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

CUL4A泛素连接酶基因在多种肿瘤类型中经常被扩增或过表达，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CUL4A E3连接酶复合物的组分和几个细胞靶点，但CUL4A在肿瘤发生中的作用在很大程度上仍然难以捉摸。我们和其他人发现CUL4A通过靶向降解DDB2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CUL4A敲除小鼠对紫外线和化学致癌物质诱导的皮肤癌具有高度抗性，并且在其他方面健康且没有异常，这表明CUL4A可能是一个有吸引力的癌症预防靶点。主要目的是开发和优化一种高通量筛选试验，从NIH化学基因组中心（NCGC）的化学文库中鉴定CUL4A泛素连接酶的小分子抑制剂。该应用程序建立在过去10年里彭波博士的实验室对CUL4A泛素连接酶进行的广泛的生化、细胞生物学和遗传表征的基础上，以及全面的体外和基于细胞的分析以及小鼠敲除模型来检查CUL4A- ddb1功能。这些研究得到了纪念斯隆-凯特琳癌症中心的李岳明博士小组和美国国立卫生研究院化学基因组中心的郑伟博士小组广泛的高通量筛查专业知识的补充。我们具有独特的优势，可以实现以下两个具体目标：(1)开发和优化基于AlphaLISA(R)的CUL4A- DDB1相互作用HTS检测；(2)配置评价命中化合物所需的二级HTS分析。拟议研究的成功完成将为CUL4A-DDB1(BPB) AlphaLISA(R)检测在HTS平台上的应用奠定基础，以筛选NCGC 300K化学文库并鉴定CUL4A-DDB1泛素连接酶的小分子抑制剂。这些研究是评估CUL4A药物抑制作为癌症预防和干预有效方法的有效性的第一步。

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.