【作者】 谭乘方；

【导师】 沈旭； 黄瑾；

【作者基本信息】 华东理工大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 雌激素受体(Estrogen Receptor, ER)是核受体超家族的成员之一,是一类重要的转录调控因子,是治疗乳腺癌、骨质疏松等疾病的重要药物作用靶标。选择性雌激素受体调节剂在治疗ER相关疾病上具有很高的临床应用价值。我们通过计算机虚拟筛选和生物实验相结合的手段来寻找新的选择性ER配体。首先我们采用分子动力学结合虚拟筛选的策略,从商业数据库中搜寻选择性ER配体。通过分子动力学模拟得到ER晶体结构的平均平衡构象作为虚拟筛选的结构基础,然后进行三轮基于分子对接的虚拟筛选,最后确定了70个候选化合物。在活性测试中,我们利用ER与其共激活因子的相互作用构建了酵母双杂交系统,通过检测报告基因活性的方式评价待测化合物的活性和作用类型。结果发现,在这70个化合物中至少有18个表现出良好的ER配体活性。其中有两个双效配体：同时表现为ERa拮抗剂及ERp激动剂；还有11个ER激动剂和5个ER拮抗剂,且不少化合物表现出较高的ER亚型选择性。通过细胞转染和报告基因检测实验,我们验证了这些化合物在细胞水平上能够改变ER的转录激活活性。MTT实验表明,具有ER拮抗活性的化合物能够抑制乳腺癌细胞增殖。我们还较深入地探讨了ER拮抗剂JB042对乳腺癌细胞的生长抑制作用。通过western blot和流式细胞周期分析实验,发现JB042可以下调雌二醇诱导的雌激素效应基因cyclin D1的表达,并将细胞周期阻滞于G1期,最终抑制癌细胞增殖。更多还原

【Abstract】 Estrogen receptor (ER), which belongs to the nuclear receptor super family, is a kind of important transcriptional regulator and is an essential drug target involving in breast cancer and osteoporosis. More and more research showed that selective estrogen receptor modulators had higher value in clinical application. We use computer virtual screening (VS) and bioassay to discover new selective ER ligands.First, we employed a strategy that combined molecular dynamics (MD) simulation with virtual screening to discover novel ER ligands from a commercial database. The average structure taken from equilibrated stage of ER crystal structure by MD simulation was prepared as the structural basis, and three rounds of VS were subsequently performed. Then 70 candidate compounds were selected for bioassay. We constructed yeast two-hybrid system to screen ligands and determine the capacity of compounds, which was based on the interaction of ER and its activator. As a result, more than 18 potent ligands were discovered. Among them, dual profile was observed in two ligands, as antagonists for ERαand agonists for ERβ; 11 agonists and 5 antagonists were discovered as well, and some of them showed high ER subtype selectivity.We performed cell transfection and luciferase activity assays and MTT assays to check the role of the novel ligands on ER transcriptional activation and antiproliferative potencies on breast cancer cell line. The novel ligands could effects ER transcription and the compounds that showed antagonistic activities on ERβor ERαdisplayed certain antiproliferative activities. We dissected the action of ER antagonist JB042 on suppressing breast caner cells proliferation. By western blot and flow cytometry analysis, we found that treatment with JB042 could down-regualte the expression of cycline D1 indued by estrogen and suppress the progression of G1 to S, subsequently inhibited cell proliferation.更多还原