In silico discovery and validation of potent small-molecule inhibitors targeting the activation function 2 site of human oestrogen receptor aα

Introduction: Current approaches to inhibit oestrogen receptor-alpha (ERaα) are focused on targeting its hormone-binding pocket and have limitations. Thus, we propose that inhibitors that bind to a coactivator-binding pocket on ERaα, called activation function 2 (AF2), might overcome some of these limitations. Methods: In silico virtual screening was used to identify small-molecule ERaα AF2 inhibitors. These compounds were screened for inhibition of ERaα transcriptional activity using stably transfected T47D-KBluc cell line. A direct physical interaction between the AF2 binders and the ERaα protein was measured using biolayer interferometry (BLI) and an ERaα coactivator displacement assay. Cell viability was assessed by MTS assay in ERaα-positive MCF7 cells, tamoxifen-resistant (TamR) cell lines TamR3 and TamR6, and ERaα-negative MDA-MB-453 and HeLa cell lines. In addition, ERaα inhibition in TamR cells and the effect of compounds on mRNA and protein expression of oestrogen-dependent genes, pS2, cathepsin D and cell division cycle 2 (CDC2) were determined. Results: Fifteen inhibitors from two chemical classes, derivatives of pyrazolidine-3,5-dione and carbohydrazide, were identified. In a series of in vitro assays, VPC-16230 of the carbohydrazide chemical class emerged as a lead ERaα AF2 inhibitor that significantly downregulated ERaα transcriptional activity (half-maximal inhibitory concentration = 5.81 μM). By directly binding to the ERaα 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 ERaα-positive breast cancer cells. Furthermore, it significantly inhibited ERaα 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 ERaα AF2-specific inhibitor that demonstrated promising antiproliferative effects in breast cancer cell lines, including TamR cells. VPC-16230 reduced the expression of ERaα-inducible genes, including CDC2, which is involved in cell division. We anticipate that the application of ERaα AF2 inhibitors will provide a novel approach that can act as a complementary therapeutic to treat ERaα-positive, tamoxifen-resistant and metastatic breast cancers.