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

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