Ma Yongxian, Preet Anju, Tomita York, De Oliveira Eliseu, Zhang Li, Ueda Yumi, Clarke Robert, Brown Milton, Rosen Eliot M
Department of Oncology, Georgetown University School of Medicine, Washington, DC, USA.
Department of Center for Drug Discovery, Georgetown University School of Medicine, Washington, DC, USA.
Oncotarget. 2015 Dec 1;6(38):40388-404. doi: 10.18632/oncotarget.6323.
Previous studies indicate that BRCA1 protein binds to estrogen receptor-alpha (ER) and inhibits its activity. Here, we found that BRCA1 over-expression not only inhibits ER activity in anti-estrogen-resistant LCC9 cells but also partially restores their sensitivity to Tamoxifen. To simulate the mechanism of BRCA1 inhibition of ER in the setting of Tamoxifen resistance, we created a three-dimensional model of a BRCA1-binding cavity within the ER/Tamoxifen complex; and we screened a pharmacophore database to identify small molecules that could fit into this cavity. Among the top 40 "hits", six exhibited potent ER inhibitory activity in anti-estrogen-sensitive MCF-7 cells and four of the six exhibited similar activity (IC50 ≤ 1.0 μM) in LCC9 cells. We validated the model by mutation analysis. Two representative compounds (4631-P/1 and 35466-L/1) inhibited ER-dependent cell proliferation in Tamoxifen-resistant cells (LCC9 and LCC2) and partially restored sensitivity to Tamoxifen. The compounds also disrupted the association of BRCA1 with ER. In electrophoretic mobility shift assays, the compounds caused dissociation of ER from a model estrogen response element. Finally, a modified form of compound 35446 (hydrochloride salt) inhibited growth of LCC9 tumor xenografts at non-toxic concentrations. These results identify a novel group of small molecules that can overcome Tamoxifen resistance.
先前的研究表明，BRCA1蛋白与雌激素受体α（ER）结合并抑制其活性。在此，我们发现BRCA1的过表达不仅抑制抗雌激素耐药的LCC9细胞中的ER活性，还部分恢复了它们对他莫昔芬的敏感性。为了模拟在他莫昔芬耐药情况下BRCA1抑制ER的机制，我们构建了ER/他莫昔芬复合物中BRCA1结合腔的三维模型；并筛选了一个药效团数据库以鉴定能够契合该腔的小分子。在排名前40的“命中”分子中，有6个在抗雌激素敏感的MCF-7细胞中表现出强效的ER抑制活性，其中4个在LCC9细胞中表现出类似活性（IC50≤1.0μM）。我们通过突变分析验证了该模型。两种代表性化合物（4631-P/1和35466-L/1）抑制了他莫昔芬耐药细胞（LCC9和LCC2）中ER依赖的细胞增殖，并部分恢复了对他莫昔芬的敏感性。这些化合物还破坏了BRCA1与ER的结合。在电泳迁移率变动分析中，这些化合物导致ER从模型雌激素反应元件上解离。最后，化合物35446的一种修饰形式（盐酸盐）在无毒浓度下抑制了LCC9肿瘤异种移植物的生长。这些结果鉴定出了一组能够克服他莫昔芬耐药性的新型小分子。
