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

细胞凋亡是多细胞生物体用于维持组织稳态和消除不需要的或受损的细胞的程序性细胞死亡的主要形式。它在发育，免疫反应和许多其他生理事件中起着关键作用。在哺乳动物中，一个重要的凋亡途径是线粒体细胞色素c介导的半胱天冬酶激活途径。在该途径中，各种凋亡刺激诱导细胞色素c从线粒体释放。释放的细胞色素c结合并激活必需介质Apaf-1，以触发多聚体蛋白复合物（即核糖体）的组装，其进而激活细胞凋亡的下游分子执行器（半胱天冬酶）。 细胞色素c凋亡途径的失调可导致疾病，如癌症、免疫紊乱和神经退行性疾病。相反，通过增强和抑制细胞凋亡来靶向细胞凋亡组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希望的靶向癌症疗法被设计为特异性激活细胞色素c凋亡途径。另一方面，该途径的抑制在治疗具有病理性增强的细胞凋亡的症状如神经变性和缺血性损伤中也应该是有效的。重要的是，抑制该途径也是减少放射性癌症治疗的不良副作用的有希望的方法，放射性癌症治疗通过非凋亡机制作用于许多实体瘤，但在正常组织中诱导大量凋亡，特别是在造血系统和胃肠道中，从而对患者造成严重的和偶尔致命的损伤。在这项资助中，我们将进行高通量筛选（HTS），以确定Apaf-1-抑制化合物从各种化学库，并随后表征的作用机制，确定的抑制剂和他们的潜力，在保护辐射损伤。 到目前为止，我们已经成功地开发和优化了HTS的Apaf-1功能测定。作为该HTS测定的验证，我们筛选了一系列药物样化合物，并获得了抑制Apaf-1介导的半胱天冬酶活化的多个阳性命中。这一进展将为我们在ARRA基金支持的两年内及时实现该研究资助的最终目标铺平道路。在这两年里，我们将从一个独特的化学库中筛选额外的抑制化合物，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。我们还将确定已鉴定的Apaf-1小分子抑制剂和Apaf-1途径中其他组分的抑制剂的作用机制。对于这一机制的调查，在体外生物化学研究和分析使用特定的细胞模型将进行深入了解的分子基础上确定的抑制剂和它们的潜在影响，在保护与放射治疗癌症相关的凋亡损伤。所鉴定的Apaf-1抑制性化合物将是用于治疗诸如癌症和脑/心肌缺血性损伤的疾病的有前景的药物先导。它们也将是研究细胞色素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。

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.