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.

第一个项目领域探索基于体外研究提出的对非复制 (NR)-MTb 很重要的代谢途径。我们正在探索辅因子生物素、辅酶 A 和吡哆醛生物合成、肽聚糖周转的重要性，假定的 F420 结合和基因注释的吡哆醛生成酶、β-氧化和铁获取的作用，并通过化学和遗传手段在非复制 (NR)-MTb 中验证这些作用。我们已经证明 Rv2607 是 MTb 的典型磷酸吡哆醇氧化酶，并对该酶进行了酶学表征。相比之下，Rv1155（也被注释为磷酸吡哆醇氧化酶家族蛋白）已用其 F420 辅因子进行表达、纯化和结晶，并在有或无结合辅因子的情况下进行生物物理表征，我们正试图通过分析与来自被鉴定为该蛋白质结合物的片段的已知代谢物的共享化学型来鉴定该蛋白质的天然底物。另一种 F420 依赖性酶 Rv2991 已被结晶，其化学成分与黄素酶的已知代谢物相似，分析其在有或没有 F420 的情况下与 Rv2991 的结合。通过分析已知代谢物和通过这种基于片段的方法鉴定的结合物之间的共同药效团，我们试图探索这种未知蛋白质的酶功能。我们还证明了生物素合成对于 MTb 体外和体内活力的重要性。 我们已经报道，泛酸合酶的条件性下调使得 Mtb 对辅酶 A 生物合成抑制剂高度敏感，并且正在使用这种方法来识别该代谢途径中的脆弱靶点。 我们使用美罗培南作为探针对分枝杆菌细胞壁合成的研究使我们能够结合冷冻电子显微镜、透射电子显微镜和扫描电子显微镜来追踪分枝杆菌细胞壁在组装过程中各层的形成。我们已经证明美罗培南对 D,D-羧肽酶以及新合成的肽聚糖上的转肽酶的双重作用导致观察到的细胞极性裂解。 该项目的第二个主要重点领域从不同的角度出发，并使用处于临床开发阶段的化合物（PA-824 和 SQ109），这些化合物已知具有抗复制和 NR-TB 活性。 我们利用最近确定的 Ddn（负责 PA824 生物还原激活的硝基还原酶）的晶体结构来了解该酶与硝基咪唑结合的差异以及这种结合与负责杀死 Mtb 的活性氮中间体形成的关系。我们正在尝试了解 Ddn 的天然底物是什么，因为这将使我们能够探测在非复制持久性过程中重要的酶促过程。初步研究已确定甲基萘醌是这种酶的底物。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，我们将其确定为海藻糖单分菌酸转运蛋白。为了进一步揭示细胞壁分枝菌基-阿拉伯半乳聚糖合成中的关键事件，我们对三种分枝菌基转移酶（抗原 85 A、B 和 C）进行了酶学表征。我们发现这些酶在动力学上不同，Ag85C 的酶活性最强，并且存在于第二配体结合位点中的某些氨基酸残基通过影响连接两个配体结合袋的螺旋中的蛋白质确认来控制酰基转移速率。 该项目的第三个主要重点涉及了解 NR-TB 代谢的全球方法。使用 MTb 的恒化器模型结合代谢组学研究，我们证明 NADH/NAD+ 比值作为氧浓度的函数而变化，TCA 循环的方向在缺氧下逆转，同时伴随细胞外琥珀酸盐积累，这与氧诱导的停滞模型一致，其中通过将 TCA 循环的还原分支耦合到 琥珀酸分泌。该过程中一个重要的非冗余步骤是延胡索酸酶，我们已启动研究，通过使用基于延胡索酸酶晶体结构的结构设计来设计该靶点的抑制剂，以在体外和体内验证正向 TCA 循环相对于反向 TCA 循环的作用。结核分枝杆菌延胡索酶与结合抑制剂的共晶结构、酶促以及延胡索酶抑制的原位演示证实了我们的模型，体内研究需要进一步优化抑制剂。 在第四种方法中，我们通过在各种体内相关环境条件下进行的高通量筛选方法来鉴定代谢抑制剂。这些筛选结果提供了一种有用的工具，可以在存在或不存在亚硝化胁迫的情况下绘制 MTb 代谢随碳源、氧浓度或低 pH 值变化的函​​数图，目前正在研究以确定目标。在靶标识别过程中，进行平行研究以快速推进体内概念验证研究，以便在药物发现过程的早期验证靶标对于体内发病机制的重要性。我们正在与南非的多位研究人员合作，研究从 BioFocus 收集的 35,000 种化合物中鉴定出的一些热门化合物。此外，合作者从 100,000 个化合物库筛选中筛选出的结果已经产生了 12 种正在研究的不同支架。基于 SAR 研究为我们提供特定靶点证据的支架，通过组合方法进一步用于靶点识别，包括抗性突变体生成，然后进行全基因组测序以识别单核苷酸多态性、转录谱、大分子掺入测定和代谢组学研究。对于两种化学上不同的支架，确定了分枝杆菌细胞壁合成中的相同靶点，并且功效研究证实，抑制体内某些细胞壁生物合成基因可产生轻微的抑菌作用。其他十一个支架的目标也已确定。对于其他几种支架，MmpL3（我们之前确定为 SQ109 靶标的蛋白质）的突变赋予了抗性，表明这种转运蛋白结合不同配体的能力是混杂的。对于几种支架，不可能产生抗性突变体，并且在其中一些情况下，无法产生抗性突变体与哺乳动物细胞毒性相关，这表明存在非特异性作用机制。一类化合物被证明可以靶向结核分枝杆菌的氧依赖性呼吸。基于在针对厌氧持久性结核分枝杆菌的筛选中鉴定出的抑制剂，我们已经证明了 NR-Mtb 脆弱点中呼吸与能量产生的耦合。目前正在通过分析呼吸敲除突变体、生化测定和互补研究来探索呼吸抑制的精确点。对另一种打击的抵抗力映射到叶酸代谢中的酶。我们已经能够证明这种药物具有代谢毒物的功能，因为它具有模拟底物的能力，并通过代谢组学和生化分析结合到叶酸样代谢物中。我们与威尔康奈尔医学院的合作者一起，使用这种抑制剂以及其他已知的叶酸生物合成酶抑制剂来探索叶酸依赖性反应的扰动如何导致结核分枝杆菌复制的抑制。