Development of high throughput assays to identify small molecule inhibitors of th

描述（由申请人提供）：CUL 4A泛素连接酶基因在多种肿瘤类型中经常扩增或过表达，包括乳腺癌、肝细胞癌、间皮瘤和鳞状细胞癌。虽然最近的研究已经确定了多聚体CUL 4A E3连接酶复合物的组分和几个细胞靶点，但CUL 4A在肿瘤发生中的作用在很大程度上仍然难以捉摸。我们和其他人表明，CUL 4A通过靶向降解DDB 2和XPC DNA损伤传感器以及G1/S DNA损伤检查点蛋白p21在核苷酸切除修复中起抑制作用。重要的是，我们的皮肤特异性CUL 4A敲除小鼠对UV和化学致癌物诱导的皮肤癌具有超抗性，并且在其他方面是健康的并且没有显示出异常，这表明CUL 4A可能是癌症预防的有吸引力的靶标。主要目的是开发和优化高通量筛选测定，以从NIH化学基因组中心（NCGC）的化学文库中鉴定CUL 4A泛素连接酶的小分子抑制剂。该应用建立在过去10年中在Pengbo Zhou博士实验室进行的CUL 4A泛素连接酶的广泛生物化学，细胞生物学和遗传学表征，以及体外和基于细胞的测定的全面阵列以及用于检查CUL 4A-DDB 1功能的小鼠敲除模型基础上。这些研究得到了Memorial Sloan-Kettering Cancer的Yueming Li博士小组和NIH化学基因组中心的Wei Zheng博士小组的广泛高通量筛选专业知识的补充。我们处于独特的地位，追求以下两个具体目标：（1）开发和优化基于AlphaLISA（R）的HTS测定法，用于CUL 4A-DDB 1相互作用;（2）配置评估命中化合物所需的二级HTS测定法。拟议研究的成功完成将为在HTS平台上应用CUL 4A-DDB 1（BPB）AlphaLISA（R）测定法筛选NCGC 300 K化学文库并鉴定CUL 4A-DDB 1泛素连接酶的小分子抑制剂奠定基础。这些研究代表了评估药理学CUL 4A抑制作为癌症预防和干预的有效方法的功效的第一步。 公共卫生相关性：CUL 4A基因在多种肿瘤类型中经常被发现扩增或过表达，包括乳腺癌、肝细胞癌、间皮瘤和鳞状细胞癌。最近的研究表明，CUL 4A的废除在增强DNA修复和DNA损伤反应方面具有显著的益处，并防止致癌。然而，目前没有可用于阻断CUL 4A泛素连接酶活性的药理学试剂。我们的总体目标是开发针对DDB-CUL 4A（或CRL 4）泛素连接酶的药理学抑制剂作为抗癌剂。在这里，我们建议采取关键的第一步，开发和优化的高通量筛选试验，并建立二级验证试验，目的是实现大的化学库的高通量筛选，以确定小分子抑制剂的CUL 4A泛素连接酶。该R21项目预计将为开发选择性靶向CUL 4A的药理学试剂作为新型癌症预防和/或治疗剂奠定基础。

DESCRIPTION (provided by applicant): The CUL4A ubiquitin ligase gene is frequently amplified or overexpressed in a wide variety of tumor types, including breast cancer, hepatocellular carcinomas, mesotheliomas, and squamous cell carcinomas. While recent studies have identified the components of the multimeric CUL4A E3 ligase complex and several cellular targets, the role of CUL4A in tumorigenesis remains largely elusive. We and others showed that CUL4A plays an inhibitory role in nucleotide excision repair through targeted degradation of the DDB2 and XPC DNA damage sensors, as well as the G1/S DNA damage checkpoint protein p21. Importantly, our skin-specific CUL4A knockout mice are hyper- resistant to UV- and chemical carcinogen-induced skin cancer, and are otherwise healthy and display no abnormalities, suggesting that CUL4A may be an attractive target for cancer prevention. The primary objective is to develop and optimize a high- throughput screening assay to identify small molecule inhibitors of the CUL4A ubiquitin ligase from chemical libraries at the NIH Chemical Genomic Center (NCGC). This application is built upon the extensive biochemical, cell biological, and genetic characterization of the CUL4A ubiquitin ligase performed in Dr. Pengbo Zhou's lab during the last 10 years, and the comprehensive array of in vitro and cell-based assays as well as the mouse knockout models to examine CUL4A-DDB1 functions. The studies are complemented by the extensive high-throughput screening expertise of Dr. Yueming Li's group at Memorial Sloan-Kettering Cancer and Dr. Wei Zheng's group at the NIH Chemical Genomic Center. We are uniquely positioned to pursue the following two specific aims: (1) to develop and optimize an AlphaLISA(R)-based HTS assay for CUL4A- DDB1 interaction; (2) to configure secondary HTS assays required to evaluate the hit compounds. Successful completion of the proposed studies will set the stage for application of the CUL4A-DDB1(BPB) AlphaLISA(R) assay on the HTS platform to screen the NCGC 300K chemical libraries and identify small molecule inhibitors of the CUL4A- DDB1 ubiquitin ligase. These studies represent the first step towards evaluating the efficacy of pharmacological CUL4A inhibition as an effective approach for cancer prevention and intervention. PUBLIC HEALTH RELEVANCE: The CUL4A gene is frequently found amplified or overexpressed in a wide variety of tumor types, including breast cancer, hepatocellulat carcinomas, mesotheliomas and squamous cell carcinomas. Recent studies suggest that abrogation of CUL4A presents significant benefits in enhancing DNA repair and DNA damage response, and protects against carcinogenesis. However, there is currently no pharmacological agent available for blocking the CUL4A ubiquitin ligase activity. Our overall goal is to develop pharmacological inhibitors against the DDB-CUL4A (or CRL4) ubiquitin ligase as anticancer agents. Here we propose to take the critical first step to develop and optimize a high throughput screening assay and to establish secondary validation assays, with the aim of implementing a high throughput screening of large chemical libraries to identify small molecule inhibitors of the CUL4A ubiquitin ligase. This R21 project expected to set the stage for developing pharmacological agents that selectively target CUL4A as novel cancer prevention and/or therapeutic agents.