Identification of ligands with bicyclic scaffolds provides insights into mechanisms of estrogen receptor subtype selectivity

Estrogen receptors α (ERα) and β (ERβ) have distinct functions and differential expression in certain tissues. These differences have stimulated the search for subtype-selective ligands. Therapeutically, such ligands offer the potential to target specific tissues or pathways regulated by one receptor subtype without affecting the other. As reagents, they can be utilized to probe the physiological functions of the ER subtypes to provide information complementary to that obtained from knock-out animals. A fluorescence resonance energy transfer-based assay was used to screen a 10,000-compound chemical library for ER agonists. From the screen, we identified a family of ERβ-selective agonists whose members contain bulky oxabicyclic scaffolds in place of the planar scaffolds common tomost ER ligands. These agonists are 10-50-fold selective for ERβ in competitive binding assays and up to 60-fold selective in transactivation assays. The weak uterotrophic activity of these ligands in immature rats and their ability to stimulate expression of an ERβ regulated gene in human U2OS osteosarcoma cells provides more physiological evidence of their ERβ-selective nature. To provide insight into the molecular mechanisms of their activity and selectivity, we determined the crystal structures of the ERα ligand-binding domain (LBD) and a peptide from the glucocorticoid receptor-interacting protein 1 (GRIP1) coactivator complexed with the ligands OBCP-3M, OBCP-2M, and OBCP-1M. These structures illustrate how the bicyclic scaffolds of these ligands are accommodated in the flexible ligand-binding pocket of ER. A comparison of these structures with existing ER structures suggests that the ERβ selectivity of OBCP ligands can be attributed to a combination of their interactions with Met-336 in ERβ and Met-421 in ERα. These bicyclic ligands show promise as lead compounds that can target ERβ. In addition, our understanding of the molecular determinants of their subtype selectivity provides a useful starting point for developing other ER modulators belonging to this relatively new structural class.