Ubiquitin E3 ligases and apoptosis in cancer drug discovery

翻译后摘要：CARP 2是一种抗凋亡的RING结构域蛋白，特异性结合和负调控 死亡结构域（DED）半胱天冬酶8和10，作为E3泛素连接酶，引起泛素化， 最终蛋白酶体降解靶半胱天冬酶。这种连接酶在许多肿瘤中过度表达， 使它们能够逃避凋亡并存活下来。因此，预期CARP 2的抑制剂具有 除了促进诸如TRAIL的药剂的抗肿瘤活性之外，还具有内在的抗肿瘤活性，所述药剂通过 死亡受体通路该提案描述了高通量筛选的发展， 基于酵母的测定，其中人CARP 2、其p53融合底物、半胱天冬酶8和p53报告基因 （β-半乳糖苷酶）在S.啤酒。当所有这些组件都起作用时，CARP 2， 与内源性酵母泛素-蛋白酶体途径元件（E1，E2，蛋白酶体）的协作， 使Caspase 8-p53的水平保持在最低水平，导致没有报道活性。CARP 2的抑制允许 Caspase融合p53，与其反应元件结合并激活报告基因，产生阳性信号 （荧光）。在克隆和构建必要的 通过转化CARP 2，其底物半胱天冬酶8与p53融合，以及p53-应答性半胱天冬酶， 报告员，在酵母中。在调整测定条件以产生可接受的信噪比和其他参数之后， 高通量筛选的参数，有限数量的化合物和天然产物提取物将被 筛选以评价该测定法用于抗癌药物全规模高通量筛选的适用性 发展此外，二次筛选试验，包括基于酵母的CARP 1试验，将在 开发用于筛选主要命中，以进行临床前开发。建议的最终目标 工作是将该测定转化为高通量筛选作为单一药剂或作为药物组合物具有活性的药物。 针对难治性癌症的组合疗法的组分

Abstract: CARP2 is an anti-apoptotic RING-domain protein that specifically binds to and negatively regulates death domain (DED) caspases 8 and 10 by acting as an E3 ubiquitin ligase, causing ubiquitination and ultimately proteasomal degradation of the target caspase. This ligase is over-expressed in many tumors, permitting them to escape apoptosis and survive. Inhibitors of CARP2 would thus be expected to have intrinsic antitumor activity in addition to facilitating antitumor activity of agents such as TRAIL, which act via the death receptor pathway. This proposal describes the development for high throughput screening of a yeast-based assay in which human CARP2, its p53-fused substrate, caspase 8, and a p53 reporter (p-galactosidase) are expressed in S. cerevisiae. Wnen all of these components are functional, CARP2, in collaboration with the endogenous yeast ubiquitin-proteasomal pathway elements (E1, E2, proteasomes), keeps the level pf Caspase 8-p53 at a minimum, resulting in no reporter activity. Inhibition of CARP2 permits Caspase-fused p53 to bind to its response element and activate the reporter, producing a positive signal (fluorescence). The assay will be configured, following cloning and construction of the necessary components, by transformation of CARP2, its substrate caspase 8 fused to p53, and a p53-responsive reporter, in yeast. After adjusting assay conditions to produce an acceptable signal:noise ratio and other parameters of high throughput screening, limited numbers of compounds and natural product extracts will be screened to evaluate the suitability of the assay for full scale high throughput screening for anticancer drug development. In addition, secondary screening assays, including a yeast based assay for CARP1, will be developed to filter primary hits for progression to preclinical development. The ultimate goal of the proposed work is to translate the assay to high throughput screening for a drug that is active as a single agent or a component of combination therapy against refractory cancers