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.

细胞凋亡是程序性细胞死亡的主要形式，多细胞生物利用它来维持组织稳态并消除不需要的或受损的细胞。它在发育、免疫反应和许多其他生理事件中发挥着关键作用。在哺乳动物中，一个重要的细胞凋亡途径是线粒体细胞色素 c 介导的 caspase 激活途径。在此途径中，各种细胞凋亡刺激诱导线粒体释放细胞色素 c。释放的细胞色素 c 结合并激活重要介质 Apaf-1，以触发多聚蛋白复合物（凋亡体）的组装，进而激活细胞凋亡的下游分子执行者 caspase。 细胞色素 c 凋亡途径的失调可导致癌症、免疫紊乱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细胞凋亡成分代表了治疗各种人类疾病的重要治疗方法。临床试验中的几种有前途的靶向癌症疗法旨在特异性激活细胞色素 c 凋亡途径。另一方面，抑制该途径也应该能有效治疗病理性细胞凋亡增强的症状，例如神经变性和缺血性损伤。重要的是，抑制该途径也是减少放射癌症治疗不良副作用的一种有前景的方法，放射癌症治疗通过非凋亡机制作用于许多实体瘤，但会诱导正常组织尤其是造血系统和胃肠道的大量细胞凋亡，从而对患者造成严重甚至致命的损害。在这笔资助中，我们将进行高通量筛选 (HTS)，以从各种化学库中识别 Apaf-1 抑制化合物，随后表征所识别抑制剂的作用机制及其在保护辐射损伤方面的潜力。 迄今为止，我们已经成功开发和优化了 HTS 的 Apaf-1 功能测定。作为对该 HTS 测定的验证，我们筛选了一系列药物样化合物，并获得了多个抑制 Apaf-1 介导的 caspase 激活的阳性结果。这一进展将为我们在 ARRA 基金支持的两年时间内及时实现这项研究拨款的最终目标铺平道路。在这两年中，我们将从一个独特的化学库中筛选更多的抑制化合物，该化学库包含大量类似天然产物的合成分子，这些分子更有可能通过不同的机制靶向 Apaf-1。我们还将确定已确定的 Apaf-1 小分子抑制剂和 Apaf-1 途径其他成分抑制剂的作用机制。对于这一机制研究，将进行体外生化研究和使用特定细胞模型的分析，以深入了解已确定的抑制剂的分子基础及其在保护与放射癌症治疗相关的细胞凋亡损伤方面的潜在作用。已鉴定的 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希望的先导药物。它们也将成为研究细胞色素c介导的细胞凋亡在各种生理和疾病过程中的机制和作用的重要药理学工具。