Exploring the metabolism of non-replicating and drug-resistant TB

探索非复制性和耐药结核病的代谢

• 批准号: 8555825
• 负责人: Clifton Barry
• 金额: $ 65.51万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8555825
• 关键词: Aerobic Bacteria; Affect; Amino Acids; Anabolism; Antigens; Area; Bacillus (bacterium); Bacteria; Binding; Binding Sites; Biochemical; Biological Assay; Biotin; Carbon; Cell Wall; Cells; Citric Acid Cycle; Clinical; Coenzyme A; Collaborations; Collection; Complex; Coupling; Cytolysis; DNA Sequence; Development; Disease; Down-Regulation; Drug resistance; Electrons; Enzymes; Event; Folate; Folic Acid Antagonists; Fumarate Hydratase; Generations; Genes; Genomics; Glucose; Growth; Hypoxia; Immune response; In Situ; In Vitro; Infection; Iron; Knock-out; Laboratories; Libraries; Life; Ligand Binding; Ligands; Lipids; Maps; Membrane; Meropenem; Metabolic; Metabolic Pathway; Metabolism; Modeling; Mutation; Mycobacterium tuberculosis; NADH; Nature; Nitrogen; Nitroimidazoles; Nitroreductases; Oxidases; Oxygen; Pathogenesis; Peptidoglycan; Peptidyltransferase; Pharmaceutical Preparations; Physiology; Process; Protein Family; Proteins; Pyridoxal; Reaction; Reporting; Research Personnel; Resistance; Respiration; Role; Scanning Transmission Electron Microscopy Procedures; Single Nucleotide Polymorphism; Source; South Africa; Structure; Succinates; Textbooks; Transferase; Trehalose; Virulent; Vitamin K 2; arabinogalactan; base; chemical genetics; cofactor; cytotoxicity; design; drug discovery; enzyme substrate; extracellular; flasks; folic acid metabolism; genome sequencing; high throughput screening; in vivo; inhibitor/antagonist; killings; medical schools; metabolic poison; metabolomics; mutant; mycobacterial; nitrosative stress; oxidation; pantothenate; pharmacophore; pyridoxine 5-phosphate; respiratory; scaffold; serine-type D-Ala-D-Ala carboxypeptidase; tool

The first project area explores metabolic pathways that have been proposed based on in vitro studies to be important in non-replicating (NR)-MTb. We are exploring the importance of the biosynthesis of the cofactors biotin, coenzyme A and pyridoxal, peptidoglycan turnover, the role of putative F420-binding and genetically annotated pyridoxal-generating enzymes, beta-oxidation and iron acquisition and validating these by chemical and genetic means in non-replicating (NR)-MTb. We have shown that Rv2607 is the canonical pyridoxine phosphate oxidase of MTb and have enzymatically characterized this enzyme. In contrast, Rv1155, which is also annotated as a pyridoxine phosphate oxidase family protein has been expressed, purified, crystalized with its F420 cofactor, biophysically characterized with and without bound cofactor and we are attempting to identify the natural substrate of this protein by analyzing shared chemotypes with known metabolites from fragments identified as binders to this protein. Another F420-dependent enzyme, Rv2991, has been crystalized and fragments chemically similar to known metabolites of flavoenzymes analyzed for binding to Rv2991 with and without F420. By analyzing common pharmacophores between known metabolites and the binders identified by this fragment-based approach, we are attempting to probe the enzymatic function of this unknown protein We have also demonstrated the importance of biotin synthesis for the viability of MTb in vitro and in vivo. We have reported that conditional downregulation of pantothenate synthase makes Mtb hypersusceptible to inhibitors of coenzyme A biosynthesis and are using this approach to identify vulnerable targets in this metabolic pathway. Our studies of mycobacterial cell wall synthesis using meropenem as probe have allowed us to track the formation of the various layers of the mycobacterial cell wall during its assembly using a combination of cryo-electron, transmission and scanning electron microscopy. We have shown that the dual action of meropenem on both the D,D-carboxypeptidases as well as the transpeptidases on newly synthesized peptidoglycan leads to the observed polar lysis of cells. The second major focus area of this project starts from a different perspective and uses compounds that are in clinical development (PA-824 and SQ109) which are known to possess activity against replicating as well as NR-TB. We capitalized our recently determined crystal structure of Ddn, the nitroreductase responsible for the bioreductive activation of PA824 to understand the differences in binding of the enzyme to nitroimidazoles and the relationship of this binding to the formation of the reactive nitrogen intermediates responsible for killing of Mtb. We are attempting to understand what the natural substrate is for the Ddn, since this will allow us to probe the enzymatic processes that are important during non-replicating persistence. Preliminary studies have identified menaquinone as a substrate for this enzyme. For SQ109 we were able to demonstrate that the mechanism by which this drug kills Mtb is by inhibition of the MmpL3 protein which we identified as a trehalose monomycolate transporter. To further unravel the key events in cell wall mycolyl-arabinogalactan synthesis, we have enzymatically characterized the three mycolyl transferase enzymes (Antigens 85 A, B and C). We have found that the enzymes are kinetically distinct with Ag85C being enzymatically the most active and that certain amino acid residues residing in a secondary ligand binding site control rates of acyl transfer by affecting protein confirmation in a helix connecting the two ligand binding pockets. The third major focus of this project involves global approaches to understanding the metabolism in NR-TB. Using a chemostat model of MTb combined with metabolomic studies, we demonstrated that the NADH/NAD+ ratio changed as a function of oxygen concentration, that the direction of the TCA cycle reverses under hypoxia with concomitant extracellular succinate accumulation which is consistent with a model of oxygen-induced stasis in which an energized membrane is maintained by coupling the reductive branch of the TCA cycle to succinate secretion. An essential non-redundant step in this process is fumarase and we have initiated studies to validate the role of the forward as opposed to reverse TCA cycle in vitro as well as in vivo by using structure-based design based on the fumarase crystal structure to design inhibitors of this target. Co-crystal structures of Mtb fumarase with bound inhibitors, enzymatic as well as in situ demonstration of fumarase inhibition have corroborated our model with further inhibitor optimization being required for in vivo studies. In a fourth approach, we are identifying inhibitors of metabolism by high-throughput screening approaches performed under a variety of in vivo relevant environmental conditions. Hits from these screens have provided a useful tool to map metabolism of MTb as a function of carbon source, oxygen concentration or presence of low pH in the presence or absence of nitrosative stress and are currently being studied to identify the target. In the process of target identification, parallel studies are done to rapidly progress the hits to in vivo proof of concept studies so that the importance of the target for in vivo pathogenesis can be validated early on in the drug discovery process. We are studying some of the hits that were identified from a 35,000 compound BioFocus collection in collaboration with various researchers in South Africa. In addition, hits from a 100,000 compound library screen from a collaborator have yielded 12 different scaffolds that are being pursued. The scaffolds that gave us evidence of a specific target based on SAR studies were taken further into target identification by a combination of approaches including resistant mutant generation followed by whole genome sequencing to identify single nucleotide polymorphisms, transcriptional profiling, macromolecular incorporation assays and metabolomics studies. For 2 chemically different scaffolds, the same target in mycobacterial cell wall synthesis was identified and efficacy studies confirmed that inhibition of some cell wall biosynthetic genes in vivo, led to a mild bacteriostatic effect. The targets of eleven other scaffolds were identified. For several other scaffolds, mutations in MmpL3, a protein we previously identified as the SQ109 target, conferred resistance suggesting that this transporter is promiscuous in its ability to bind diverse ligands. For several scaffolds, generation of resistant mutants was impossible and in several of these cases, inability to generate resistant mutants was correlated with mammalian cytotoxicity suggesting a non-specific mechanism of action. One class of compounds was shown to target oxygen-dependent respiration in Mtb. We have demonstrated that the coupling of respiration to energy generation in a vulnerable point in NR-Mtb based on inhibitors identified in a screen against anaerobically persisting Mtb.The precise point in inhibition of respiration is currently being explored by analysis of respiratory knockout mutants, biochemical assays and complementation studies. Resistance to another hit mapped to an enzyme in folate metabolism. We have been able to show that this drug functions as a metabolic poison by its ability to mimic substrates and become incorporated into folate-like metabolites by a combination of metabolomics and biochemical analyses. With collaborators at Weill Cornell Medical College, we have used this inhibitor as well as other known inhibitors of folate biosynthetic enzymes to explore how perturbation of folate-dependent reactions leads to inhibition of Mtb replication.

第一个项目区域探讨了基于体外研究提出的代谢途径，对非复制（NR）-MTB很重要。我们正在探索辅因子生物素，辅酶A和吡ido糖，肽聚糖的生物合成的重要性，假定的F420结合和遗传学注释的吡idoxal生成酶的作用，β-氧化和铁的续集和铁的方法 - 通过化学和遗传（这些方法） - 在化学和遗传上进行了验证 - 在化学和概括性上均在化学和概括（NOCE）。我们已经表明，RV2607是MTB的典型吡ido醇氧化酶，并具有酶促表征该酶。相反，RV1155也被注释为磷酸磷酸氧化酶家族蛋白，已被表达，纯化，及其F420辅助因子，以有和没有结合的辅助作用的生物物理，我们试图通过与已知的Metseine的自然化学物质鉴定该蛋白质的自然化化学物质，从而鉴定出该蛋白质的质量鉴定，从而鉴定出了与已知的Metseents的自然化化合物相结合。另一种依赖F420的酶RV2991已被结晶，并且碎片在化学上类似于已知与RV2991结合的黄酮酶的已知代谢产物，并没有F420。通过分析已知代谢产物与通过这种基于片段的方法鉴定的粘合剂之间的共同药物浮动能，我们试图探测这种未知蛋白的酶促功能，我们还证明了生物素合成对MTB在体外和体内生存能力的重要性。 我们已经报道说，有条件的泛酸合酶的条件下调使辅酶A抑制剂的MTB超敏感性可能是生物合成的，并正在使用这种方法来识别该代谢途径中的脆弱靶标。 我们使用MeropeNem作为探针对分枝杆菌细胞壁合成的研究使我们能够在支原体细胞壁组装过程中使用冷冻电子，透射和扫描电子显微镜的组合跟踪其组装过程中的各个层的形成。我们已经表明，美培元对D，D-羧肽酶以及新合成的肽聚糖的双肽酶的双重作用都导致细胞的极性裂解。 该项目的第二个主要焦点区域从不同的角度开始，并使用临床开发中的化合物（PA-824和SQ109），这些化合物已知具有反对复制和NR-TB的活性。 我们资本化了我们最近确定的DDN晶体结构，DDN是负责PA824生物补充激活的硝化化合酶，以了解酶与硝基咪唑与硝基咪唑的结合差异以及这种结合与负责杀死MTB的反应性氮中间体的结合的关系。我们试图了解DDN的自然底物是什么，因为这将使我们能够探测在不复制持久性过程中重要的酶促过程。初步研究已经确定了梅纳金酮是该酶的底物。对于SQ109，我们能够证明该药物杀死MTB的机制是通过抑制MMPL3蛋白的抑制，我们将其识别为海藻糖单霉菌转运蛋白。为了进一步揭示细胞壁霉菌基 - 阿拉伯分离素的合成中的关键事件，我们已经酶上表征了三种霉菌基因转移酶（抗原85 A，B和C）。我们发现，这些酶在动力学上是不同的，而AG85C在酶上是最活跃的，并且某些驻留在二次配体结合位点控制酰基转移的氨基酸残基，通过影响连接两个配体结合口袋的螺旋中的蛋白质确认，通过影响蛋白质确认。 该项目的第三个主要重点涉及了解NR-TB中新陈代谢的全球方法。 Using a chemostat model of MTb combined with metabolomic studies, we demonstrated that the NADH/NAD+ ratio changed as a function of oxygen concentration, that the direction of the TCA cycle reverses under hypoxia with concomitant extracellular succinate accumulation which is consistent with a model of oxygen-induced stasis in which an energized membrane is maintained by coupling the reductive branch of the TCA cycle to琥珀酸酯分泌。在此过程中，基本的非冗余步骤是富马酶，我们通过使用基于基于富马酶晶体结构的基于结构的设计来设计基于结构的设计来设计该靶标的抑制剂，从而启动了研究，以验证前向而不是体外反向TCA周期的作用。 MTB富马酶的共结构结构具有结合的抑制剂，酶促和原位抑制作用，已证实了我们的模型，并需要进一步的抑制剂优化体内研究。 在第四种方法中，我们通过在各种体内相关的环境条件下执行的高通量筛选方法来鉴定代谢的抑制剂。这些筛选的命中提供了一种有用的工具，可以在存在或不存在亚硝化应力的情况下将MTB代谢作为碳源，氧浓度或低pH的函数的函数，目前正在研究以识别目标。在目标识别过程中，进行平行研究是为了快速进行概念研究证明的打击，以便可以在药物发现过程的早期验证目标对体内发病机理的重要性。我们正在研究与南非的各种研究人员合作，从35,000种化合物生物对焦收集中鉴定出的一些热门单曲。此外，来自合作者的100,000个复合图书馆屏幕的命中率产生了12种不同的脚手架。通过包括抗性突变体产生（全基因组测序）的组合，将基于SAR研究的特定靶标的证据的支架进一步鉴定为靶标识别，以鉴定单核苷酸多态性，转录分析，大分子分子掺入分析测定和代谢组学研究。对于2个化学上不同的支架，鉴定了分枝杆菌细胞壁合成中的相同靶标，功效研究证实，体内对某些细胞壁生物合成基因的抑制导致了轻度的抑菌作用。确定了其他11个支架的目标。对于其他几个脚手架，MMPL3中的突变是我们先前被鉴定为SQ109靶标的蛋白质的突变，表明该转运蛋白在结合多种配体的能力上是混杂的。对于几个脚手架，抗性突变体的产生是不可能的，在其中一些情况下，无法产生抗性突变体与哺乳动物的细胞毒性相关，表明非特异性作用机理。显示了一类化合物靶向MTB中的氧气依赖性呼吸。我们已经证明，基于在筛查中鉴定在厌氧上持续的MTB中鉴定的抑制剂的易害点，呼吸与能量产生的偶联。目前正在通过分析呼吸敲除突变体，生物化学分析和补体研究来探索呼吸抑制的精确点。对叶酸代谢中映射到酶的另一个命中的抗性。我们已经能够通过模仿底物的能力来证明该药物可以作为代谢毒物作用，并通过代谢组学和生物化学分析的结合将其掺入叶酸样代谢产物中。与Weill Cornell医学院的合作者一起，我们使用了该抑制剂以及其他已知的叶酸生物合成酶抑制剂，以探索叶酸依赖性反应的扰动如何导致MTB复制的抑制作用。