In silico discovery and validation of potent small molecule inhibitors targeting the activation function-2 site of human estrogen receptor-¿ (Breast Cancer Research)

IntroductionCurrent approaches to inhibit estrogen receptor-alpha (ERα) are focused on targeting its hormone binding pocket and face limitations. Thus, we have proposed that inhibitors which bind to a coactivator binding pocket on ERα, called Activation Function-2 (AF2), might overcome some of these limitations. Methods: In silico virtual screening was used to identify small-molecule ERα AF2 inhibitors. These compounds were screened for inhibition of ERα transcriptional activity using stably transfected T47D-KBluc cell line. A direct physical interaction between the AF2 binders and the ERα protein was measured using Biolayer Interferometry (BLI) and an ERα coactivator displacement assay. Cell viability was assessed by MTS assay in ERα-positive MCF7 cells, tamoxifen-resistant (TamR) cell lines, TamR3, TamR6 and ERα-negative MDA-MB-453 and HeLa cell lines. In addition, ERα inhibition in TamR cells and effect of compounds on mRNA and protein expression of estrogen dependent genes, pS2, Cathepsin-D and cell division cycle 2 (CDC2) was determined. Results: Fifteen inhibitors from two chemical classes, derivatives of pyrazolidine-3,5-dione and carbohydrazide, were identified. Through a series of in vitro assays, VPC-16230 of the carbohydrazide chemical class emerged as a lead ERα AF2 inhibitor that significantly downregulated ERα transcriptional activity (half maximal inhibitory concentration (IC50) = 5.81 μM). By directly binding to the ERα protein as confirmed by BLI, VPC-16230 effectively displaced coactivator peptides from the AF2 pocket confirming its site-specific action. VPC-16230 selectively suppressed the growth of ERα-positive breast cancer cells. Furthermore, it significantly inhibited ERα mediated transcription in TamR cells. More importantly, it reduced mRNA and protein levels of pS2, Cathepsin-D and CDC2, validating its ER-directed activity. Conclusion: We identified VPC-16230 as an ERα AF2 specific inhibitor that demonstrated promising anti-proliferative effect in breast cancer cell lines including TamR cells. VPC-16230 reduced the expression of ERα-inducible genes including CDC2, which is involved in cell division. We anticipate that the application of ERα AF2 inhibitors will provide a novel approach that can act as complementary therapeutics to treat ERα-positive tamoxifen-resistant and metastatic breast cancers.