Assays for Estogen and Progesterone Receptor Antagonists

雌激素和孕激素受体拮抗剂的测定

• 批准号: 7094064
• 负责人: DAVID J SHAPIRO
• 金额: $ 26.93万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-15 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7094064
• 关键词: RNA interference; breast neoplasms; chromatin immunoprecipitation; estrogen inhibitor; estrogen receptors; fluorescence polarization; heat shock proteins; high throughput technology; hormone inhibitor; hormone related neoplasm /cancer; molecular chaperones; neoplastic cell; progesterone receptors; receptor binding; small molecule; technology /technique development; tissue /cell culture; transfection

DESCRIPTION (provided by applicant): Estrogens and progestins, acting through the estrogen and progesterone receptors (ER and PR) are implicated in the growth and metastases of many breast cancers. Tamoxifen, and other drugs that modify ER and PR action bind in the receptor's ligand binding pocket and lead to development of resistant tumors. We recently developed a novel fluorescence anisotropy microplate assay (FAMA). We used the FAMA to analyze the interaction of liganded ER and PR with their DNA response elements and with a full-length co-activator (SRC1) and demonstrated FAMA's potential for high throughput screening (HTS) assays. We will use the FAMA to develop HTS assays for identification of new classes of ER and PR antagonists targeted at different steps in ER and PR action. The Specific Aims are: 1. We will develop an HTS assay for small molecules that interfere with binding of ER and PR to their respective DNA response elements (EREs and the PRE/GRE) and that exhibit selectivity for ER and PR. 2. We will develop an HTS assay for compounds that interfere with binding of the co-activators SRC1 and Amplified in Breast Cancer 1 (AIB1) to ERE-E2: ER complexes. 3. We will develop HTS assays for small molecules that stimulate the ability of heat shock proteins and chaperones to disassemble complexes of ER and PR with DMA response elements, and with co-activators. HTS suitability studies and assay validation will include analysis of FAMA's precision, quality (Z1 and Z factor), spiking with known inhibitors, and controls for compound fluorescence. For each assay, we will screen approximately 3,000 compounds. Verification assays on candidate and control small molecules will include EMSA, effects on estrogen-dependent growth of breast cancer cells, transient transfections, quantitative RT-PCR of ER and PR-induced cell mRNAs, chromatin IP and RNAi of target proteins. These studies target novel sites for antagonizing cancer cell growth and metastases, and are prototypes for FAMA-based HTS assays.

描述（由申请人提供）：雌激素和孕激素通过雌激素和孕激素受体（ER和PR）起作用，与许多乳腺癌的生长和转移有关。他莫昔芬和其他改变ER和PR作用的药物结合在受体的配体结合口袋中，并导致耐药肿瘤的发展。 我们最近开发了一种新的荧光各向异性微孔板分析（法马）。我们使用法马来分析配体ER和PR与它们的DNA响应元件和全长共激活因子（SRC 1）的相互作用，并证明法马用于高通量筛选（HTS）测定的潜力。我们将使用法马开发HTS测定法，用于鉴定针对ER和PR作用中不同步骤的新型ER和PR拮抗剂。具体目标是：1。我们将开发一种HTS检测方法，用于检测干扰ER和PR与其各自的DNA反应元件（ERE和PRE/GRE）结合的小分子，并对ER和PR表现出选择性。我们将开发一种HTS检测方法，用于检测干扰共激活剂SRC 1和乳腺癌扩增因子1（AIB 1）与ERE-E2：ER复合物结合的化合物。3.我们将开发用于刺激热休克蛋白和分子伴侣分解ER和PR与DMA反应元件和共激活剂的复合物的能力的小分子的HTS测定。 HTS适用性研究和试验验证将包括法马精密度、质量（Z1和Z因子）、已知抑制剂加标和化合物荧光对照的分析。对于每项检测，我们将筛选约3，000种化合物。候选和对照小分子的验证试验将包括EMSA、对乳腺癌细胞雌激素依赖性生长的影响、瞬时转染、ER和PR诱导细胞mRNA的定量RT-PCR、染色质IP和靶蛋白的RNAi。这些研究针对拮抗癌细胞生长和转移的新位点，并且是基于FAMA的HTS测定的原型。