Interactions of synthetic estrogens with human estrogen receptors G N Nikov, M Eshete, R V Rajnarayanan and W L Alworth Department of Chemistry, Tulane University, New Orleans, Louisiana 70118, USA (Requests for offprints should be addressed to W L Alworth; Email: alworth@tulane.edu) Abstract Synthetic estrogens have diverse chemical structures and may either positively or negatively affect the estrogenic signaling pathways through interactions with the estrogen receptors (ERs). Modeling studies suggest that 4-(1-adamantyl)phenol (AdP) and 4,4-(1,3- adamantanediyl)diphenol (AdDP) can bind in the ligand binding site of ER. We used fluorescence polarization (FP) to compare the binding affinities of AdP, AdDP and 2-(1-adamantyl)-4-methylphenol (AdMP) for human ER and ER with the binding affinities of the known ER ligands, diethylstilbestrol (DES) and 4- hydroxytamoxifen (4OHT). Competition binding experi- ments show that AdDP has greater affinity for both ERs than does AdP, while AdMP does not bind the receptor proteins. The relative binding affinities of AdDP and AdP are weaker than the affinity of DES or 4OHT for both ERs with the exception of AdDP, which binds ER with higher affinity than does 4OHT. We also found that AdDP and AdP cause differential conformational changes in ER and ER, which result in altered affinities of the ERs for fluorescein-labeled estrogen response elements (EREs) using a direct binding FP assay. The results show that ER liganded with either AdDP or AdP has greater affinity for human pS2 ERE than the ER–4OHT complex. The data suggest that synthetic molecules like adamantanes may function as biologically active ligands for human ERs. This demonstrates the importance of consid- ering the potential of novel classes of synthetic compounds as selective ER modulators. Journal of Endocrinology (2001) 170, 137–145 Introduction The estrogen receptor (ER) is a ligand-dependent tran- scriptional factor with domain structure (Kumar et al. 1987) and belongs to a supergene family that includes receptors for steroid and thyroid hormones, vitamin D 3 and retinoic acid (Evans 1988). In the absence of a ligand the ER resides in the cell nucleus associated with heat- shock proteins (Joab et al. 1984, Sanchez et al. 1990). Binding of the hormone estrogen causes the ER to undergo conformational changes leading to dissociation of the heat-shock proteins and formation of stable receptor dimers (Kumar & Chambon 1988). The ligand-occupied receptor dimers interact with estrogen response elements (EREs) located within the regulatory region of target genes, and then are able to interact with other cellular components to either activate or suppress transcription of a target gene in a promoter- and cell-specific manner (Tora et al. 1989). Recently it has been discovered that a second ER (ER) exists, in addition to the traditional ER, now called ER. ER and ER share common physical and func- tional properties; they also have high degrees of homology in their ligand binding domains (LBDs) and DNA binding domains (Kuiper et al. 1996, Mosselman et al. 1996). Both ERs have similar affinities for 17-estradiol (E 2 ), recognize the same EREs and are expressed in distinct and overlapping tissues (Couse et al. 1997). In addition to the endogenous estrogens, a group of exogenous chemicals called xenoestrogens can display estrogen-like functions in estrogen responsive tissues. The source of xenoestrogens can be dietary in nature including phytoestrogens (Kurzer & Xu 1997) or industrial chemi- cals (pharmaceuticals, pesticides, pollutants) (Korach et al. 1997). The xenoestrogens interact with the ERs and can either induce a response that mimics endogenous estrogen stimulation or produce an inactive receptor–ligand complex that inhibits the transcription of ER regulated genes. The synthetic xenoestrogens have diverse chemical structures but there are some common structural motifs among these compounds. The most essential structural motif that elicits estrogenic activity is a phenol that is relatively unhindered, attached to a rather bulky hydro- phobic structure (Katzenellenbogen 1995). Adlercreutz & Mazur (1997) have noted that although the length and the width of the E 2 molecule fit compactly within the LBD of the ER protein, binding of E 2 within this domain leaves large unoccupied regions opposite the B- and C-rings. Endo et al. (1999) have recently described binding of 137 Journal of Endocrinology (2001) 170, 137–145 0022–0795/01/0170–137  2001 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology.org