Synthesis of an estrogen receptor beta-selective radioligand: 5-[18F]fluoro-(2R,3S)-2,3-bis(4-hydroxyphenyl)pentanenitrile and comparison of in vivo distribution with 16alpha-[18F]fluoro-17beta-estradiol

Estrogen receptor beta (ERbeta), a less active ER subtype that appears to have a restraining effect on the more active ERalpha, could be a factor that determines the level of estrogen action in certain estrogen target tissues. ERbeta is found in breast cancer, and its levels relative to ERalpha decline with disease progression. Thus, the independent quantification of ERalpha and ERbeta levels in breast cancer by imaging might be predictive of responses to different hormone therapies. To develop an imaging agent for ERbeta, we synthesized a fluoroethyl analogue of DPN (2,3-bis(4-hydroxyphenyl)propanonitrile), a known ERbeta-selective ligand. This analogue, FEDPN (5-fluoro-(2R,3S)-2,3-bis(4-hydroxyphenyl)pentanenitrile), has an 8.3-fold absolute affinity preference for ERbeta. [18F]Fluoride-labeled FEDPN was prepared from a toluenesulfonate precursor, which provided [18F]FEDPN with a specific activity greater than 3100 Ci/mmol after HPLC purification. Biodistribution studies in immature female rats using estradiol as a blocking agent revealed specific uptake of [18F]FEDPN in the uterus and ovaries. Experiments using ERalpha- and ERbeta-knockout mice demonstrated the expected ERalpha-subtype dependence in the tissue uptake of the known 16alpha-[18F]fluoro-17beta-estradiol ([18F]FES), which has a 6.3-fold preference for ERalpha. The tissue uptake of [18F]FEDPN in the ER knockout mice showed some evidence of mediation by ERbeta, but the levels of specific uptake of this agent were relatively modest. Based on our results, imaging of ERalpha can be done effectively with [18F]FES, but imaging of ERbeta will likely require agents with more optimized ERbeta binding affinity and selectivity than [18F]FEDNP.     

A beta-lactamase-dependent Gal4-estrogen receptor beta transactivation assay for the ultra-high throughput screening of estrogen receptor beta agonists in a 3456-well format

Estrogen action is mediated via two estrogen receptor (ER) subtypes, ERalpha and ERbeta. Selective ER modulators with balanced high affinity for ERalpha and ERbeta have been developed as therapeutics for the treatment of a variety of diseases, including hormone-responsive breast cancer and osteoporosis. Recent data based primarily on the evaluation of ER-knockout mice have revealed that ERalpha and ERbeta may regulate separate and distinct biological processes. The identification of ERbeta specific ligands could further enhance our understanding of ERbeta biology. In addition, compounds targeting ERbeta may prove useful as therapeutic agents with activity profiles distinguishable from that of estradiol. To discover novel selective ligands for ERbeta, we developed and characterized a cell-based Gal4-ERbeta beta-lactamase reporter gene assay (GERTA) in CHO cells for the ligand-induced activation of the human ERbeta. The sensitivity and selectivity of this assay were found to be comparable to those of an ER ligand-binding assay. The assay was optimized for screening in an ultra high throughput 3456-well nanoplate format and was successfully used to screen a large compound collection for ERbeta agonists. Compounds identified in a primary screen were tested in an in vitro ligand-binding assay to characterize further the selectivity and potency for ERbeta.     

Effect of in vitro estrogenic pesticides on human oestrogen receptor alpha and beta mRNA levels

Nine widely distributed pesticides were recently demonstrated to possess potential estrogenic properties in oestrogen receptor (ER) transactivation and/or E-screen assays. We tested the effect of these nine pesticides on the human ERalpha and ERbeta mRNA steady state levels in the mamma cancer fibroblast MCF-7BUS cells using on-line RT-PCR. Like 17beta-oestradiol (E2), fenarimol significantly decreased the ERalpha and increased the ERbeta mRNA level. Endosulfan and pirimicarb alone decreased the ERalpha mRNA level weakly. After co-exposure with E2, all the tested pesticides counteracted the E2-induced decrease of the ERalpha mRNA level, but only significantly for prochloraz, dieldrin, and tolchlofos-methyl. Alone no pesticides affected the ERbeta mRNA level significantly, but chlorpyrifos increased the mRNA level weakly. Co-exposure with E2 elicited a significant increased ERbeta mRNA level by prochloraz, fenarimol, endosulfan, dieldrin, and tolchlofos-methyl, whereas no significant effect of the carbamate pesticides on the ERbeta mRNA level was observed. This study demonstrated that organochlor and organophosphorous pesticides possess the ability to interfere with the ERalpha and ERbeta mRNA steady state levels.     

Adult female wildtype, but not oestrogen receptor beta knockout, mice have decreased depression-like behaviour during pro-oestrus and following administration of oestradiol or diarylpropionitrile

Studies in people and animal models suggest that depression is influenced by natural fluctuations in the levels of 17beta-oestradiol (E(2)), as well as administration of E(2)-based therapies, such as selective oestrogen receptor modulators (SERMs). Elucidating the effects and mechanisms of E(2) is important to improve future E(2)-based therapeutics. An important question is whether effects of E(2) or SERMs for mood regulation act at the alpha or beta isoform of the oestrogen receptor (ER) because some of the unwanted trophic effects of E(2)-based therapies may involve actions at ERalpha, rather than ERbeta. In the present study, whether there are sex differences in depression-like behaviour of adult mice (experiment 1), and the effects of natural fluctuations in E(2) (experiment 2), or administration of E(2) or a SERM that has higher affinity for ERbeta than for ERalpha (diarylpropionitrile; DPN) to ovariectomised (experiment 3) wildtype and ERbeta knockout (betaERKO) mice were investigated. Results of this study supported our hypotheses that: there would be sex differences favouring males for depression-like behaviour and endogenous increases in, or exogenous administration of, E(2) or administration of an ERbeta SERM would decrease depression-like behaviour in wildtype, but not betaERKO, mice. In experiment 1, adult male mice spent less time immobile in the forced swim test (i.e., showed less depression-like behaviour) compared with female mice. In experiment 2, pro-oestrous (higher circulating E(2) levels), compared with dioestrous (lower circulating E(2) levels), mice had reduced immobility in the forced swim test; this effect was not observed in betaERKO mice. In experiment 3, administration of E(2) or DPN to ovariectomised wildtype, but not betaERKO, mice decreased immobility compared with vehicle administration, these data suggest that ERbeta may be required for some of the anti-depressant-like effects of E(2).     

Functional characterization of estrogen receptor subtypes, ERalpha and ERbeta, mediating vitellogenin production in the liver of rainbow trout

The estrogen-dependent process of vitellogenesis is a key function on oviparous fish reproduction and it has been widely used as an indicator of xenoestrogen exposure. The two estrogen receptor (ER) subtypes, ERalpha and ERbeta, are often co-expressed in the liver of fish. The relative contribution of each ER subtype to modulate vitellogenin production by hepatocytes was studied using selected compounds known to preferentially interact with specific ER subtypes: propyl-pyrazole-triol (PPT) an ERalpha selective agonist, methyl-piperidino-pyrazole (MPP) an ERalpha selective antagonist, and diarylpropionitrile (DPN) an ERbeta selective agonist. First, the relative binding affinity of the test compounds to estradiol for rainbow trout hepatic nuclear ER was determined using a competitive ligand binding assay. All the test ligands achieved complete displacement of specific [(3)H]-estradiol binding from the nuclear ER extract. This indicates that the test ligands have the potential to modify the ER function in the rainbow trout liver. Secondly, the ability of the test compounds to induce or inhibit vitellogenin production by primary cultures of rainbow trout hepatocytes was studied. Estradiol and DPN were the only compounds that induced a dose-dependent increase on vitellogenin synthesis. The lack of vitellogenin induction by PPT indicates that ERalpha could not have a role on this reproductive process whereas the ability of DPN to induce vitellogenin production supports the participation of ERbeta. In addition, this hypothesis is reinforced by the results obtained from MPP plus estradiol. On one hand, the absence of suppressive activity of MPP in the estradiol-induced vitellogenin production does not support the participation of ERalpha. On the other hand, once blocked ERalpha with MPP, the only manifestation of agonist activity of estradiol would be achieved via ERbeta. In conclusion, the present results indicate that vitellogenin production is mainly mediated through ERbeta, implying, furthermore that compounds which only exhibit ERalpha selectivity are not detected by vitellogenin bioassay.     

Regulation of cell growth by estrogen signaling and potential targets in thyroid cancer

Thyroid cancer occurs three times more frequently in females than in males, and in females the incidence decreases after menopause. This gender difference suggests that the growth and progression of thyroid cancer may be influenced by female sex hormones, particularly estrogens. Experimental data have clearly demonstrated that estrogens can influence cancer cell growth. The action of estrogens on target sites is mediated through related but distinct estrogen receptors, designated estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta), both of which are known to be expressed in thyroid cancer cells. The proliferation of thyroid cancer cells is promoted by an ERalpha agonist, whereas the proliferation is reduced by the enhanced expression of ERbeta or by an ERbeta agonist. When ERbeta is down-regulated, the proliferation of thyroid cells is significantly increased. Studies have shown that the expression of ERalpha in thyroid cancer cells is increased while the expression of ERbeta is either very low or absent. In conclusion, it appears that estrogens have opposite effects on the growth of thyroid cancer cells, depending on the balance between ERalpha and ERbeta in the cells. The modulation of ERalpha and ERbeta and the intervention of their pathways may open up new potential targets for the treatment of thyroid cancer.     

Analysis of the effects of oestrogen receptor alpha (ERalpha)- and ERbeta-selective ligands given in combination to ovariectomized rats

BACKGROUND AND PURPOSE: Studies with oestrogen receptoralpha (ERalpha)- and ERbeta-selective compounds have already shown that the effects of 17beta-estradiol (E2) on body weight, movement drive and bone-protection are mediated via ERalpha. This study was based on the hypothesis that activation of ERbeta may antagonize ERalpha-mediated effects and designed to investigate potential effects of ERalpha/ERbeta heterodimers. EXPERIMENTAL APPROACH: Ovariectomized (OVX) female Wistar rats were treated with combinations of the ERalpha-specific agonist 16alpha-LE2 (ALPHA; 1 and 10 microg kg(-1) d(-1)), the ERbeta-specific agonist 8beta-VE2 (BETA; 100 microg kg(-1) d(-1)), the phytoestrogen, genistein (10 mg kg(-1) d(-1)) and with the anti-oestrogen compound, ICI 182,780 (3 mg kg(-1) d(-1)) for three weeks. The combined effects of the substances on body weight increase, tibial bone mineral density (BMD) and the influence on running wheel activity (RWA) were investigated. KEY RESULTS: OVX-induced body weight increase was reduced by co-administration of genistein and BETA. Co-application of BETA or genistein with ALPHA had no effect on ALPHA-mediated bone-protection. The RWA of OVX animals was significantly reduced by treatment with genistein but stimulated by application of ALPHA. The stimulatory effect of ALPHA on RWA could be antagonized by co-treatment with the pure antioestrogen ICI 182,780 but also by co-administration of genistein or BETA. CONCLUSIONS AND IMPLICATIONS: Our results indicate that activation of ERbeta may modulate ERalpha-mediated physiological effects in vivo. The observation that substances with selective affinity for ERbeta are able to antagonize distinct physiological functions, like RWA, may be of great relevance to the pharmaceutical use of such drugs.     

Target specific virtual screening: optimization of an estrogen receptor screening platform

In this work, we introduce a four-step scoring and filtering procedure, furnishing target specific virtual screening (TS-VS), which serves to minimize false positives resulting from conformational artifacts of the docking process and is optimized to converge on novel chemotypes of estrogen receptor alpha (ERalpha). As a proof of concept, VS of a commercial compound database was undertaken (SPECs database release: Aug 2005, 202 054 compounds in total), resulting in the identification of both previously known and novel putative ER scaffolds. Application of distance constraints within TS-VS allowed facile identification of three novel active ligands with ERalpha binding affinities (IC50) of 1.4 microM, 57 nM, and 53 nM. Importantly, they all exhibited ERalpha over ERbeta selectivity, with the most selective being 17-fold. The ligands also displayed low micomolar antiproliferative activity (7-15 microM) in the human MCF-7 breast cancer cell line.     

Interaction of methoxychlor and related compounds with estrogen receptor alpha and beta, and androgen receptor: structure-activity studies

We previously demonstrated differential interactions of the methoxychlor metabolite 2,2-bis(p-hydroxyphenyl)-1,1, 1-trichloroethane (HPTE) with estrogen receptor alpha (ERalpha), ERbeta, and the androgen receptor (AR). In this study, we characterize the ERalpha, ERbeta, and AR activity of structurally related methoxychlor metabolites. Human hepatoma cells (HepG2) were transiently transfected with human ERalpha, ERbeta, and AR plus an appropriate steroid-responsive luciferase reporter vector. After transfection, cells were treated with various concentrations of HPTE or structurally related compounds in the presence (for detecting antagonism) and absence (for detecting agonism) of 17beta-estradiol and dihydrotestosterone. The monohydroxy analog of methoxychlor, as well as monohydroxy and dihydroxy analogs of 2, 2-bis(p-hydroxyphenyl)-1,1-dichloroethylene, had ERalpha agonist activity and ERbeta and AR antagonist activity similar to HPTE. The trihydroxy metabolite of methoxychlor displayed only weak ERalpha agonist activity and did not alter ERbeta or AR activities. Replacement of the trichloroethane or dichloroethylene group with a methyl group resulted in a compound with ERalpha and ERbeta agonist activity that retained antiandrogenic activities. This study identifies some of the structural requirements for ERalpha and ERbeta activity and demonstrates the complexity involved in determining the mechanism of action of endocrine-active chemicals that simultaneously act as agonists or antagonists through one or more hormone receptors.     

Steroids and the endometrium

Steroids are commonly employed in current clinical practice. The benefits of steroids in hormone replacement therapy, contraception and prevention or treatment of breast cancer are limited by their side effects arising from disorders in endometrial function. These side effects are complex and enclose bleeding problems and endometrial proliferation during hormone replacement therapy and antioestrogen treatment or menstrual disturbances during oral contraception. Numerous reports have identified gene targets influenced by steroids and have implicated these products as contributors to endometrial physiology or pathology. The expression of estrogen and progesterone receptors is regulated by steroids. The new estrogen receptor (ER) subtype ERbeta with different functional characteristics from ERalpha was recently described in endometrium. In addition, there is now increasing evidence that the functionally distinct progesterone receptor (PR) isoforms A and B are differentially expressed in this tissue. The relative proportions of these steroid receptors and their interaction determine the expression of specific genes upon steroidal stimulation. Steroids induce endometrial expression of various growth and angiogenic factors. Dysregulations of this steroid modulated expression is believed to be involved in the pathogenesis of many endometrial diseases. Irregular bleeding induced by steroidal contraception, for example, is thought to involve aberrant endometrial vascular development and expression of angiogenic growth factors. The antioestrogen tamoxifen induces growth factors like vascular endothelial growth factor and adrenomedullin which may be key mediators of endometrial neoplastic effects. This review describes recent advances regarding the mechanism of action of steroids on endometrium. The expression of oestrogen and progesterone receptors as well as steroid hormone dependent growth factors and angiogenic modulators are going to be discussed.     

Design and synthesis of aryl diphenolic azoles as potent and selective estrogen receptor-beta ligands

New diphenolic azoles as highly selective estrogen receptor-beta agonists are reported. The more potent and selective analogues of these series have comparable binding affinities for ERbeta as the natural ligand 17beta-estradiol but are >100-fold selective over ERalpha. Our design strategy not only followed a traditional SAR approach but also was supported by X-ray structures of ERbeta cocrystallized with various ligands as well as molecular modeling studies. These strategies enabled us to take advantage of a single conservative residue substitution in the ligand-binding pocket, ERalpha Met(421) --> ERbeta Ile(373), to optimize ERbeta selectivity. The 7-position-substituted benzoxazoles (Table 5) were the most selective ligands of both azole series, with ERB-041 (117) being >200-fold selective for ERbeta. The majority of ERbeta selective agonists tested that were at least approximately 50-fold selective displayed a consistent in vivo profile: they were inactive in several models of classic estrogen action (uterotrophic, osteopenia, and vasomotor instability models) and yet were active in the HLA-B27 transgenic rat model of inflammatory bowel disease. These data suggest that ERbeta-selective agonists are devoid of classic estrogenic effects and may offer a novel therapy to treat certain inflammatory conditions.     

Estrogenic activity of cosmetic components in reporter cell lines: parabens, UV screens, and musks

In this work, the estrogenic effects of three classes of substances included in cosmetic formulations-parabens, ultraviolet (UV) screens, and musk fragrances-were studied. Their estrogenic activity was measured with the use of three reporter cell lines: HELN, HELN ERalpha, and HELN ERbeta. These three cell lines allowed for the measurement of estrogenic activity toward estrogen receptors alpha and beta (ERalpha and ERbeta, while taking nonspecific interactions into account. Eight of the 15 substances tested showed specific estrogenic activity with the following degree of potency on ERalpha butylparaben > propylparaben > homosalate = octyl-dimethyl-PABA = 4-methyl-benzylidenecamphor = octyl-methoxycinnamate > ethylparaben = galaxolide. Among these active substances, parabens activated ERalpha and ERbeta similarly, UV screens activated ERalpha moderately and had almost no effect on ERbeta, and fragrances did not activate ERbeta. Methylparaben, ethylparaben, musk moskene, celestolide, and cashmeran did not activate estrogenic responses up to 10(-5) M. Musk ketone and benzophenone-3 were not considered estrogenic at 10(-5) M.