Rat Uterine Estrogen Receptor Research Articles

Abstract 44: The discovery of ARV-471, an orally bioavailable estrogen receptor degrading PROTAC for the treatment of patients with breast cancer

Abstract ARV-471, an estrogen receptor (ER) alpha PROTAC® protein degrader, is a hetero-bifunctional molecule that facilitates the interactions between estrogen receptor alpha and an intracellular E3 ligase complex, leading to the ubiquitylation and subsequent degradation of estrogen receptors via the proteasome. ARV-471 robustly degrades ER in ER-positive breast cancer cell lines with a half-maximal degradation concentration (DC50) of ~ 1 nM. PROTAC® mediated ER degradation decreases the expression of classically regulated ER-target genes and inhibits cell proliferation of ER-dependent cell lines (MCF7, T47D). Additionally, ARV-471 degrades clinically relevant ESR1 variants (Y537S and D538G) and inhibits growth of cell lines expressing those variants. In an immature rat uterotrophic model, ARV-471 degrades rat uterine ER and demonstrates no agonist activity. Daily, oral-administration of single agent ARV-471 (3, 10, and 30 mpk) leads to significant anti-tumor activity of estradiol-dependent MCF7 xenografts and concomitant tumor ER protein reductions of >90% at study termination. Moreover, when a CDK4/6 inhibitor is combined with ARV-471 in the MCF7 model, even more pronounced tumor growth inhibition is observed (131% TGI), accompanied by significant reductions in ER protein levels. In an ESR1 Y537S, hormone-independent patient-derived xenograft model, ARV-471 at 10 mpk completely inhibited growth and also significantly reduced mutant ER protein levels. Taken together, the preclinical data of ARV-471 supports its continued development as a best-in-class oral ER PROTAC® protein degrader. These preclinical data supported the clinical development of ARV-471 for the treatment of patients with breast cancer. The discovery, chemical structure and initial clinical data of ARV-471 will be presented. Citation Format: Lawrence B. Snyder, John J. Flanagan, Yimin Qian, Sheryl M. Gough, Monica Andreoli, Mark Bookbinder, Gregory Cadelina, John Bradley, Emma Rousseau, Julian Chandler, Ryan Willard, Jennifer Pizzano, Craig M. Crews, Andrew P. Crew, John Houston, Marcia Dougan Moore, Ron Peck, Ian Taylor. The discovery of ARV-471, an orally bioavailable estrogen receptor degrading PROTAC for the treatment of patients with breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 44.

Abstract P5-04-18: ARV-471, an oral estrogen receptor PROTAC degrader for breast cancer

Abstract ARV-471, an estrogen receptor (ER) alpha PROTAC, is a hetero-bifunctional molecule that facilitates the interactions between estrogen receptor alpha and an intracellular E3 ligase complex, leading to the ubiquitylation and subsequent degradation of estrogen receptors via the proteasome. ARV-471 robustly degrades ER in ER-positive breast cancer cell lines with a half-maximal degradation concentration (DC50) of ˜ 2 nM. PROTAC-mediated ER degradation decreases the expression of classically-regulated ER-target genes (PR, GREB1, TFF) and inhibits cell proliferation of ER-dependent cell lines (MCF7, T47D). Additionally, ARV-471 degrades clinically-relevant ESR1 variants (Y537S and D538G) and inhibits growth of cell lines expressing those variants. In an immature rat uterotrophic model, ARV-471 degrades rat uterine ER and demonstrates no agonist activity. Daily, oral-administration of single agent ARV-471 (3, 10, and 30 mpk) leads to significant tumor volume regressions of estradiol-dependent MCF7 xenografts and concomitant tumor ER protein reductions of >90% at study termination. Moreover, when a CDK4/6 inhibitor is combined with ARV-471 in the MCF7 model, even more pronounced tumor growth inhibition is observed (˜130% TGI), accompanied by significant reductions in ER protein levels. In an ESR1 Y537S, hormone-independent patient-derived xenograft model, ARV-471 at 10 mpk completely inhibited growth and also reduced mutant ER protein levels. Taken together, the preclinical data of ARV-471 supports its continued development as a best-in-class oral ER PROTAC-degrader. Citation Format: Flanagan JJ, Qian Y, Gough SM, Andreoli M, Bookbinder M, Cadelina G, Bradley J, Rousseau E, Willard R, Pizzano J, Crews CM, Crew AP, Taylor I, Houston J. ARV-471, an oral estrogen receptor PROTAC degrader for breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-04-18.

Neonatal exposure to herbicide acetochlor alters pubertal development in female wistar rats

Objectives: The purpose of this study was to evaluate the effects of neonatal exposure to the herbicide acetochlor (ACT) on pubertal development and reproductive functions in female Wistar rats and to investigate capability of ACT to interfere with estradiol binding to rat uterine estrogen receptors (ERs) ex vivo.Methods: Acetochlor (7.68 and 15.36 mg/kg/day) was administered by subcutaneous injection from postnatal day (PND) 4–7, and vaginal opening, and estrous cyclicity were evaluated from PND 8-159. A second group of adult ovariectomized female rats was dosed for 6 days with ACT (153.6 mg/kg/day, oral gavage). The interference of ACT with the binding of [3H]Estradiol -17β to uterine nuclear and cytoplasmic estrogen receptors was analyzed ex vivo in receptor binding assay.Results: Both doses of ACT caused acceleration of the age at eye opening and vaginal patency that were significantly different from the control. In addition, altered estrous cyclicity was observed in the ACT (15.36 mg/kg/day) group with 54% of the female rats displaying irregular cycles at PND 159. While uterine weights were not altered, a significant accumulation of uterine nuclear estrogen receptors was observed in the ACT group.Conclusion: These results indicate that acetochlor can act as the endocrine disruptor and that endpoints related to pubertal development and reproductive functions sensitive sites are targeted with this persistent pollutant.

Development of methods for extraction and in vitro quantification of estrogenic and androgenic activity of wastewater samples

Chemicals released into the environment by anthropogenic activities have been linked to estrogenic or androgenic effects in exposed wildlife, and there is a need to develop and validate rapid and cost-effective methods to quantify the total estrogenic and androgenic activity of environmental water samples. In this study, estrogen receptors (ER) were isolated from sheep ( Ovis aries) uteri and rainbow trout ( Oncorhynchus mykiss) livers and androgen receptors (AR) were isolated from rainbow trout brains. The isolated receptors were used in competitive receptor binding assays to test the affinity of known estrogenic and androgenic chemicals for the receptor binding site, and results were compared with literature values for the rat uterine ER binding assay and the E-Screen. The relative binding affinities of the tested compounds to ER from different species were similar, and binding to the ER was a more responsive endpoint than the cellular effect measured in the E-Screen. Using the sheep ER binding assay in combination with solid-phase extraction, the estrogenic activity in a raw sewage sample from a municipal treatment plant in Brisbane (Queensland, Australia) was measured at 51–73 ng/L estradiol equivalents (EEq).

Binding of Flavonoids fromSophora flavescensto the Rat Uterine Estrogen Receptor

Prenylflavonoids and lavandulylflavonoids were isolated from the roots of Sophora flavescens Aiton (Fabaceae). The ability of 8-prenylkaempferol (1), kushenol X (2), norkurarinone (3), leachianone A (4), kushenol C (5), maackiain (6) and a root-extract of S. flavescens to displace 17beta-estradiol (E2) from rat uterine estrogen receptor (ER) was determined. Relative binding affinities (RBA) of prenylated flavonoids were weak with RBA values between 0.004 and 0.072. A lavandulyl or prenyl group at the position 8 enhanced binding to rat uterine ER.

Binding of Phytoestrogens to Rat Uterine Estrogen Receptors and Human Sex Hormone-binding Globulins

The interaction of phytoestrogens with the most important binding sites of steroid hormones, i.e. sex hormone-binding globulin and estrogen receptors, was investigated. Relative binding affinities and association constants for 21 compounds among them isoflavones, flavones, flavonols, flavanones, chalcones and lignans were determined. The lignan nordihydroguaiaretic acid weakly displaced 17beta-[3H]-estradiol from estrogen receptor and Scatchard analysis suggests non-conformational changes. Compounds from Glycyrrhiza glabra, liquiritigenin and isoliquiritigenin, showed estrogenic affinities to both receptors. 18beta-Glycyrrhetinic acid displaced 17beta-[3H]-estradiol from sex hormone-binding globulin but not from the estrogen receptor. Phytoestrogens compete with 17beta-estradiol much stronger than with 5alpha-dihydrotestosterone for binding to sex hormone-binding globulin.

Confirmation of uterotrophic activity of 3-(4-methylbenzylidine)camphor in the immature rat.

In this study we found that the ultraviolet sunscreen component 3-(4-methylbenzylidine)camphor (4MBC) is uterotrophic in immature rats when administered by either subcutaneous injection or oral gavage. These data confirm earlier reports of uterotrophic activity for this agent when administered to immature rats in the diet or by whole-body immersion; however, they are in contrast to negative unpublished immature rat uterotrophic assay results. Data also indicate that 4MBC binds to isolated rat uterine estrogen receptors and shows activity in a human estrogen receptor yeast transactivation assay; however, we considered both of these effects equivocal. In this study, we confirmed the original observation that 4MBC was active as a mitogen to MCF-7 breast cancer cells. We evaluated and discounted the possibility that the estrogenic activity of 4MBC is related to its bulky camphor group, which is of similar molecular dimensions to that of the weak estrogen kepone. Uncertainty remains regarding the mechanism of the uterotrophic activity of 4MBC.

Bisphenol a binds to the low-affinity estrogen binding site.

Environmental estrogens are suspected of being involved in the current increase in the incidence of human reproductive malfunctions, such as a decrease in male reproductive capacity and an increased incidence of breast cancer in women. The influences of these compounds have been proposed to be mediated through binding to macromolecules, such as estrogen receptor alpha or beta. In this study we examined whether the low-affinity Type II estrogen binding site (Type II EBS), originally identified in the rat uterus, is a possible mediator of environmental estrogens such as bisphenol A (BPA). Analysis of BPA's binding to an enriched fraction of Type II EBS, using a competition assay, indicated that BPA was able to compete with estradiol in binding to this site. At a concentration of 10-15 microM (comparable to that required to induce uterine proliferation), BPA inhibited the binding of estradiol to Type II EBS by greater than 50%. The binding affinity of BPA for the Type II EBS was only 8-10-fold lower than that of the synthetic estrogen diethylstilbestrol. The binding of BPA to Type II EBS appeared specific to BPA, in that endosulfan, another environmental estrogen, failed to displace estradiol from the site. A comparison of the relative binding affinities of BPA for rat uterine estrogen receptor alpha to that of the Type II EBS implies that BPA preferentially binds to the Type II EBS.

Safety evaluation of phytosterol esters. Part 1. Assessment of oestrogenicity using a combination of in vivo and in vitro assays

Phytosterols are natural constituents of the human diet, and as part of an extensive programme of safety evaluation studies investigating their use as a novel food ingredient, the possible oestrogenic effects of phytosterols have been investigated using a combination of in vitro and in vivo assays. Competitive binding with the immature rat uterine oestrogen receptor (ER) has been used to measure the ability of phytosterols to bind to ERs while the transcriptional activation of oestrogen-responsive genes has been examined in an oestrogen-inducible yeast screen. Phytosterols did not display any activity in these in vitro assays. Uterotrophic assays have been conducted to investigate the potential for phytosterols to elicit an oestrogenic response when administered orally to immature female rats (n=10) at doses of 0, 5, 50 or 500 mg/kg/day for 3 consecutive days. Phytosterols (a well characterized mixture of β-sitosterol, campesterol and stigmasterol) and phytosterol esters (the previous phytosterol mixture esterified with fatty acids from sunflower oil) did not exhibit oestrogenic activity in the immature female rat using uterine wet weight as the endpoint. β-oestradiol (0.4 mg/kg/day) consistently produced a significant increase in uterus weights. Coumestrol (a known phytoestrogen) was also tested as a weak positive control and produced a dose response at doses of 20, 40 and 80 mg/kg/day in the uterotrophic assay. In conclusion, we have shown that phytosterols do not bind to the ER and do not stimulate transcriptional activity of the human ER in a recombinant yeast strain. In addition, there was no indication of oestrogenicity from the uterotrophic assay when the material was administered by oral gavage to immature female rats.

Ah Receptor and Estrogen Receptor-Dependent Modulation of Gene Expression by Extracts of Diesel Exhaust Particles

The ability of a methylene chloride extract of diesel exhaust particle (EDEP) to activate the aryl hydrocarbon receptor (AhR), bind to and activate the estrogen receptor (ER), and induce gene expression mediated via these nuclear receptors was examined in Hepa1c1c7 mouse hepatoma and MCF-7 human breast cancer cells. EDEP was able to induce a protein–DNA complex by gel retardation assays using a [γ-32P]dATP-labeled dioxin response element (DRE). This complex could be effectively competed by a 150-fold excess of unlabeled DRE but not by a 150-fold excess of unlabeled mutated DRE. In Hepa1c1c7 cells that were transiently transfected with a DRE-regulated luciferase reporter gene, 4.6 ng/μl EDEP treatment for 24 h resulted in a 22-fold induction of luciferase activity. In the same cell line, ethoxyresorufin-O-deethylase activity was significantly induced 20-fold following 24 h treatment with 4.6 ng/μl EDEP. Using a competitive ligand binding assay, EDEP displaced bound tritiated E2 from the rat uterine ER in a dose-dependent manner with an IC50of approximately 100 ng/μl compared to the IC50of E2, which was approximately 4.4×10−4ng/μl (1.6 nM). In MCF-7 human breast cancer cells transiently transfected with a Gal4-regulated luciferase reporter gene (17m5-G-Luc) and a chimeric ER (Gal4-HEG0), treatment with 4.6 ng/μl EDEP for 24 h resulted in a three-fold increase in luciferase activity (P<0.01) compared with the seven-fold increase observed with E2. This study demonstrates that EDEP is able to activate the AhR and ER and induce transcription of reporter genes regulated by these receptors' DNA response elements. Further study is required to identify the individual compound(s) responsible for the observed activity.

Pharmacokinetics and pharmacodynamics of a novel estrogen Δ 8-estrone in postmenopausal women and men

Recently a tenth equine estrogen, identified as the sulfate ester of Δ 8-estrone has been reported to be present in Premarin (a conjugated equine estrogen preparation), and because of its unique ring B unsaturated structure (conjugated double bond in the B ring), we have, in the present study, determined its pharmacokinetics in postmenopausal women and men, its interaction with uterine estrogen receptors and its uterotropic activity. After the administration of [ 14C]Δ 8-estrone, blood was drawn at various time intervals, and the plasma fractionated into the unconjugated sulfate and glucuronide fractions. The disappearance of radioactivity as Δ 8-estrone from plasma can be described as a function of two exponentials. The half-lives of the first and second components were 5±0.2 and 40.4 min, respectively. The mean metabolic clearance rate calculated (MCR), was 1711±252 l/d m 2. From the unconjugated fraction, Δ 8-17 β-estradiol was also isolated and identified. From the sulfate conjugated fraction, Δ 8-estrone sulfate and Δ 8-17 β-estradiol sulfate were isolated in almost equal amounts. No other metabolites of Δ 8-estrone was detectable in the plasma. Both Δ 8-estrone and Δ 8-17 β-estradiol bind with human endometrial and rat uterine estrogen receptors with high affinity. The binding affinities of Δ 8-17 β-estradiol for human endometrial and rat uterine cytoplasmic receptors were 4 and 25 times higher than those of the parent estrogen Δ 8-estrone, respectively. Administration of Δ 8-estrone and Δ 8-17 β-estradiol (2 μg/100 g body weight) to immature rats significantly ( P<0.05) increased the uterine weight compared to the controls. These data demonstrate that Δ 8-estrone has estrogenic activity, and that it is further metabolized in man to a single more potent estrogen, Δ 8-17 β-estradiol. The extent of this activation by 17 β-reduction appears to be greater than that observed with other estrogens. Both estrogens circulate as sulfate conjugates and are very slowly eliminated from the circulation. These data further suggest that Δ 8-estrone and its major metabolite Δ 8-17 β-estradiol can contribute to the overall in vivo biological effects of Premarin.

Binding characteristics of novel nonsteroidal antiestrogens to the rat uterine estrogen receptors

Tamoxifen (TAM), the only antiestrogen currently available for the endocrine therapy of breast cancer behaves as a mixed agonist/antagonist of estrogen action, thus limiting its therapeutic potential. We report the binding characteristics of a novel series of nonsteroidal antiestrogens to the rat uterine estrogen receptor. As measured by competition studies, the affinity of EM-652, the active metabolite of the prodrug EM-800, for the estrogen receptor is 7–11 times higher than that of 17 β-estradiol (E 2), ICI 182780, and hydroxy-tamoxifen (OH-TAM), the active metabolite of Tamoxifen. EM-652 is 20× more potent than ICI 164384 and Droloxifene while it is 400 times more potent than Toremifene in displacing [ 3H]E 2 from the rat uterine estrogen receptor. On the other hand, the prodrug EM-800 and Tamoxifen have respectively 150-fold and 410-fold less affinity for the estrogen receptor than the pure antiestrogen EM-652. No significant binding of EM-652, EM-800, TAM or OH-TAM was observed to the rat uterine progesterone receptor at concentrations up to 10 000 nM except for TAM that caused a 50% displacement of labeled R5020 at 4000 nM. No significant binding of EM-652 or EM-800 was observed on the rat ventral prostate androgen receptor or the rat uterine progesterone receptor. The present data demonstrate the high affinity and specificity of the new antiestrogen, EM-652, for the rat uterine estrogen receptor. The antiestrogen EM-652 thus becomes the compound having the highest known affinity for the estrogen receptor. Due to its unique potency and its pure antiestrogenic activity already demonstrated in many systems, this antiestrogen could well offer an important advance for the endocrine therapy of breast cancer, uterine cancer, and other estrogen-sensitive diseases in women.

Two populations of the estrogen receptor separated and characterized using aqueous two-phase partitioning.

Two populations of the rat uterine estrogen receptor (ER) were separated and characterized using aqueous two-phase partitioning. Countercurrent distribution of rat uterine cytosolic ER allowed rapid and efficient separation of two populations, one population partitioned preferentially into the top phase (T, K(obs) = 3-6) and the other into the bottom phase (B, K(obs) = 0.01-0.03). The majority of unoccupied cytosolic ER is in the T population. Upon estrogen binding and/or heating to 30 degrees C in vitro the T population is converted to the B population. The transition from T to B does not exclusively involve loss of heat shock protein 90 and does not alter the ligand binding ability of the steroid binding domain. Using the human ER steroid binding domain overproduced in Escherichia coli and the steroid binding domain generated by partial trypsinization of the rat uterine ER, we demonstrate that the characteristic distinguishing T and B populations is not localized to this domain alone but may be associated with the amino terminal half of the ER (the A/B and DNA binding domains). The T to B transition of the ER also occurs in human MCF-7 breast cancer cells upon treatment with estrogen at 37 degrees C.

Interactions between endosulfan and dieldrin on estrogen-mediated processes in vitro and in vivo

There is growing concern that estrogenic chemicals, both natural and human-made, may be causing a variety of reproductive disorders in wildlife and human populations. Recent in vitro data suggest that the interaction between some weakly estrogenic organochlorines, dieldrin, endosulfan, toxaphene, and chlordane, causes a synergistic increase in their estrogenic potency, an effect due to joint action on estrogen receptors (ER). As these studies were conducted using models of estrogen action derived from cells that are not physiologically controlled by estrogens, the relevance of these findings to human health are not clear. The present studies were conducted to examine the interaction between endosulfan and dieldrin in the activation of ER in or extracted from mammalian cells. Endosulfan and dieldrin showed no synergism in displacing 3H-E 2 from rat uterine ER or in inducing the proliferation of MCF-7 breast cancer cells, an estrogen-dependent response. Furthermore, endosulfan (0.1 mg per animal per d) or dieldrin (0.1 mg), alone or in combination, injected intraperitoneally daily for 3 d, did not stimulate any uterotrophic activity nor had any effect on pituitary prolactin or other endocrine-related endpoints in immature female rats. These studies demonstrate that these weakly estrogenic compounds do not interact in a synergistic fashion in binding to ER or in activating ER-dependent responses in mammalian tissues or cells. Thus, these results suggest that coexposure to these weakly estrogenic environmental contaminants likely will not cause human reproductive toxicity related to estrogen action.

Étude fonctionnelle de mutations du gène du récepteur V2 responsables d'un diabète insipide néphrogénique congénital

In previous reports the amount of rat uterine progesterone receptor detected in crude nuclear pellets was only 50% of that which was depleted from the cytosol after an injection of progesterone. We have previously shown that this discrepancy is due to a 50% decrease in receptor binding in the nuclear pellets during the post-assay washing procedure (Walters & Clark, 1978). If this loss were due to the dissociation of receptor-progesterone complexes from nuclear sites, it should be possible to detect and recover them by adding hydroxylapatite to the nuclear pellets at the end of the incubation. This modification of the nuclear exchange assay resulted in 83% greater recovery of specific [3H]-progesterone binding than in the nuclear pellet alone and accounted for the remainder of the translocated nuclear receptors. Thus, quantitation of nuclear progesterone receptors can be achieved in the rat uterus with this nuclear exchange-HAP assay. This method of trapping solubilized hormone complexes also increased recovery of progesterone receptors from the chick oviduct. In contrast, this procedure did not significantly increase the amount of rat uterine estrogen receptor detected, suggesting that little solubilization of nuclear bound estrogen receptors takes place during incubation and nuclear washing procedures.This simple modification of the exchange assay permits measurement of the total quantity of nuclear bound progesterone receptors in the rat uterus. These results emphasize that receptors may dissociate from nuclear pellets under in vitro conditions which results in an underestimate of the nuclear receptor level that actually existed in vivo.

Calmodulin Decreases the Estrogen Binding Capacity of the Estrogen Receptor

We previously demonstrated the ability of calmodulin (CaM) to decrease the binding affinity of estradiol (E2) to the rat uterine estrogen receptor (ER). We show now that CaM induces a loss of E2binding capacity especially when ER molecules exhibit a lower binding affinity for the hormone. By Western blotting and [125I]tamoxifen aziridine covalent labeling we found that this CaM-induced loss is not associated with a disappearance of the ER protein. In addition, we were able to demonstrate a CaM-mediated decrease in E2binding of a human recombinant ER expressing solely its hormone binding domain (HBD, aa 282-595). Hence, CaM can modulate the structure of the HBD of the ER without any involvement of a degradative process, this conformational change is not mediated by other domains of the receptor and / or components of the native ER heterocomplex.

4′-Substituted analogues of idoxifene: Antiestrogens and calmodulin antagonists

4′-substituted analogues of the antiestrogen idoxifene have been prepared. All the compounds were assayed for antagonism of calmodulin dependent c-AMP phosphodiesterase and for binding to rat uterine estrogen receptor. The 4′-amino compound was the most potent antiestrogen (RBA = 47) whilst retaining a similar calmodulin antagonism to idoxifene.

Modulation of Estradiol and DNA Binding to Estrogen Receptor upon Association with Calmodulin

The ability of calmodulin (CaM) to modify the conformation of the hormone binding domain (HBD) rat uterine estrogen receptor (ER) was investigated. Dissociation constant of estradiol (E2) binding to HBD increased almost 2-3-fold when CaM bound to ER ; two other Ca++-binding proteins failed to show this property, demonstrating the specificity of the phenomenon. Elution on CaM-Sepharose of truncated ER molecules located the CaM binding domain of ER at the left edge of HBD. Association of CaM with activated [3H]E2-ER complexes increased the ability of the latter to interact with an estrogen response element (ERE) as demonstrated by DNA-cellulose competition assay ; CaM also reinforces the potency of ERE to dissociate the [3H]E2-ER complex. We hypothesize that these CaM-induced conformational changes of ER may play a role in transcriptional activity of the latter.

Chloro‐s‐triazine antagonism of estrogen action: Limited interaction with estrogen receptor binding

In an accompanying article (see pp. 183-196), it was reported that administration of very high doses of the chlorotriazine herbicides atrazine, simazine, and diaminochlorotriazine (DACT), a common metabolite, expressed antiestrogenic activity in uteri of female Sprague-Dawley rats without expressing intrinsic estrogenic activity. In the present article, studies of chlorotriazine interaction with rat uterine estrogen receptors (ER) are reported. Under equilibrium conditions, none of the triazine compounds showed an ability to compete against binding of radiolabeled estradiol to ER. A weak competition was evident only if cytosols were preincubated with triazines at 25 degrees C prior to introduction of tracer. Competition was very weak, with kl estimates of 10-100 microM. A limited Scatchard analysis suggested a competitive type of inhibition. Sucrose gradient analysis of cytosol incubations showed that triazine interaction with the 4S isoform of ER may be greater than with the 8S form. When administered to ovariectomized rats for 2 d at 300 mg/kg/d, atrazine, simazine, or DACT all reduced uterine ER binding capacity by approximately 30%. Results from the receptor binding studies indicated that triazine competition against ER binding occurred to a much lesser degree than inhibition of estrogen-mediated responses reported in accompanying articles. This suggests that the complete responses to triazines may include inhibition of events other than or in addition to ER binding of estrogen.

Hexestrol diazirine photoaffinity labeling reagent for the estrogen receptor.

3-Azibutyl (2R*,3S*)-2,3-bis(4-hydroxyphenyl)pentyl sulfide (1), a photoaffinity labeling reagent for the estrogen receptor (ER), has been prepared in unlabeled and in high specific activity tritium-labeled form (32 Ci/mmol) and has been shown to undergo selective and efficient photocovalent attachment to rat uterine ER. Diazirine 1 demonstrates high binding affinity for ER, as determined by both a competitive binding assay and a direct binding assay (relative binding: estradiol = 100; (1) = 17. Kd: estradiol = 0.19 nM; (1) = 0.98 nM, respectively). It is efficient in site-specific photoinactivation of ER, reaching the level of 31% after 5 min of irradiation at > 315 nm. The tritium-labeled diazirine [3H]-1 undergoes specific photocovalent attachment to ER with an attachment efficiency of 29% and a selectivity of 90%. Both of these values are quite high for a photoaffinity reagent. SDS-polyacrylamide gel electrophoretic analysis of the photolabeled proteins shows specific labeling of a major species at M(r) 65,000, the same species that is labeled by [3H]tamoxifen aziridine, a well-characterized affinity label for ER. Hexestrol diazirine 1 is the first carbene-generating photoaffinity label that covalently labels ER with high efficiency and selectivity, and it should be useful in further studies on the hormone-binding domain of ER.