280 Chem. Res. Toxicol. 2001, 14, 280-294 Structure-Activity Relationships for a Large Diverse Set of Natural, Synthetic, and Environmental Estrogens Hong Fang,† Weida Tong,*,† Leming M. Shi,†,‡ Robert Blair,§ Roger Perkins,† William Branham,§ Bruce S. Hass,§ Qian Xie,† Stacy L. Dial,§ Carrie L. Moland,§ and Daniel M. Sheehan§ R.O.W. Sciences, Inc., 3900 NCTR Road, MC 910, Jefferson, Arkansas 72079, and Division of Genetic and Reproductive Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079 Received October 3, 2000 Understanding structural requirements for a chemical to exhibit estrogen receptor (ER) binding has been important in various fields. This knowledge has been directly and indirectly applied to design drugs for human estrogen replacement therapy, and to identify estrogenic endocrine disruptors. This paper reports structure-activity relationships (SARs) based on a total of 230 chemicals, including both natural and xenoestrogens. Activities were generated using a validated ER competitive binding assay, which covers a 106-fold range. This study is focused on identification of structural commonalities among diverse ER ligands. It provides an overall picture of how xenoestrogens structurally resemble endogenous 17â-estradiol (E2) and the synthetic estrogen diethylstilbestrol (DES). On the basis of SAR analysis, five distinguishing criteria were found to be essential for xenoestrogen activity, using E2 as a template: (1) H-bonding ability of the phenolic ring mimicking the 3-OH, (2) H-bond donor mimicking the17â-OH and O-O distance between 3- and 17â-OH, (3) precise steric hydrophobic centers mimicking steric 7R- and 11â-substituents, (4) hydrophobicity, and (5) a ring structure. The 3-position H-bonding ability of phenols is a significant requirement for ER binding. This contributes as both a H-bond donor and acceptor, although predominantly as a donor. However, the 17â-OH contributes as a H-bond donor only. The precise space (the size and orientation) of steric hydrophobic bulk groups is as important as a 17â-OH. Where a direct comparison can be made, strong estrogens tend to be more hydrophobic. A rigid ring structure favors ER binding. The knowledge derived from this study is rationalized into a set of hierarchical rules that will be useful in guidance for identification of potential estrogens. Introduction turally diverse chemicals have estrogenic activity like the There is a growing body of evidence that some man- endogenous hormone estradiol. made chemicals, now called endocrine-disrupting chemi- SARs for estrogens date back more than six decades cals (EDCs),1 have the potential to disrupt the endocrine to the early work of Dodds et al. (4, 5). The succeeding system by mimicking endogenous hormones such as two decades saw the discovery of nonsteroidal estrogens, estrogens and androgens (1). Recent legislation mandates such as DES, based on understanding of the important that the Environmental Protection Agency develop a structural features governing potency for steroidal es- screening and testing program for potential EDCs, of trogens. Recently, a number of SAR studies have been which xenoestrogens figure predominately (2). Xenoestro- reported for steroidal estrogens (6) and nonsteroidal gens contain a number of chemical classes that display estrogens (7). These are generally focused on identifica- a broad range of structural diversity (3). For example, tion of structural characteristics for chemicals within DES, DDTs, polychlorinated biphenyls (PCBs), alkylphe- similar two-dimensional (2D) structural frameworks, nols, phthalates, and parabens have been found to be including E2 derivatives (6), DES derivatives (8), PCBs estrogenic. It has long been an enigma why such struc- (9), phytoestrogens (10), alkylphenols (11), raloxifenes (12), and others. Modern computer-based tools have * To whom correspondence should be addressed: R.O.W. Sciences, enabled the development of quantitative structure- Inc., 3900 NCTR Road, MC 910, Jefferson, AR 72079. Telephone: (870) 543-7142. Fax: (870) 543-7382. E-mail: [email protected]. activity relationship (QSAR) models for identifying steric † R.O.W. Sciences, Inc. and electrostatic features of a molecule in three-dimen- ‡ Current address: BASF Corp., P.O. Box 400, Princeton, NJ 08543. sional (3D) space for estrogenic activity (8, 13-19). § National Center for Toxicological Research. 1 Abbreviations: SAR, structure-activity relationship; QSAR, quan- Recent crystallographic structures of the human ER R - R titative structure activity relationship; ER, estrogen receptor; hER , subtype (hERR) with a number of ligands, including E2, human estrogen receptor R subtype; EDCs, endocrine disrupting chemicals; RBA, relative binding affinity; NA, not active; NCTR, DES, raloxifene, and 4-OH-tamoxifene, have also been National Center for Toxicological Research; 2D, two-dimensional; 3D, reported (20, 21). By aligning these four ligands on the three-dimensional; E2,17â-estradiol; E1, estrone; E3, estriol; EE, basis of the superposition of their ER binding sites, we ethynylestradiol; DES, diethylstilbestrol; DMS, dimethylstilbestrol; DDTs, 1,1,1-trichloro-2,2′-bischlorophenylethane derivatives; PCBs, have been able to demonstrate the common binding polychlorinated biphenyls; log P, hydrophobicity. characteristics among these ligands (22). 10.1021/tx000208y CCC: $20.00 © 2001 American Chemical Society Published on Web 02/10/2001 SARs of Diverse Estrogens Chem. Res. Toxicol., Vol. 14, No. 3, 2001 281 In principle, chemicals with similar biological activity and multiplying by 100 (E2 RBA ) 100). The validated assay share common structural features. This implies that incubation conditions were 20 h at 4 °C using 17 mg of uterine ) 3 structurally diverse estrogens possess a certain degree tissue/mL (Bmax 0.22 nM) with 1 nM [ H]E2. The competing of structural commonality essential to eliciting estrogenic chemical concentrations ranged from 1 nM to 1 mM. Chemicals 3 activity. Although a number of chemical classes are that failed to compete for [ H]E2 binding to the ER were designated as “not active” (NA). Chemicals that exhibited known to be estrogenic, little thorough structural evalu- binding, but did not reach 50% inhibition in the designed ation has been presented for their structural resemblance concentration range, were designated as “slight binders”. All to the E2 or the strong synthetic estrogen DES. Moreover, assays were repeated at least twice; the IC50 values of positive only limited efforts have explored the structural similari- chemicals are the means of the replicate values. The standard ties between different chemical classes of xenoestrogens. deviation of IC50 for each chemical was reported (23), and only To better understand the structural requirements for ER the mean RBA value was used for this study. The purity of binding, it is important to have a reliable data set, chemicals as well as their effect on RBAs was also studied by obtained with consistent assay design, covering a broad Blair et al. (23). The largest fold difference (∼10-fold) was found range of chemical classes. Recently, we reported ER for nonylphenol from different commercial sources due to the binding activity data for a large number of chemicals, impurity of the sample. including natural, synthetic, and environmental estro- Molecular Modeling. The crystal structures of E2, DES, raloxifene, and 4-OH-tamoxifen bound to the ER were obtained gens, using a validated rat ER competitive binding assay from the Protein Data Bank (PDB) as entries 1A52, 3ERD, (23, 24). This data set, called the NCTR data set, 1ERR, and 3ERT, respectively. The alignment of these four currently contains 230 chemicals. It was designed a priori ligands, based on root-mean-square (RMS) fitting of their to cover broad structural diversity and a wide range of receptor coordinates, was performed using the InsightII software binding activities for elucidating the structural charac- package (Molecular Simulations, Inc., San Diego, CA). Log P teristics of xenoestrogens and natural estrogens. was calculated using the atom/fragment contribution method The rat uterine cytosol ER competitive binding assay (27). Pharmacophore searching was performed with the CATA- is the gold standard for in vitro ER assays. When our LYST package (Molecular Simulations, Inc.). The energy dif- results are compared to results from other ER binding ferences for E2 and DES between the conformation in their binding modes and that in the minimum conformation of the assays, there is a general consistency between relative free ligands were calculated using the AM1 model Hamiltonian ER activities across different assay methods and species of the AMPAC/MOPAC module in InsightII (Molecular Simula- (25). For example, we found a high linear correlation for tions, Inc.). The atom-atom distance was also measured using ER binding affinities among a diverse group of chemicals InsightII (Molecular Simulations, Inc.). assayed with the ER from rat uterine cytosol and hERR.2 Further, we also found that ER assay results correlated Results very well with those from a yeast-based reporter gene assay and a MCF-7 cell proliferation assay. These find- We determined the ER RBA of 230 chemicals, of which ings demonstrate that ER binding is the major determi- 130 were active and 100 inactive. To the best of our nant across three levels of biological complexity (receptor knowledge, this is the largest published ER competitive binding, a yeast reporter gene response, and cell