Identification of Inhibitory Compounds for Apaf-1 by High Throughput Screening

通过高通量筛选鉴定 Apaf-1 抑制化合物

• 批准号: 7560117
• 负责人: HAKIM DJABALLAH
• 金额: $ 39.34万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-08 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7560117
• 关键词: Action Potentials; Adverse effects; Apoptosis; Apoptotic; Applications Grants; Binding; Biochemical; Biological Assay; Biological Factors; Cancer Patient; Caspase; Cell model; Cerebrum; Chemicals; Clinical Trials Design; Collection; Complex; Development; Disease; Dissection; Event; Funding; Gastrointestinal tract structure; Goals; Grant; Hand; Hematopoietic System; Homeostasis; Immune System Diseases; Immune response; In Vitro; Injury; Investigation; Kinetics; Lead; Malignant Cerebral Neoplasm; Malignant Neoplasms; Mammals; Mediating; Mediator of activation protein; Mitochondria; Molecular; Multiprotein Complexes; Myocardial; Nerve Degeneration; Neurodegenerative Disorders; Normal tissue morphology; Organism; Pathologic Processes; Pathway interactions; Patients; Pharmaceutical Preparations; Physiological; Play; Process; Radiation; Research; Research Project Grants; Research Proposals; Role; Screening procedure; Solid Neoplasm; Stimulus; Symptoms; Therapeutic; Tissues; Validation; apoptotic protease-activating factor 1; base; cancer radiation therapy; cancer therapy; cell injury; cytochrome c; high throughput screening; human disease; inhibitor/antagonist; insight; novel; protein complex; protein protein interaction; research study; scaffold; small molecule; small molecule libraries; tool

Apoptosis is a major form of programmed cell death that multicellular organisms utilize to maintain tissue homeostasis and to eliminate unwanted or damaged cells. It plays a critical role in development, immune responses and many other physiological events. In mammals, a crucial apoptotic pathway is the mitochondrial cytochrome c-mediated caspase activation pathway. In this pathway, various apoptotic stimuli induce cytochrome c release from mitochondria. Released cytochrome c binds to and activates the essential mediator Apaf-1 to trigger assembly of a multimeric protein complex, the apoptosome, which in turn activates the downstream molecular executioners of apoptosis, caspases. Deregulation of the cytochrome c apoptotic pathway can lead to diseases such as cancer, immune disorders, and neurodegenerative diseases. Conversely, targeting apoptotic components by both enhancing and inhibiting apoptosis represents important therapeutic approaches to treat various human diseases. Several promising targeted cancer therapies under clinical trials are designed to specifically activate the cytochrome c apoptotic pathway. On the other hand, inhibition of this pathway should also be effective in treating symptoms with pathologically enhanced apoptosis such as neurodegeneration and ischemic injuries. Importantly, inhibition of this pathway is also a promising approach for reducing adverse side effects of radiation cancer therapy, which acts on many solid tumors via non-apoptotic mechanisms but induces massive apoptosis in normal tissues especially in the hematopoietic system and gastrointestinal tract, thus results in severe and occasionally fatal damage to patients. In this grant, we will perform high throughput screening (HTS) to identify Apaf-1-inhibitory compounds from various chemical libraries, and subsequently to characterize the mechanism of action of the identified inhibitors and their potential in protecting radiation damage. To date, we have succeeded in developing and optimizing an Apaf-1 functional assay for HTS. As a validation of this HTS assay, we have screened a collection of drug-like chemical compounds and obtained multiple positive hits that inhibit Apaf-1-mediated caspase activation. This progress should pave the way for us to achieve the eventual goals of this research grant in a timely fashion, within a two-year period supported by the ARRA fund. In these two years, we will screen for additional inhibitory compounds from a distinct chemical library containing large number of natural product-like synthetic molecules that more likely target Apaf-1 via different mechanisms. We will also determine the mechanisms of action of the identified small molecule inhibitors for Apaf-1 and inhibitors for other components in the Apaf-1 pathway. For this mechanistic investigation, both in vitro biochemical studies and analyses using specific cell models will be conducted to gain insights into the molecular basis of the identified inhibitors and their potential effect in protecting apoptotic damage associated with radiation cancer therapy. The identified Apaf-1 inhibitory compounds will be promising drug leads for treating diseases such as cancer and cerebral/myocardial ischemic injuries. They will also be important pharmacological tools for studying mechanisms and roles of cytochrome c-mediated apoptosis in various physiological and disease processes.

凋亡是多细胞生物用于维持组织稳态并消除不需要或受损细胞的编程细胞死亡的一种主要形式。它在发育，免疫反应和许多其他生理事件中起着至关重要的作用。在哺乳动物中，至关重要的凋亡途径是线粒体细胞色素c介导的胱天蛋白酶激活途径。在这一途径中，各种凋亡刺激会诱导线粒体释放细胞色素c。释放的细胞色素c与基本介体APAF-1结合并激活了多聚体蛋白复合体的组装，即凋亡小体，这又激活了凋亡的下游分子执行者，caspase。 细胞色素C凋亡途径的放松管制会导致癌症，免疫疾病和神经退行性疾病等疾病。相反，通过增强和抑制细胞凋亡来靶向凋亡成分是治疗各种人类疾病的重要治疗方法。在临床试验下，设计了几种有前途的靶向癌症疗法，以专门激活细胞色素C凋亡途径。另一方面，对该途径的抑制也应有效治疗病理增强的凋亡（例如神经变性和缺血性损伤）的症状。重要的是，抑制该途径也是一种减少放射癌治疗的不良副作用的有前途的方法，该方法通过非凋亡机制作用于许多实体瘤，但诱导正常组织中的大量凋亡，尤其是在造血系统和胃肠道中，因此会导致严重和有时对患者的致命损害。在这笔赠款中，我们将执行高吞吐量筛选（HTS），以鉴定来自各种化学文库的APAF-1抑制化合物，然后表征已鉴定抑制剂的作用机理及其在保护辐射损伤方面的潜力。 迄今为止，我们已经成功地开发和优化了HTS的APAF-1功能分析。作为对该HTS测定法的验证，我们筛选了一系列类似药物的化合物，并获得了抑制APAF-1介导的caspase激活的多个正命中。这一进步应该为我们及时在ARRA基金支持的两年内及时实现这项研究赠款的最终目标铺平道路。在这两年中，我们将从不同的化学文库中筛选出额外的抑制性化合物，其中包含大量天然产物样合成分子，这些分子更有可能通过不同的机制靶向APAF-1。我们还将确定已鉴定出的小分子抑制剂对APAF-1和APAF-1途径中其他组件的抑制剂的作用机理。为了进行这项机械研究，将进行体外生化研究和使用特定细胞模型的分析，以了解识别抑制剂的分子基础，及其在保护与放射癌治疗相关的凋亡损伤方面的潜在影响。鉴定出的APAF-1抑制化合物将成为治疗癌症和脑/心肌缺血性损伤等疾病的有前途的药物铅。它们还将是研究细胞色素c介导的凋亡在各种生理和疾病过程中的机制和作用的重要药理工具。