Identification of Ligands with Bicyclic Scaffolds Provides Insights into Mechanisms of Estrogen Receptor Subtype Selectivity

Abstract

Estrogen receptors ␣ (ER␣) and ␤ (ER␤) 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 ER␤-selective agonists whose members contain bulky oxabicyclic scaffolds in place of the planar scaffolds common to most ER ligands. These agonists are 10 -50-fold selective for ER␤ 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 ER␤ regulated gene in human U2OS osteosarcoma cells provides more physiological evidence of their ER␤-selective nature. To provide insight into the molecular mechanisms of their activity and selectivity, we determined the crystal structures of the ER␣ ligandbinding 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 ER␤ selectivity of OBCP ligands can be attributed to a combination of their interactions with Met-336 in ER␤ and Met-421 in ER␣. These bicyclic ligands show promise as lead compounds that can target ER␤. 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. Estrogen receptors ␣ (ER␣)4 and ␤ (ER␤) are ligand-inducible transcription factors that are involved in regulating cell growth, proliferation, and differentiation in various normal and cancerous tissues (1-3). Although the two subtypes of ER bind the endogenous estrogen, 17␤-estradiol (E2), with similar affinity (4), they differ in size, share modest sequence identity (47%), and are encoded by different genes (5, 6). Studies of knock-out mice have also shown that the two subtypes have distinct functions and are differentially expressed in certain tissues (1). These differences have stimulated the search for subtype-specific ligands that can elicit tissue-or cell-specific ER activity. In particular, the dominance of ER␣ expression in the breast and uterus (7) suggests that ER␤-selective ligands may offer some of the benefits of hormone replacement therapy such as a decrease in the risk of colorectal cancer (8) without increasing the risk of breast ...

Molecular characterization of a B-ring unsaturated estrogen: Implications for conjugated equine estrogen components of Premarin

“…pCMV-βgal was obtained from Invitrogen. Other plasmids used for protein expression have been described previously [12].…”

“…Steps for the expression and purification of ERα LBD, as well as the initial screening for crystallization conditions were performed as previously described [12]. For NCI 122, optimization of initial screen conditions yielded diffraction-quality crystals of ER in ~ 1 week at 16 °C in 0.2 M ammonium sulfate, 0.1 M Tris-HCl, 25 % (w/v) PEG 3350, pH 8.5.…”

“…Plasmids used in this assay, and methods for the expression of FLAG-SRC1 and GST-ERα LBD were previously described [12]. For the HTRF assay, FLAG-SRC1 and GST-ERα LBD were dialyzed in HTRF buffer (50 mM HEPES, pH 7.4, 50 mM KCl, 0.1% (v/v) Tween 20, 1 mM EDTA, 1 mM dithiothreitol and 1 mg/ml bovine serum albumin) and separately preincubated with anti-FLAG-XL665 and anti-GST-Eu(K) (Cisbio Internation (Gif/Yvette Cedex, France)), respectively, for 1 h at 4°C.…”

“…Plasmids used in this assay and methods for expressing His-tagged ERα LBD and ERβ LBD have been previously described [12]. His-tagged ERα LBD/ERβ LBD was diluted with binding buffer (50 mM Tris pH 8.0, 100 mM NaCl, 0.01% beta-octyl glucoside, and 0.1% (v/v) glycerol) to 2-8 nM and added to the wells of nickel-coated FlashPlates (PerkinElmer).…”

“…We have identified NCI 122 (17β-methyl-17α-dihydroequilenin), from a screen of approximately 10,000 compounds [12] that shares many similarities with some of the estrogens in CEE. For example, the unsaturated B-ring and 17α-hydroxyl (17α-OH) group of NCI 122 are also present in half the estrogens found in CEE ( Figure 1).…”