Identification of active and inactive agonists/antagonists of estrogen receptor based on Tox21 10K compound library: Binomial analysis and structure alert.

Endocrine disrupting chemicals can mimic, block, or interfere with hormones in organisms and subsequently affect their development and reproduction, which has raised significant public concern over the past several decades. To investigate (quantitative) structure-activity relationship, 8280 compounds were compiled from the Tox21 10K compound library. The results show that 50% activity concentrations of agonists are poorly related to that of antagonists because many compounds have considerably different activity concentrations between the agonists and antagonists. Analysis on the chemical classes based on mode of action (MOA) reveals that estrogen receptor (ER) is not the main target site in the acute toxicity to aquatic organisms. Binomial analysis of active and inactive ER agonists/antagonists reveals that ER activity of compounds is dominated by octanol/water partition coefficient and excess molar refraction. The binomial equation developed from the two descriptors can classify well active and inactive ER chemicals with an overall prediction accuracy of 73%. The classification equation developed from the molecular descriptors indicates that estrogens react with the receptor through hydrophobic and π-n electron interactions. At the same time, molecular ionization, polarity, and hydrogen bonding ability can also affect the chemical ER activity. A decision tree developed from chemical structures and their applications reveals that many hormones, proton pump inhibitors, PAHs, progestin, insecticides, fungicides, steroid and chemotherapy medications are active ER agonists/antagonists. On the other hand, many monocyclic/nonaromatic chain compounds and herbicides are inactive ER compounds. The decision tree and binomial equation developed here are valuable tools to predict active and inactive ER compounds.

Evaluation of ligand selectivity using reporter cell lines stably expressing estrogen receptor alpha or beta.

Estrogens control transcriptional responses through binding to two different nuclear receptors, estrogen receptor alpha (ERalpha) and beta (ERbeta). Since these two ER subtypes are thought to mediate different biological effects, there is intense interest in designing subtype-selective ER ligands. In this study, we evaluated the ERalpha and ERbeta selectivity of 19 known estrogens and antiestrogens using reporter cell lines previously developed in our laboratory. The HELN-ERalpha and HELN-ERbeta cells stably express full-length ERalpha and ERbeta, respectively, and are derived from HELN cells (HeLa cells stably transfected with an ERE-driven luciferase plasmid). We report that 16alpha-LE2, PPT and 3beta,5alpha-GSD have a high ERalpha-selective agonist potency while 8beta-VE2, DPN, genistein and biochanin A show ERbeta selectivity with 8beta-VE2 being the most potent and selective ERbeta agonist. We also tested ER antagonists and we showed that raloxifene and RU486 are ERalpha and ERbeta-selective antiestrogens, respectively. In all cases, selectivity is due to differences in binding affinities as indicated by whole-cell ligand-binding assays. Very interestingly, we demonstrate that a combination of genistein and raloxifene produces a full-ERbeta specific response. Together these results demonstrate the usefulness of our stably transfected cell lines to characterize ER ligands and indicate that treatments combining agonist/antagonist ligands produce full-ERbeta selectivity.

Human estrogen receptor ligand activity inversion mutants: receptors that interpret antiestrogens as estrogens and estrogens as antiestrogens and discriminate among different antiestrogens.

The estrogen receptor (ER) is a transcription factor whose activity is normally activated by the hormone estradiol and inhibited by antiestrogen. It has been found that certain mutational changes in the activation function-2 region in the hormone-binding domain of the human ER result in ligand activity inversion mutants, i.e. receptors that are now activated by antiestrogen and inhibited by estrogen. The ER point mutant L540Q is activated by several antiestrogens (the more pure antiestrogens ICI 164,384 and RU 54,876 or the partial antiestrogen trans-hydroxytamoxifen) but not by estradiol. The presence of the F domain and an intact activation function-i in the A/B domain are required for this activity, as is the DNA-binding ability of the receptor. This inverted ligand activity is observed with several estrogen-responsive promoters, both simple and complex; however, the activating ability of antiestrogens is observed only in some cells, highlighting the important role of cell-specific factors in ligand interpretation. The introduction of two additional amino acid changes close to 540 results in receptors that are still not activated by estradiol but are now able to distinguish between partial antiestrogens (which remain agonistic) and pure antiestrogens (which show a greatly reduced stimulatory activity). These ligand activity inversion mutants remain stable in cells in the presence of the antiestrogen ICI 164,384, as does a related ER mutant receptor that shows the normal, wild type ER ligand activity profile in which ICI 164,384 is transcriptionally inactive. Thus, the presence of adequate levels of mutant ER may be necessary but not sufficient for ICI 164,384 to elicit transcriptional activity. These findings highlight the means by which the carboxyl-terminal region in domain E functions to interpret the activity of a ligand, and they demonstrate that rather minimal changes in the ER can result in receptors with inverted response to antiestrogen and estrogen. Such point mutations, if present in estrogen target cells, would result in antiestrogens being seen as growth stimulators, rather than suppressors, with potentially detrimental consequences in terms of breast cancer treatment with antiestrogens.

Analysis of estrogen receptor function in vitro reveals three distinct classes of antiestrogens.

We have developed a series of in vitro models with which to evaluate the biological activity of estrogen receptor (ER) agonists and antagonists. Using a protease digestion assay we show that the conformational changes induced within ER are distinct for agonists and antagonists. However, this assay is unable to discriminate between pure antagonists like ICI164,384 and partial agonists such as 4-OH tamoxifen or keoxifene. Using a chimeric ER-VP16 construct, we demonstrate that both pure antagonists and partial agonists deliver ER to its DNA target within cells. However, the ability of the DNA-bound receptor to activate transcription in the presence of a given antagonist is dependent on cell and promoter context. These data, suggesting functional differences among ER antagonists, were confirmed by additional experiments demonstrating that their ability to modulate the transcriptional activity of a series of ER mutants is dramatically different. Depending on the cell and promoter context and the particular ER form expressed, 4-OH tamoxifen and the related compound, keoxifene, functioned as partial agonists. Importantly, the transcriptional profiles of these two compounds were dissimilar, suggesting that they are functionally different from each other and from ICI164,384, which does not display agonist activity under any context examined. Our results reveal functional differences between these clinically important antiestrogens and suggest that the distinct biologies manifest by these compounds in vivo relate to their ability to differentially regulate ER function.

Docking-based classification models for exploratory toxicology studies on high-quality estrogenic experimental data.

BACKGROUND: The ethical and practical limitation of animal testing has recently promoted computational methods for the fast screening of huge collections of chemicals. RESULTS: The authors derived 24 reliable docking-based classification models able to predict the estrogenic potential of a large collection of chemicals provided by the US Environmental Protection Agency. Model performances were challenged by considering AUC, EF1% (EFmax = 7.1), -LR (at sensitivity = 0.75); +LR (at sensitivity = 0.25) and 37 reference compounds comprised within the training set. Moreover, external predictions were made successfully on ten representative known estrogenic chemicals and on a set consisting of >32,000 chemicals. CONCLUSION: The authors demonstrate that structure-based methods, widely applied to drug discovery programs, can be fairly adapted to exploratory toxicology studies.

A new interpretation of antiestrogen action.

Despite differences in their pharmacological behavior, type I and type II antiestrogens have certain important properties in common. Both differ from estradiol in that they enhance the immunoreactivity of estrogen receptors, apparently by inducing conformational change that exposes an additional epitope for a particular monoclonal antibody. Moreover, both types of antihormones not only compete with estradiol for its binding to the receptor but they also react with another domain not recognized by the hormone. The binding capacity for either type of antiestrogen is nearly twice that for estradiol, providing definitive evidence for the existence of specific antiestrogen-binding sites that are postulated to be important in antagonist action. These findings suggest a unified two-site model which helps explain how the same substance can be both an agonist and an antagonist; why there may be species variations in the agonist/antagonist relationship of type I compounds; and why type II agents show only antagonistic properties. It is suggested that interaction with secondary, antagonist-specific binding sites may provide a useful screen in the search for new and improved antihormonal agents.

Basic guide to the mechanisms of antiestrogen action.

Forty years ago, Lerner and coworkers (1958) discovered the first nonsteroidal antiestrogen and Jensen (Jensen and Jacobson, 1960) identified a target for drug action, the ER. This knowledge opened the door for the clinical development of tamoxifen which we now know provides a survival advantage in both node-positive and node-negative patients with ER-positive disease (Early Breast Cancer Trialists Collaborative Group, 1992, 1998). The drug has been studied extensively, and the results have provided an invaluable insight into possible ancillary advantages of "antiestrogens", i.e., maintenance of bone density and the prevention of coronary heart disease, and possible disadvantages, i.e., rat liver carcinogenesis and an increased risk of endometrial cancer. Most importantly, the identification of the target site-specific actions of tamoxifen caused a paradigm shift in the prospective uses of antiestrogens from a direct exploitation of the antitumor properties to the broader application as a preventative for osteoporosis, but with the beneficial side effects of preventing breast and endometrial cancer. Raloxifene, a second-generation SERM, has all the properties in the laboratory that would encourage development as a safe preventative for osteoporosis (Jordan et al., 1997). As a result, raloxifene has been evaluated in more than 11,000 postmenopausal women and found to maintain bone density with significant decreases in breast cancer incidence and no increase in endometrial thickness. Raloxifene is now available as a preventative for osteoporosis in postmenopausal women. There is every reason to believe that a multifaceted agent like raloxifene will find widespread use, and there will be continuing interest by the pharmaceutical industry in the development of new agents with even broader applications. The extensive clinical effort is augmented by past molecular innovations in the laboratory and the future promise of new discoveries. The cloning and sequencing of the ER (Green et al., 1986; Greene et al., 1986) has allowed the development of an ER knock-out mouse (Lubahn et al., 1993) that compliments Jensen's pioneering work (Jensen and Jacobson, 1962) and describes the consequences of the loss of ER alpha. However, ER beta (Kuiper et al., 1996), the second ER, has provided an additional dimension to the description of estrogen and antiestrogen action. For the future, the development of ER beta monoclonal antibodies, the classification of target sites for the protein around the body, and the creation of ER beta and ER alpha, beta knock-out mice will identify new therapeutic targets to modulate physiological functions. Clearly, the successful crystallization of ER alpha with raloxifene (Brzozowski et al., 1997) must act as a stimulus for the crystallization of ER beta. The central issue for research on antiestrogen pharmacology is the discovery of the mechanism (or mechanisms) of target site-specificity for the modulation of estrogenic and antiestrogenic response. The description of a stimulatory pathway for antiestrogens through an AP-1 ER beta signal transduction pathway (Paech et al., 1997), although interesting, may not entirely explain the estrogenicity of antiestrogens. The model must encompass the sum of pharmacological consequences of signal transduction through ER alpha and ER beta with the simultaneous competition from endogenous estrogens at both sites. This is complicated because estradiol is an antagonist at ER beta through AP-1 sites (Paech et al., 1997), so this is clearly not the pathway for estrogen-induced bone maintenance in women. Estrogen is stimulatory through ER alpha, but antiestrogens are usually partial agonists and may either block or stimulate genes. However, we suggest that the ER alpha stimulatory pathway could be amplified through selective increases in coactivators. The principle is illustrated with the MDA-MB-231 cells stably transfected with the cDNAs for the wild-type and the amino acid 351 mutan

Differential DNA-binding abilities of estrogen receptor occupied with two classes of antiestrogens: studies using human estrogen receptor overexpressed in mammalian cells.

We have developed a transient transfection system using the Cytomegalovirus (CMV) promoter to express the human estrogen receptor (ER) at very high levels in COS-1 cells and have used it to study the interaction of agonist and antagonist receptor complexes with estrogen response element (ERE) DNA. ER can be expressed to levels of 20-40 pmol/mg or 0.2-0.3% of total soluble protein and all of the soluble receptor is capable of binding hormone. The ER binds estradiol with high affinity (Kd 0.2 nM), and is indistinguishable from native ER in that the receptor is capable of recognizing its cognate DNA response element with high affinity, and of transactivating a transgene in an estradiol-dependent manner. Gel mobility shift assays reveal interesting ligand-dependent differences in the binding of receptor complexes to ERE DNA. Receptors occupied by estradiol or the type I antiestrogen transhydroxytamoxifen bind to DNA response elements when exposed to the ligand in vitro or in vivo. Likewise, receptors exposed to the type II antiestrogen ICI 164,384 in vitro bind to ERE DNA. However, when receptor exposure to ICI 164,384 is carried out in vivo, the ER-ICI 164,384 complexes do not bind to ERE DNA, or do so only weakly. This effect is not reversed by subsequent incubation with estradiol in vitro, but is rapidly reversible by in vivo estradiol exposure of intact COS-1 cells. This suggests there may be some cellular process involved in the mechanism of antagonism by the pure antiestrogen ICI 164,384, which is not observed in cell-free extracts.

Estrogenic and antiestrogenic activities of flavonoid phytochemicals through estrogen receptor binding-dependent and -independent mechanisms.

Members of the flavonoid class of phytochemicals have previously been demonstrated to possess estrogenic activity in a number of hormonally responsive systems. We have performed the present study to characterize the estrogenic and antiestrogenic activity of flavonoids in the estrogen receptor (ER)-positive MCF-7 human breast cancer cell line. Using an ER-dependent reporter gene assay and an ER competition binding assay, we have identified phytochemicals possessing estrogenic and antiestrogenic activities, which appeared to correlate directly with their capacity to displace [3H]estradiol from ER. Several flavonoids, including kaempferide, apigenin, and flavone, were distinct, in that their antiestrogenic activity did not appear to correlate with binding to ER, and therefore their suppression of estrogen-mediated gene transactivation and proliferation may occur independent of direct antagonism of the receptor. Further examination in HEK-293 cells transfected with ERalpha or ERbeta demonstrated potent antagonism with kaempferide and apigenin, while flavone was weakly antagonistic only toward ERP. These results suggest that the receptor binding-independent antiestrogenic chemicals may function through alternate signaling pathways as indirect ER modulators in a receptor- and cell type-specific manner. We conclude that antiestrogenic activities of flavonoid phytochemicals may occur through ER binding-dependent and -independent mechanisms and that the binding-independent antiestrogen activity of certain flavonoids is biologically significant in regulation of breast cancer cell proliferation.

Concept evaluation: an assay for receptor-mediated and biochemical antiestrogens using pubertal rats.

At present, assessment of chemicals for receptor-mediated antiestrogenic activity involves inhibition of uterine growth stimulated by coadministration of a reference estrogen in either ovariectomized or immature rodents. In the present paper, we describe an alternative assay for both receptor-mediated and biochemical antiestrogens. The assay involves treatment of immature rats from postnatal (pnd) 25 or 26 for either 7 or 14 days and monitors two benchmarks of puberty, the mean day of vaginal opening and the weight of the uterus, that require estrogen activity. The receptor-mediated antiestrogens ZM 189,154 and Faslodex (ICI 182,780), the aromatase inhibitor Arimidex (Anastrozole), and the GnRH inhibitor Antarelix were each effective in preventing uterine growth and in delaying vaginal opening for the course of the experiments. The 5alpha-reductase inhibitor Finasteride was inactive in the assay indicating assay specificity for antiestrogens. Delays in uterine growth were clearly evident in the 7-day experiments, but assessment of vaginal opening required the 14-day protocol. No significant changes in body weight were observed in any of the experiments. It is concluded that the assay holds promise as a simple method of detecting antiestrogens and that it is worthy of further study.

Antiestrógenos: revisión químico-farmacéutica / Antiestrogens: chemical-pharmaceutical revision

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