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: 8745359
• 负责人: Clifton Barry
• 金额: $ 59.91万
• 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/8745359
• 关键词: ATP Synthesis Pathway; Acids; Active Sites; Aerobic Bacteria; Affinity; Anabolism; Arabinose; Area; Bacillus (bacterium); Bacteria; Binding; Binding Proteins; Biotin; Carbon; Cell Wall; Cells; Chemical Structure; Citric Acid Cycle; Coenzyme A; Complex; Computer Simulation; Culture Media; DNA Sequence; Data; Disease; Docking; Drug Targeting; Drug resistance; Enzymes; Escherichia coli; Folate; Folate Biosynthesis Pathway; Fumarate Hydratase; Fumarates; Genes; Genetic; Genomics; Glucose; Goals; Growth; Homologous Gene; Hydroquinones; Hypoxia; Immune response; In Vitro; Infection; Iron; Laboratories; Libraries; Life; Ligand Binding; Ligands; Lipids; Malates; Maps; Membrane Potentials; Messenger RNA; Metabolic; Metabolic Pathway; Metabolism; Methanobacteria; Modeling; Mycobacterium tuberculosis; Nature; Organism; Oxidation-Reduction; Oxygen; Pantothenate kinase; Pathogenesis; Pathway interactions; Peptidoglycan; Pharmaceutical Preparations; Physiology; Play; Poison; Poisoning; Process; Proteins; Pyridoxal; Reaction; Reporting; Repression; Respiration; Ribose; Role; Source; Structure; Succinate Dehydrogenase; Succinates; System; Textbooks; Transferase; Validation; Virulent; Vitamin K 2; analog; arabinogalactan mycolate; base; chemical genetics; cofactor; design; drug discovery; epimerase; epimerization; flasks; folic acid metabolism; high throughput screening; in vivo; inhibitor/antagonist; interest; killings; macromolecular assembly; metabolomics; mutant; mycolate; nitrosative stress; oxidation; p-Aminosalicylic Acid; pantothenate; pathogen; pharmacophore; protein complex; respiratory; small molecule; tool; tuberculosis treatment

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 are continuing our studies to understand the role of the deazaflavin cofactor F420 in metabolism of this pathogen. We have identified several proteins that use F420 for unique redox reactions. In several of these, menaquinone can be reduced to its quinol by these enzymes in an F420-dependent manner which suggests that F420 may play a role in respiration of this pathogen reminiscent of F420-dependent respiratory complexes in methanobacteria. The substrate of some of these F420-binding proteins is also been identified by fragment-based approaches where we are identifying the binding affinity of commercially available metabolites to these proteins with and without bound F420. Small molecules that bind in the presence of reduced F420 are further characterized after incubation with the reduced cofactor and protein to determine whether these are enzymatically reduced. Finally, co-crystal structures of small ligands of interest bound to F420-protein complexes are determined with the ultimate goal of using the ligand-binding, enzymatic and crystallographic data to determine the true substrate of the proteins as well as enabling potential design of high-affinity inhibitors that can be used to probe the importance of the identified process in cellular metabolism. We are continuing our systematic analysis of potential bottlenecks in the coenzyme A metabolic pathway. The gene for each enzymatic step has been genetically manipulated in the pathogen to generate a strain where its expression is controlled by anhydrotetracycline levels in the culture media. In this way, we have identified those genes in the pathway that are permissive to fluctuation in their mRNA levels whereas repression of other coenzyme A biosynthetic genes have led to strains with poor growth or genetic revertants of the transcriptional regulatory systems suggesting that these may be chokepoints in coenzyme A biosynthesis. The metabolic changes in these cells with particular emphasis on metabolties in the coenzyme A super-pathway are analyzed in these mutants in combination with inhibitors of pantothenate synthase or pantothenate kinase. The second major focus area of this project starts from a different perspective and uses compounds that have activity against targets in cell wall biosynthesis to identify vulnerable steps in assembly of the macromolecular peptidoglycan-arabinogalactan-mycolate complex. We have identified inhibitors of several of these steps including the epimerase required for ribose-arabinose epimerization, synthesis of the peptidoglycan component, export of the mycolate precursors and inhibitors of the mycolyl transferases and are assessing the enzymatic activities associated with each step as well as their validation in terms of innate drug target potential during in vivo relevant growth and persistence of the organism. The third major focus of this project involves global approaches to understanding the metabolism in NR-TB. WE had previously demonstrated that reduction of fumarate to succinate plays a key role in reoxidation of reduced cofactors under hypoxic conditions. Although fumarate reductase/succinate dehydrogenase plays the key role in this process, MTb has 3 homologs of this enzyme complicating efforts to design inhibitors of this step in the organism. However, the interconversion of malate and fumarate precedes this step in the reductive pathway and is encoded by a single essential enzyme raising the potential of fumarase as a drug target under hypoxic survival of the pathogen. We have used the crystal structure of pyromellitic acid bound to the E. coli bound fumarase and used this to dock into the corresponding MTb fumarase crystal structure to generate a pharmacophore model of a MTb fumarase active site inhibitor. This pharmacophore model was used to screen 3 million compounds from the ZINC library to identify potential high affinity binders. The most promising of these were confirmed for their in silico ability to bind to the fumarase and subsequently these and their analogs were synthesized. The synthesized compounds were evaluated for their ability to bind to and enzymatically inhibit the MTb fumarase. The most promising of these were found to kill Mtb surviving under anaerobic conditions in a target-specific manner. Co-crystal structures of these inhibitors bound to fumarase were generated and are currently being refined to allow us to design MTb-specific fumarase inhibitors to validate the importance of this pathway in vivo. The reductive branch of the TCA cycle plays a role in reoxidizing reduced cofactors but the processes that play a role in maintaining the membrane potential of the organism under hypoxia remain poorly understood. We are using inhibitors of ATP synthesis and respiration to gain a better understanding of the interplay between respiratory, fermentative and energy generating processes under hypoxic growth and survival of the pathogen. 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. One class of compounds were found to target folate biosynthesis. To further understand the vulnerable steps in folate biosynthesis we performed metabolomics of MTb exposed to a variety of inhibitors of folate metabolism and enzymatic analyses of the corresponding proteins in this pathway. We reported that para-aminosalicylic acid, a second line drug for the treatment of tuberculosis, poisons this pathway by acting as a substrate analog that gets incorporated into the folate pathway hereby poisoning folate pools. Other analogs of para-aminosalicylic acid are similarly incorporated but their metabolic fate depends on their chemical structure. The metabolic consequences of inhibitors of folate biosynthesis are being explored to understand the vulnerability of folate-dependent enzymes in this pathogen.

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。我们正在继续研究，以了解脱氮黄素辅因子 F420 在这种病原体代谢中的作用。我们已经鉴定出几种使用 F420 进行独特氧化还原反应的蛋白质。在其中一些中，甲基萘醌可以通过这些酶以 F420 依赖性方式还原为对苯二酚，这表明 F420 可能在这种病原体的呼吸中发挥作用，让人想起甲烷细菌中 F420 依赖性呼吸复合物。其中一些 F420 结合蛋白的底物也通过基于片段的方法进行了鉴定，我们正在鉴定市售代谢物与这些具有或不具有结合 F420 的蛋白质的结合亲和力。与还原的辅因子和蛋白质一起孵育后，进一步表征在还原的 F420 存在下结合的小分子，以确定它们是否被酶促还原。最后，确定与 F420-蛋白质复合物结合的目标小配体的共晶结构，最终目标是使用配体结合、酶学和晶体学数据来确定蛋白质的真正底物，并实现高亲和力抑制剂的潜在设计，该抑制剂可用于探测细胞代谢中已识别过程的重要性。 我们正在继续对辅酶 A 代谢途径中的潜在瓶颈进行系统分析。每个酶促步骤的基因都在病原体中进行了基因操作，以产生菌株，其表达受培养基中的脱水四环素水平控制。通过这种方式，我们鉴定了该途径中允许其 mRNA 水平波动的基因，而其他辅酶 A 生物合成基因的抑制导致菌株生长不良或转录调控系统的遗传回复体，表明这些可能是辅酶 A 生物合成中的阻塞点。在这些突变体中与泛酸合酶或泛酸激酶抑制剂结合分析这些细胞的代谢变化，特别强调辅酶A超级途径中的代谢。 该项目的第二个主要重点领域从不同的角度出发，使用对细胞壁生物合成中的靶标具有活性的化合物来确定大分子肽聚糖-阿拉伯半乳聚糖-分枝菌酸酯复合物组装过程中的脆弱步骤。我们已经鉴定了其中几个步骤的抑制剂，包括核糖-阿拉伯糖差向异构化所需的差向异构酶、肽聚糖成分的合成、霉菌酸前体的输出和霉菌基转移酶抑制剂，并且正在评估与每个步骤相关的酶活性以及它们在生物体体内相关生长和持久性期间的先天药物靶点潜力方面的验证。 该项目的第三个主要重点涉及了解 NR-TB 代谢的全球方法。 WE之前已经证明，富马酸盐还原成琥珀酸盐在缺氧条件下还原辅因子的再氧化中起着关键作用。尽管富马酸还原酶/琥珀酸脱氢酶在此过程中发挥着关键作用，但 MTb 具有该酶的 3 个同源物，这使得在生物体中设计该步骤的抑制剂的工作变得复杂。然而，苹果酸和富马酸的相互转化先于还原途径中的这一步骤，并由单一必需酶编码，从而提高了富马酸酶作为病原体缺氧生存下的药物靶标的潜力。我们使用了与大肠杆菌结合的延胡索酸酶结合的均苯四酸的晶体结构，并将其对接到相应的 MTb 延胡索酸酶晶体结构中，以生成 MTb 延胡索酸酶活性位点抑制剂的药效团模型。该药效团模型用于从 ZINC 库中筛选 300 万种化合物，以识别潜在的高亲和力结合剂。其中最有前途的物质因其与延胡索酶结合的计算机能力而得到证实，随后合成了这些物质及其类似物。评估了合成化合物结合并酶抑制 MTb 延胡索酸酶的能力。其中最有希望的是以特定目标的方式杀死厌氧条件下存活的结核分枝杆菌。这些抑制剂与延胡索酶结合的共晶结构已经生成，目前正在完善，以便我们能够设计 MTb 特异性延胡索酶抑制剂，以验证该途径在体内的重要性。 TCA循环的还原分支在再氧化还原的辅助因子中发挥作用，但在缺氧条件下维持生物体膜电位的过程仍然知之甚少。我们正在使用 ATP 合成和呼吸抑制剂来更好地了解病原体缺氧生长和生存下呼吸、发酵和能量产生过程之间的相互作用。 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。发现一类化合物以叶酸生物合成为目标。为了进一步了解叶酸生物合成中的脆弱步骤，我们对暴露于各种叶酸代谢抑制剂的 MTb 进行了代谢组学，并对该途径中相应的蛋白质进行了酶分析。我们报道，对氨基水杨酸是治疗结核病的二线药物，它通过充当底物类似物而毒害该途径，该底物类似物掺入叶酸途径中，从而使叶酸库中毒。对氨基水杨酸的其他类似物也以类似方式掺入，但它们的代谢命运取决于它们的化学结构。正在探索叶酸生物合成抑制剂的代谢后果，以了解该病原体中叶酸依赖性酶的脆弱性。