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

开发高通量测定法来鉴定小分子抑制剂

• 批准号: 8103587
• 负责人: Pengbo Zhou
• 金额: $ 16.9万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8103587
• 关键词: Affinity; Antineoplastic Agents; Binding; Biochemical; Biological; Biological Assay; CUL4A gene; Cancer Biology; Cancer Prevention Intervention; Carcinoma; Cells; Chemicals; Complement; Complex; DNA Damage; DNA Repair; DNA Repair Pathway; DNA damage checkpoint; Defect; Development; Diagnosis; Dimethyl Sulfoxide; Disease; Genetic; Genomics; Goals; Human; In Vitro; Incidence; Knockout Mice; Laboratories; Learning; Libraries; Malignant Neoplasms; Memorial Sloan-Kettering Cancer Center; Mesothelioma; Modeling; Molecular; Mus; Nucleotide Excision Repair; Ozone; Pathway interactions; Pharmaceutical Preparations; Play; Positioning Attribute; Prevention; Primary carcinoma of the liver cells; Regulation; Reporter; Resistance; Roentgen Rays; Role; Skin; Skin Cancer; Skin Carcinogenesis; Specificity; Speed; Squamous cell carcinoma; Staging; Structure; Therapeutic Agents; Translating; UV induced; UV induced DNA damage; UV sensitive; Ubiquitin; Ubiquitin Ligase Gene; Ubiquitination; United States; United States National Institutes of Health; Validation; Xeroderma Pigmentosum; base; cancer prevention; cancer therapy; carcinogenesis; cellular targeting; chemical carcinogen; counterscreen; high throughput screening; in vivo; inhibitor/antagonist; malignant breast neoplasm; miniaturize; multicatalytic endopeptidase complex; novel; oncoprotein p21; overexpression; reconstitution; response; sensor; small molecule; small molecule libraries; sunlight-induced; tumor; tumorigenesis; ubiquitin ligase; ubiquitin-protein ligase

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.

描述（由申请人提供）：CUL4A泛素连接酶基因经常在多种肿瘤类型中放大或过表达，包括乳腺癌，肝细胞癌，间皮瘤和鳞状细胞癌。尽管最近的研究确定了多聚体Cul4a E3连接酶复合物和几个细胞靶标的成分，但CUL4A在肿瘤发生中的作用仍然难以捉摸。我们和其他人表明，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诱导的皮肤癌具有过度抗性，否则健康，没有异常，这表明CUL4A可能是预防癌症的有吸引力的靶标。主要目的是开发和优化高吞吐量筛选测定法，以鉴定NIH化学基因组中心（NCGC）的化学文库中CUL4A泛素连接酶的小分子抑制剂。该应用建立在过去10年中Pengbo Zhou博士实验室中进行的CUL4A泛素连接酶的广泛的生化，细胞生物学和遗传表征，以及全面的基于细胞和细胞的测定阵列以及检查CUL4A-DDB1的小鼠敲除模型的功能。这项研究得到了纪念斯隆 - 凯特林癌症的Yueming li小组的广泛的高通量筛查专业知识，以及NIH化学基因组中心的Wei Zheng小组。我们有独特的位置来追求以下两个特定目标：（1）开发和优化基于α（R）基于alphalisa（R）的HTS分析，以进行CUL4A-DDB1相互作用； （2）配置评估HIT化合物所需的辅助HTS分析。拟议研究的成功完成将为应用HTS平台上的CUL4A-DDB1（BPB）α（R）测定奠定阶段，以筛选NCGC 300K化学文库的筛选，并识别CUL4A-A-DDB1泛素依比喹啉ligase的小分子抑制剂。这些研究代表了评估药理学CUL4A抑制作用作为预防癌症和干预的有效方法的第一步。 公共卫生相关性：经常发现CUL4A基因在各种肿瘤类型中被放大或过表达，包括乳腺癌，肝纤维素癌，间皮瘤和鳞状细胞癌。最近的研究表明，Cul4a的废除在增强DNA修复和DNA损伤反应方面具有重大好处，并防止致癌作用。但是，目前尚无药理学剂可阻止CUL4A泛素连接酶活性。我们的总体目标是开发针对DDB-CUL4A（或CRL4）泛素连接酶作为抗癌剂的药理抑制剂。在这里，我们建议采取关键的第一步，以开发和优化高吞吐量筛选测定法并建立二级验证测定法，以实施对大型化学文库的高吞吐量筛选，以鉴定CUL4A泛素蛋白小酶的小分子抑制剂。该R21项目预计将为开发药理学剂奠定阶段，这些药理学剂选择性地将CUL4A作为新型癌症预防和/或治疗剂旨在靶向。