Characterization of novel small molecule inhibitors of estrogen receptor-activation function 2 (ER-AF2)

Up to 40% of patients with estrogen receptor (ER)-positive breast cancer will develop resistance against the majority of current ER-directed therapies. Resistance can arise through various mechanisms such as increased expression levels of coregulators, and key mutations acquired in the receptor's ligand binding domain rendering it constitutively active. To overcome these resistance mechanisms, we explored targeting the ER Activation Function 2 (AF2) site, which is essential for coactivator binding and activation. Using artificial intelligence and the deep docking methodology, we virtually screened > 1 billion small molecules and identified 290 potential AF2 binders that were then characterized and validated through an iterative screening pipeline of cell-based and cell-free assays. We ranked the compounds based on their ability to reduce the transcriptional activity of the estrogen receptor and the viability of ER-positive breast cancer cells. We identified a lead compound, VPC-260724, which inhibits ER activity at low micromolar range. We confirmed its direct binding to the ER-AF2 site through a PGC1α peptide displacement experiment. Using proximity ligation assays, we showed that VPC-260724 disrupts the interaction between ER-AF2 and the coactivator SRC-3 and reduces the expression of ER target genes in various breast cancer models including the tamoxifen resistant cell line TamR3. In conclusion, we developed a novel ER-AF2 binder, VPC-260724, which shows antiproliferative activity in ER-positive breast cancer models. The use of an ER-AF2 inhibitor in combination with current treatments may provide a novel complementary therapeutic approach to target treatment resistance in ER-positive breast cancer.