Ubiquitin E3 ligases and apoptosis in cancer drug discovery

描述（由申请人提供）：CARP 2是一种抗细胞凋亡的RING结构域蛋白，其特异性结合死亡结构域（DED）半胱天冬酶8和10，并通过作为E3泛素连接酶起负调节作用，导致靶半胱天冬酶的泛素化和最终蛋白酶体降解。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。因此，除了促进通过死亡受体途径起作用的药剂如TRAIL的抗肿瘤活性之外，预期CARP 2的抑制剂还具有内在抗肿瘤活性。该提案描述了基于酵母的检测的高通量筛选的开发，其中人CARP 2、其p53融合底物、半胱天冬酶8和p53报告基因（半乳糖苷酶）在S.啤酒。当所有这些组分都有功能时，CARP 2与内源性酵母泛素-蛋白酶体途径元件（E1，E2，蛋白酶体）合作，将Caspase 8-p53的水平保持在最低水平，导致没有报告活性。CARP 2的抑制允许半胱天冬酶融合的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组分后，通过在酵母中转化CARP 2、与p53融合的其底物半胱天冬酶8和p53响应性报告基因来配置测定。在调整试验条件以产生可接受的信噪比和高通量筛选的其他参数后，将筛选有限数量的化合物和天然产物提取物，以评价该试验用于抗癌药物开发的全规模高通量筛选的适用性。此外，将开发二次筛选试验，包括基于酵母的CARP 1试验，以过滤初步命中，用于临床前开发。这项工作的最终目标是将该检测方法转化为高通量筛选药物，该药物作为单一药物或联合治疗难治性癌症的组成部分具有活性

DESCRIPTION (provided by applicant): 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 (¿-galactosidase) are expressed in S. cerevisiae. When all of these components are functional, CARP2, in collaboration with the endogenous yeast ubiquitin-proteasomal pathway elements (E1, E2, proteasomes), keeps the level of 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