Ubiquitin E3 ligases and apoptosis in cancer drug discovery

癌症药物发现中的泛素 E3 连接酶和细胞凋亡

• 批准号: 7073879
• 负责人: Michael R Mattern
• 金额: $ 21.41万
• 依托单位: PROGENRA, INC.
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-01 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7073879
• 关键词: apoptosis; cysteine endopeptidases; drug discovery /isolation; high throughput technology; neoplasm /cancer; ubiquitin; ubiquitin protein ligase; yeasts

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

描述(申请人提供)：CARP2是一种抗凋亡环域蛋白，通过作为E3泛素连接酶特异性结合并负调控死亡结构域(DED)caspase 8和10，导致目标caspase泛素化并最终蛋白酶体降解。这种连接酶在许多肿瘤中过度表达，使它们能够逃脱细胞凋亡并存活下来。因此，CARP2的抑制剂除了促进TRAIL等通过死亡受体途径发挥作用的药物的抗肿瘤活性外，还有望具有内在的抗肿瘤活性。这项建议描述了一种基于酵母的高通量筛选方法的发展，其中人CARP2、其与P53融合的底物caspase 8和P53报告基因(β-半乳糖苷酶)在酿酒酵母中表达。当所有这些成分都起作用时，CARP2与内源酵母泛素-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。抑制CARP2允许Caspase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件后，将通过在酵母中转化CARP2、其底物caspase 8与P53融合和P53反应报告来配置该分析。在调整分析条件以产生可接受的高通量筛选的信噪比和其他参数后，将对有限数量的化合物和天然产物提取物进行筛选，以评估该分析方法是否适合于抗癌药物开发的全面高通量筛选。此外，将开发二次筛选试验，包括基于酵母的CARP1试验，以筛选进展到临床前开发的一次命中。这项拟议工作的最终目标是将这种分析转化为高通量筛选，以筛选出一种有效的药物，作为联合治疗难治性癌症的单一药剂或成分。