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）：通过雌激素和孕激素受体（ER和PR）作用的雌激素和孕激素与许多乳腺癌的生长和转移有关。他莫昔芬以及其他修饰ER和PR作用的药物在受体的配体结合口袋中结合并导致耐药性肿瘤的发展。 我们最近开发了一种新型的荧光各向异性微孔板测定法（FAMA）。我们使用FAMA分析了配体ER和PR与其DNA响应元件以及全长共激活因子（SRC1）的相互作用，并证明了FAMA对高吞吐量筛选（HTS）测定的潜力。我们将使用FAMA开发HTS测定法，以识别针对ER和PR行动不同步骤的新类ER和PR拮抗剂。具体目的是：1。我们将开发针对小分子的HTS测定法，该分子干扰ER和PR与其各自的DNA响应元件（ERES和PRE/GRE）的结合，并且对ER和PR具有选择性。 2。我们将开发用于干扰共激活剂SRC1结合并在乳腺癌1（AIB1）与ERE-E2：ER-E2：ER复合物中扩增的化合物的HTS分析。 3。我们将开发针对小分子的HTS分析，以刺激热激蛋白和伴侣蛋白与DMA响应元件以及与共激活剂的ER和PR复合物的复合物的能力。 HTS的适用性研究和测定验证将包括对FAMA的精度，质量（Z1和Z因子）的分析，具有已知抑制剂的峰值以及复合荧光的对照。对于每种测定，我们将筛选大约3,000种化合物。对候选和对照小分子的验证分析将包括EMSA，对乳腺癌细胞雌激素依赖性生长的影响，瞬时转染，ER的定量RT-PCR和PR诱导的细胞mRNA，靶蛋白的染色质IP和RNAI。这些研究针对新的部位来拮抗癌细胞的生长和转移，并且是基于FAMA的HTS测定法的原型。