-tuberculosis drugs; however most studies have focussed on carbon metabolism. Nitrogen is also an essential nutrient of Mtb but few studies have attempted to elucidate fundamental questions such as the nature of the nitrogen source of the pathogen when it grows inside the host. Our previous studies have identified the principle amino acids as sources of nitrogen for Mtb when growing inside host cells. In this study we aim to take this study forward to test the hypothesis that nitrogen metabolism represents an unexplored and potentially fruitful drug target for TB drug development. We will use serine metabolism as a test case of this hypothesis. In our previous study we demonstrated that the enzyme SerC that performs the last step in the biosynthesis of serine is essential for intracellular replication of Mtb. This suggest that it is also essential for growth of the pathogen inside the host and thereby a good target for the development of novel drugs. To confirm this hypothesis, we will first measure the virulence of the SerC mutant of Mtb in a mouse model of infection. The next step will be to screen a 'library' of chemical compounds library to identify any that target SerC. Conventional drug screens identify compounds that are active against particularly enzymes, such as SerC. However, the identified compounds often fail to work in the natural host because of poor penetration or inactivation of the drug in host cells. To overcome this problem, we propose to screen compounds against whole live cells of Mtb. This is more difficult as Mtb makes hundreds of different enzymes, each of which might be inhibited by the test compounds. To identify compounds that specifically target SerC, we will search for those that are active against wild-type Mtb but not a SerC mutant strain supplemented with serine so that it no longer needs SerC to grow. We will then extend the study to other key genes involved in nitrogen metabolism and uptake of amino acids from host cells. Finally, we will develop a computer model of nitrogen and carbon metabolism in Mtb that can be used to design combinations of drugs that target nitrogen metabolism that will work effectively together and test predictions of the model experimentally.