Assays for Estrogen /Progesterone Receptor Antagonists

雌激素/孕激素受体拮抗剂的测定

• 批准号: 6958695
• 负责人: DAVID J SHAPIRO
• 金额: $ 27.14万
• 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/6958695
• 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）发挥作用，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型荧光各向异性微孔板测定（FAMA）。我们使用 FAMA 分析了配体 ER 和 PR 与其 DNA 反应元件以及全长共激活子 (SRC1) 的相互作用，并证明了 FAMA 在高通量筛选 (HTS) 分析中的潜力。我们将使用 FAMA 开发 HTS 测定法，以鉴定针对 ER 和 PR 作用不同步骤的新型 ER 和 PR 拮抗剂。具体目标是： 1. 我们将开发一种小分子 HTS 检测方法，干扰 ER 和 PR 与其各自 DNA 反应元件（ERE 和 PRE/GRE）的结合，并对 ER 和 PR 表现出选择性。 2. 我们将开发一种 HTS 检测方法，检测干扰乳腺癌共激活剂 SRC1 和 Amplified in Breast Cancer 1 (AIB1) 与 ERE-E2: ER 复合物结合的化合物。 3. 我们将开发小分子的 HTS 检测方法，以刺激热休克蛋白和分子伴侣用 DMA 反应元件和共激活剂分解 ER 和 PR 复合物的能力。 HTS 适用性研究和测定验证将包括分析 FAMA 的精度、质量（Z1 和 Z 因子）、已知抑制剂的加标以及化合物荧光的对照。对于每次检测，我们将筛选大约 3,000 种化合物。对候选小分子和对照小分子的验证测定将包括 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 诱导的细胞 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。