Development Of New Chemotherapeutics For Tuberculosis

结核病新化疗药物的开发

• 批准号: 9161485
• 负责人: Clifton Barry
• 金额: $ 75.65万
• 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/9161485
• 关键词: Affect; Anabolism; Antibiotic Therapy; Area; Bacteria; Biological; Biological Assay; Biological Factors; Biological Process; Carbon; Cell Respiration; Cell Wall; Cells; Chemicals; Chemistry; Cholesterol; Collection; Complex; Coupled; Cytolysis; DNA Damage; DNA Resequencing; Dependence; Development; Disease Resistance; Disease model; Dose; Drug Targeting; Drug resistance; Ensure; Enzymes; Equilibrium; Evaluation; Excretory function; Frequencies; Fumarate Hydratase; Generations; Genes; Genome; Goals; Granuloma; Growth; HepG2; Human; Hypoxia; IMP Dehydrogenase; In Vitro; Infection; Inhibitory Concentration 50; Kinetics; Lead; Libraries; Ligase; Lipids; Measures; Metabolic; Metabolism; Minimum Inhibitory Concentration measurement; Mitochondria; Monkeys; Mutation; Mycobacterium tuberculosis; Nitrites; Organism; Oryctolagus cuniculus; Oxygen; Pathway interactions; Pharmaceutical Preparations; Pharmacologic Substance; Phosphotransferases; Process; Property; Reporter; Reporting; Resistance; Respiration; Series; Solubility; Source; Speed; Staging; Structure; Structure-Activity Relationship; Synthesis Chemistry; Testing; Thermodynamics; Tissues; Titrations; Toxic effect; Trehalose; Triage; Tuberculosis; Validation; absorption; analog; arabinogalactan; base; chemical synthesis; chemotherapy; cytotoxic; cytotoxicity; design; drug development; epimerase; follow-up; glucose metabolism; high throughput screening; improved; in vivo; inhibitor/antagonist; killings; lead series; macrophage; member; metabolomics; mutant; mycobacterial; oxidation; pathogen; pre-clinical; respiratory; scaffold; screening; therapy duration; tuberculosis drugs

Currently this project focuses on four key areas: (1) chemical synthesis of lead molecules and series identified by high-throughput screening against whole Mycobacterium tuberculosis (MTb), (2) the synthesis and evaluation of inhibitors of fumarase from tuberculosis, (3) the synthesis and evaluation of inhibitors of the inosine 5'-monophosphate dehydrogenase and (4) structure-based design and screening of inhibitors of NAD synthetase. Most of our effort currently is devoted to Project (1) in which we are screening large compounds libraries obtained from global collaborators including several large pharmaceutical companies to identify inhibitors of MTb growth under in vivo relevant conditions, performing dose-titration follow-up of hits and synthesizing or purchasing chemically similar compounds. These series are evaluated using secondary screens with a battery of conditions that are thought to be relevant during in vivo growth of MTb. We selected carbon source as an in vivo relevant variable since numerous studies have shown the metabolism of glucose, cholesterol and/ or other lipids are critical for growth and survival of this pathogen in host tissues. Since September 2014 we have completed hit confirmation screens by dose titration screens of the primary hits from a 270,000 compound deck from Merck under restricted growth conditions including a beta-oxidation substrate at low pH and nitrite, a 200,000 compound deck from AstraZeneca under 4 different growth conditions, a 40,000 compound deck from DuPont under two growth conditions and a 100,000 natural product collection from EISAI under 3 different growth conditions. Reconfirmed hits with IC50 values less than 10uM are then assayed for their ability to completely inhibit growth of the organism under a panel of in vitro growth conditions reflecting all carbon sources thought to be in vivo relevant by determination of the minimum inhibitory concentration (MIC) and parallel determination of their cytotoxicity to HepG2 cells. Every attempt is made to progress as many chemo-types as possible to increase the likelihood of hitting a diversity of targets. Hit series with multiple members showing activity for the scaffold with low-complexity, acceptable solubility and promising physicochemical properties for profiling are prioritized for follow-up to determine if the desirable balance of potency and ADME (absorption, distribution, metabolism and excretion) properties could be achieved in Lead Optimization. In contrast, series with structural alerts suggesting toxicophores are deprioritized. To rapidly expand the SAR for the prioritized chemotypes, commercially available analogs are purchased and tested in MIC assays. Target identification for prioritized series is initiated by mutation frequency analysis, whole genome resequencing of resistant isolates, microarray and metabolomics analyses. In addition, kinetic and thermodynamic solubility determinations and microsomal stability assays are done to further develop the information that will be essential to facilitate go / no-go progression into lead optimization. To date, we have identified hundreds of active series that require follow-up evaluation. Amongst these we have found many series of compounds that block the function of MmpL3, the trehalose monomycolate transporter essential for cell wall synthesis as well as several scaffolds that target the DprE1 epimerase involved in cell wall arabinan biosynthesis. Several kinase scaffolds were identified in the screens and SAR studies on these have suggested independent targets for a few of these whereas at least five scaffolds suggested the same target based on convergent SAR. Transcriptional profiling analyses indicated that at least 5 series affected cellular respiration. This was subsequently corroborated in mutant generation studies which demonstrated that mutations in qcrB, a component of the respiratory bc1 complex involved in aerobic respiration, conferred high-level resistance to these scaffolds. A mutant in an alternative oxygen-dependent respiratory complex was found to be hyper-susceptible to QcrB inhibitors and has proved to be a valuable strain to identify additional classes of bc1 complex inhibitors. We have determined that the majority of series that progressed into hit-to-lead chemistry, were discontinued due to failing biological target validation and have expended considerable effort in attempting to optimize the process of biological triage. Target identification for compounds with potent anti-tubercular activity has shown that certain pathways are apparently susceptible to a diversity of chemical scaffolds. These apparently promiscuous targets include several enzymes involved in mycobacterial cell wall mycolyl-arabinogalactan biosynthesis as well as the respiratory bc1 complex. Neither of these targets are thought to have significant promise for treating TB since cell wall targets are already well hit by existing TB drugs and show little promise for radically changing the duration of therapy. TB bacteria are largely only slowly replicating in human infections and appear relatively insensitive to inhibitors of cell wall enzymes. The development of additional drugs which target aspects of cell wall biosynthesis would thus be expected to have little effect on the duration of chemotherapy and would only be useful as new drugs to treat drug-resistant disease. We have found that inhibitors of the respiratory bc1 complex show strain dependence as well as medium dependence in their ability to inhibit growth. Metabolic and transcriptional analyses of MTb growing under hypoxia as well as of MTb recovered from rabbit and monkey granulomas, have indicated major alterations in utilization of components of respiratory pathways. These results suggest that careful target validation is essential before progressing any inhibitors of respiration and coupled energy generation into lead optimization. To optimize our chances of identifying series with the potential to improve the speed of cure of TB, we are now applying a 4-tiered approach to accelerating the process of drug development through early application of counter-screens and other informative assays that will increase early lead candidate attrition rates while decreasing attrition rates in the expensive later stages of drug development. In the first tier, validated hits from screens are assayed for potency against MTb under growth conditions similar to those encountered during infection, as well as their activity in reporter assays that measure cytotoxicity, mitochondrial toxicity, activity on promiscuous targets and ability to cause DNA damage. Compounds that are non-specifically cytotoxic or target promiscuous processes are de-prioritized. In the second tier, further deconvolution of mechanism of action takes place with a battery of assays that report on potential known mechanisms of action but that also report on detailed aspects of drug-target interaction through macromolecular incorporation assays, transcriptional profiling, as well as assessing their ability to cause cell lysis. In the third tier, we engage in-depth analyses for target identification or understanding mechanism of action on compounds by resistant mutant generation, analyses of kinetics of kill under replicating and non-replicating conditions, intra-macrophage activity and ensuring that the compounds retain potency against a panel of strains representing all major global strain lineages. In the fourth tier, target pathways are further validated by generation of regulated strains of genes in the pathway that can be tested in the right in vitro or in vivo disease model. In Projects 2-4 we are taking a variety of approaches to develop inhibitors and potential lead series against specific enzyme targets that are considered well validated as druggable in TB.

目前该项目重点关注四个关键领域：（1）通过高通量筛选针对整个结核分枝杆菌（MTb）鉴定的先导分子和系列的化学合成，（2）结核病延胡索酸酶抑制剂的合成和评估，（3）肌苷5'-单磷酸脱氢酶抑制剂的合成和评估以及（4） NAD合成酶抑制剂的基于结构的设计和筛选。 目前我们的大部分精力都致力于项目（1），其中我们正在筛选从全球合作者（包括几家大型制药公司）获得的大型化合物库，以识别体内相关条件下的 MTb 生长抑制剂，对命中进行剂量滴定跟踪，并合成或购买化学上相似的化合物。这些系列使用二次筛选和一系列被认为与 MTb 体内生长过程相关的条件进行评估。我们选择碳源作为体内相关变量，因为大量研究表明葡萄糖、胆固醇和/或其他脂质的代谢对于这种病原体在宿主组织中的生长和存活至关重要。自 2014 年 9 月以来，我们已经通过剂量滴定屏幕完成了对来自 Merck 的 270,000 个化合物组（在限制生长条件下，包括低 pH 和亚硝酸盐的 β-氧化底物）、来自 AstraZeneca 的 200,000 个化合物组（在 4 种不同的生长条件下）、来自 DuPont 的 40,000 个化合物组（在两种生长条件下）和来自 EISAI 的 100,000 个天然产物集合的主要命中物的剂量滴定筛选。 3种不同的生长条件。然后，通过测定最小抑制浓度 (MIC) 并平行测定其对 HepG2 细胞的细胞毒性，对 IC50 值小于 10uM 的重新确认的命中进行分析，以确定它们在一组体外生长条件下完全抑制生物体生长的能力，这些条件反映了被认为与体内相关的所有碳源。我们尽一切努力开发尽可能多的化疗类型，以增加击中多种目标的可能性。多个成员的命中系列显示出具有低复杂性、可接受的溶解度和有前景的理化特性的支架活性，可用于分析，优先进行后续研究，以确定是否可以在先导化合物优化中实现效力和 ADME（吸收、分布、代谢和排泄）特性的理想平衡。相比之下，带有结构警报的系列表明毒物的优先级被降低。为了快速扩大优先化学型的 SAR，购买市售类似物并在 MIC 测定中进行测试。优先序列的目标识别是通过突变频率分析、耐药菌株的全基因组重测序、微阵列和代谢组学分析启动的。此外，还进行了动力学和热力学溶解度测定以及微粒体稳定性测定，以进一步开发对于促进通过/不通过进展到先导化合物优化至关重要的信息。 迄今为止，我们已经确定了数百个需要后续评估的活跃系列。其中，我们发现了许多系列的化合物可以阻断 MmpL3 的功能，MmpL3 是细胞壁合成所必需的海藻糖单分枝菌酯转运蛋白，以及一些靶向参与细胞壁阿拉伯聚糖生物合成的 DprE1 差向异构酶的支架。在筛选中鉴定出了几种激酶支架，对这些支架的 SAR 研究表明其中一些是独立的靶点，而至少五个支架基于收敛 SAR 表明了相同的靶点。转录谱分析表明至少有 5 个系列影响细胞呼吸。随后的突变体生成研究证实了这一点，该研究表明 qcrB（参与有氧呼吸的呼吸 bc1 复合物的一个组成部分）的突变赋予了对这些支架的高水平抵抗力。另一种氧依赖性呼吸复合物的突变体被发现对 QcrB 抑制剂高度敏感，并已被证明是鉴定其他类别 bc1 复合物抑制剂的有价值的菌株。 我们已经确定，大多数进展为先导化学的系列由于生物靶标验证失败而被终止，并且在尝试优化生物分类过程方面付出了相当大的努力。具有有效抗结核活性的化合物的靶标鉴定表明，某些途径显然对多种化学支架敏感。这些明显混杂的靶标包括参与分枝杆菌细胞壁分枝菌基-阿拉伯半乳聚糖生物合成以及呼吸 bc1 复合物的几种酶。人们认为这两个靶点都没有治疗结核病的重大前景，因为细胞壁靶点已经被现有的结核病药物很好地击中，并且几乎没有从根本上改变治疗持续时间的希望。结核病细菌在人类感染中很大程度上只是缓慢复制，并且对细胞壁酶抑制剂相对不敏感。因此，针对细胞壁生物合成方面的其他药物的开发预计对化疗持续时间几乎没有影响，并且仅可用作治疗耐药性疾病的新药。我们发现呼吸 bc1 复合物的抑制剂在抑制生长的能力方面表现出菌株依赖性和介质依赖性。对缺氧条件下生长的 MTb 以及从兔和猴肉芽肿中回收的 MTb 的代谢和转录分析表明，呼吸途径成分的利用发生了重大变化。这些结果表明，在将任何呼吸抑制剂和耦合能量产生进行先导化合物优化之前，仔细的靶标验证至关重要。 为了最大限度地提高我们识别有可能提高结核病治愈速度的系列的机会，我们现在正在采用四层方法，通过早期应用反筛选和其他信息性分析来加速药物开发过程，这将提高早期先导候选药物的流失率，同时降低昂贵的药物开发后期阶段的流失率。在第一层中，在与感染过程中遇到的生长条件类似的生长条件下，对筛选得到的经过验证的命中物进行抗MTb的效力，以及它们在报告基因测定中的活性，以测量细胞毒性、线粒体毒性、对混杂靶标的活性以及引起DNA损伤的能力。非特异性细胞毒性或目标混杂过程的化合物被取消优先级。在第二层中，通过一系列测定对作用机制进行进一步反卷积，这些测定报告潜在的已知作用机制，但也通过大分子掺入测定、转录分析以及评估其引起细胞裂解的能力来报告药物-靶标相互作用的详细方面。在第三层中，我们通过抗性突变体的产生来深入分析目标识别或了解化合物的作用机制，分析复制和非复制条件下的杀伤动力学、巨噬细胞内活性，并确保化合物保留针对代表所有主要全球菌株谱系的一组菌株的效力。在第四层中，通过生成途径中受调节的基因株来进一步验证靶途径，这些基因株可以在正确的体外或体内疾病模型中进行测试。 在项目 2-4 中，我们正在采取多种方法来开发针对特定酶靶点的抑制剂和潜在先导系列，这些酶靶点被认为在结核病中已被充分验证为可药物治疗。