雌激素相关受体α调节剂细胞筛选模型的建立及应用  

Development of a robust cellular assay for screening estrogen-related receptor α modulators

作　　者：李群益[1,2] 陈莉[3] 杜永丽[4] 张留弟 钟明康[1,2] 施孝金[1,2] 

机构地区：[1]复旦大学附属华山医院临床药学室,上海200040 [2]复旦大学附属华山医院北院临床药学室,上海201907 [3]上海市徐汇区中心医院药械科,上海200031 [4]齐鲁工业大学化工与制药系,济南250353

出　　处：《中国新药杂志》2015年第3期351-356,共6页Chinese Journal of New Drugs

基　　金：国家自然科学基金(81302853;81202389);教育部"高校博士点"基金(20110071120071)

摘　　要：目的:建立一种筛选评价雌激素相关受体α(estrogen-related receptorα,ERRα)调节剂的细胞模型,并用于计算机辅助设计、人工合成的化合物样品的评价。方法:用p CMV-h ERRα,3×ERRE-Luc及pRL-TK共同转染人胚肾细胞HEK-293,加入阳性药XCT790或筛选化合物孵育,分别检测HEK-293中萤火虫荧光素酶及海肾荧光素酶的活性,建立基于受体-应答元件的ERRα调节剂细胞筛选模型,用Z'因子评价本模型的稳定性,并测试了83个化合物对ERRα转录调节活性。用CCK-8检测DLE2-24对乳腺癌细胞MCF-7的增殖抑制效应,用RT-PCR考察DLE2-24对MCF-7中ERRα靶基因SOD2及VEGF的mRNA水平的影响。结果:成功建立ERRα-ERRE-Luc基因瞬时共转染模型,阳性药XCT790的IC50为0.24μmol·L-1,Z'因子为0.68,信号/本底为20.7。应用本模型筛选出15个ERRα反向调节剂。其中,DLE2-24剂量依赖性地抑制MCF-7的增殖,显著下调了ERRα下游靶基因SOD2及VEGF的mRNA水平。结论:本研究成功建立了一种高效、可靠的ERRα调节剂细胞筛选模型,适用于ERRα调节剂的发现与评价。Objective： To develop a robust cellular assay for screening synthetic modulators of estrogen-re- lated receptor α （ERRα） which were designed by computer. Methods： pCMV-hERRα, 3 × ERRE-Luc and pRL- TK were co-transfected into Human Embryonic Kidney 293 （ HEK 293） cells. After incubation with control com- pound, XCT790, or test compounds, HEK 293 cells were lysed and the activities of firefly luciferase and renilla lu- ciferase were detected respectively. In this way, a cellular model for screening ERRα modulators was developed based on receptor-response element reporter gene system. Z＇ factor was used to evaluate the quality of this assay system. This method was applied to a small-scale screening campaign against eighty-three synthetic compounds de- signed by computer. CCK-8 was used to evaluate the antiproliferative effects of DLE2-24 in MCF-7 ceils, a breast cancer cell line. Effects of DLE2-24 on mRNA levels of SOD2 and VECF in MCF-7 cells were detected by RT- PCR. Results： The IC50 value （0.24 μmol· L^-1） of the control compound （XCT790） assessed with the reporter gene approach was comparable and consistent with that reported in the literature. Signal to background （S/B） ratio and Z＇ factor of the assay system were evaluated to be 20.7 and 0. 68, respectively. In a small-scale screening campaign of 83 synthetic compounds, 15 hits were confirmed as potent ERRα inverse agonists, of which, DLE2-24 displayed sig-nificant antiproliferative effects and downregulated the mRNA levels of SOD2 and VEGF in MCF-7 cells. Conclusion ： The cellular ERRα reporter gene assay is an efficient and robust tool to screen potential ERRor modulators.

关 键 词：雌激素相关受体α 反向激动剂 肿瘤 增殖 

分 类 号：R965.1[医药卫生—药理学] R975.5[医药卫生—药学]