Targets in Mycobacterium Tuberculosis: Stress Resistance & Repair

描述（由申请人提供）：结核分枝杆菌（Mtb）是一种全球性的健康危机和生物恐怖主义威胁，其耐药性日益增强，但几十年来几乎没有新的化疗方法出现。一种新的化疗方法是靶向病原体在宿主巨噬细胞的代谢生态位中生存所必需的途径。这一应用是基于这样的假设，即Mtb需要3种酶才能在能量不足、氧化和亚硝化的条件下在吞噬体中存活：(1)脂酰胺脱氢酶（Lpd）在丙酮酸脱氢酶、支链酮酸脱氢酶以及过氧亚硝酸盐还原酶/过氧化物酶中起作用，因此是脂肪酸前体酰基辅酶a的净合成和对活性氮中间体（RNI）的抗性的关键。(2) a-酮戊二酸（KG）脱羧酶（KDC）将KG转化为琥珀二醛（SSA），取代Mtb缺乏的KG脱氢酶。SSA脱氢酶将SSA转化为琥珀酸盐，从而连接Mtb的柠檬酸循环（CAC）的氧化和还原分支。因此，KDC可能对Mtb产生能量、还原等价物、氨基酸和血红素很重要。(3)通过饱和转座子诱变发现，作为核苷酸切除修复途径的一部分，紫外线修复（Uvr） B是Mtb在RNI中存活和杀死小鼠所必需的。KDC和UvrB缺乏人类同源物，Lpd的晶体结构显示出与人类酶的关键差异。我们将使用等位基因替换来破坏编码这些酶的3个基因，或确定它们的必要性；利用传统和新型组合文库鉴定每种酶的化学抑制剂；对Lpd和KDC的晶体结构进行了分析；并评估这些酶作为新化疗药物的潜在靶点。迄今为止，抗生素只针对合成蛋白质、核酸、细胞壁和叶酸的酶。这项工作的基本新颖之处在于扩大了靶标范围，包括中间代谢和DNA修复酶。

DESCRIPTION (provided by applicant): M. tuberculosis (Mtb), a global health crisis and bioterrorism threat, is increasingly drug-resistant, but little new chemotherapy has emerged in decades. A fresh approach to chemotherapy is to target pathways essential for the pathogen to survive in its metabolic niche in host macrophages. This application is based on the hypothesis that Mtb requires 3 enzymes to survive under energy-poor, oxidative and nitrosative conditions in the phagosome: (1) Lipoamide dehydrogenase (Lpd) serves in pyruvate dehydrogenase and probably in branched chain ketoacid dehydrogenase as well as in peroxynitrite reductase /peroxidase and thus is key for net synthesis of acyl CoA's, the precursors of fatty acids, and for resistance to reactive nitrogen intermediates (RNI). (2) a-ketoglutarate (KG) decarboxylase (KDC) converts KG to succinic semialdehyde (SSA), replacing KG dehydrogenase, which Mtb lacks. SSA dehydrogenase converts SSA to succinate and may thereby connect the oxidative and reductive limbs of Mtb's citric acid cycle (CAC). KDC may therefore be important for Mtb's generation of energy, reducing equivalents, amino acids and heme. (3) Ultraviolet repair (Uvr) B, part of the nucleotide excision repair pathway, was found by saturation transposon mutagenesis to be essential for Mtb to survive RNI and to kill mice. KDC and UvrB lack human homologs, and Lpd's crystal structure shows key differences from the human enzyme. We will use allelic replacement to disrupt the 3 genes encoding these enzymes, or establish their essentiality; use conventional and novel combinatorial libraries to identify chemical inhibitors of each enzyme; analyze the crystal structures of Lpd and KDC with and without inhibitors; and assess these enzymes as potential targets for new chemotherapeutics. Antibiotics to date only target enzymes that synthesize protein, nucleic acids, cell walls and folate. The fundamental novelty of this work is to broaden the range of targets to include enzymes of intermediary metabolism and DNA repair.