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Departments of Molecular and Integrative Physiology (J.S., W.R.H., S.S., B.S.K.) and Chemistry (Y.R.H., J.A.K.), University of Illinois and University of Illinois College of Medicine, Urbana, Illinois 61801
Address all correspondence and requests for reprints to: Dr. Benita S. Katzenellenbogen, Department of Molecular and Integrative Physiology, 407 South Goodwin Avenue, 524 Burrill Hall, University of Illinois, Urbana, Illinois 61801-3704. E-mail: . katzenel{at}life.uiuc.edu
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Abstract |
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, but not ERß, we have added basic side-chains typically found in nonsteroidal antiestrogens to pyrazole compounds that bind with much higher affinity to ER than to ERß. In this way we have developed basic side-chain pyrazoles (BSC-pyrazoles) that are high affinity, potent, selective antagonists on ER. These BSC-pyrazoles are themselves inactive on ER and ERß, and they antagonize E2 stimulation by ER only. We investigated seven basic side-chain substituents on various alkyl-triaryl-substituted pyrazoles, and the most ER-selective compound was methyl-piperidino-pyrazole (MPP). ER-selective antagonism was observed on diverse reporter-promoter gene constructs containing estrogen response elements that are consensus, nonconsensus (pS2), or comprised of multiple half-estrogen response elements (NHERF/EBP50) and on genes in which ER works indirectly by tethering to other DNA-bound proteins (TGFß3). In contrast to these BSC-pyrazoles, the antiestrogens trans-hydroxytamoxifen, raloxifene, and ICI 182,780 suppress E2 activity via both ER and ERß. The most effective BSC-pyrazole, MPP, fully antagonized E2 stimulation of pS2 mRNA in MCF-7 breast cancer cells, consistent with the fact that these cells contain almost exclusively ER. These compounds should be useful in studying the biological functions of ER and ERß and in selectively blocking responses that are mediated through ER. |
Introduction |
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and ERß (1, 2, 3, 4, 5). Although both of these receptors function as ligand-modulated transcription factors, their tissue distributions are quite different (1, 2), and the activity of estrogens of varying structure is not always the same on the two subtypes (6, 7). Thus, it is likely that at least some of the biological roles of ER and ERß are different, but the nature and importance of these differences are still open questions.
The generation of ER and ERß knockout animals has provided much information about the function of these ER subtypes (8). Another approach has been the development of ER subtype-selective ligands. Coming to a good understanding of the key biological roles of ER and ERß will be important for understanding the action of natural estrogens and for developing synthetic selective ER modifiers (9, 10) that are needed to optimally regulate fertility and menopausal hormone replacement,and to prevent and treat hormone-responsive breast cancer.
We and others have developed compounds that are capable of stimulating ER very selectively. Members of the triarylpyrazole class, such as propylpyrazole triol (PPT), are more than 1000-fold more potent on ER than on ERß (11, 12), and A-ring reduced metabolites of 19-nor synthetic progestins also show strong ER agonist selectivity (13). Certain tetrahydrochrysenes, such as R,R-diethyl-tetrahydrochrysene, are agonists on ER, but pure antagonists on ERß (14, 15), as is a methoxychlor metabolite (16).
From what has been published, it appears that it is more difficult to develop ligands that stimulate ERß to a greater extent than ER. Certain phytoestrogens, such as genistein, are more potent on ERß than ER (6, 7), but their selectivity toward ERß is more limited than is the selectivity of PPT toward ER, for example (11). However, there is progress in this direction, in that some new synthetic bis-benzylnitriles and related compounds have up to 170-fold potency selectivity on ERß (17).
In this report we take a new approach to the development of antagonists that are more potent on ER than on ERß. We have introduced into the very ER-selective agonist pyrazole ligands, basic side-chain (BSC) substituents typical of those found in antiestrogens such as tamoxifen and raloxifene. In this manner, we have generated BSC-pyrazoles that are selective ER antagonists, and in this report we characterize their activities and the optimization of their selectivity as ER antagonists.
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Materials and Methods |
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(pCMV5-hER) was constructed as previously described (18). The expression vector pCMV5-ERß was constructed by inserting the cDNA encoding the full-length human ERß (530 residues) (19) or the short form of human ERß (477 residues), into the BamHI site of pCMV5. The estrogen-responsive reporter plasmids were (ERE)3-pS2-chloramphenicol acetyltransferase (CAT), constructed as described previously (20); (ERE)2-pS2-Luc, constructed by inserting the (ERE)2-pS2 fragment from (ERE)2-pS2-CAT into the MluI/BglII sites of pGL3-Basic vector (Promega Corp.); and NHERF/EBP50-Luc, constructed by inserting the human sodium- hydrogen exchanger regulatory factor/ezrin radixin moesin-binding protein 50 (NHERF/EBP50) promoter fragment (containing -2985 to +496 bp of the human NHERF gene) (21, 22) (Ediger, T. R., S.-E. Park, and B. S. Katzenellenbogen, manuscript submitted) into the KpnI/XhoI sites of pGL3-Basic vector. The plasmid pCMVß (CLONTECH Laboratories, Inc., Palo Alto, CA), which contains the ß-galactosidase gene, was used as an internal control for transfection efficiency. The compounds studied in this report were prepared as previously described (23, 24).
Hormone binding assays
Binding affinities of each compound for ER and ERß were determined in a radiometric competitive binding assay, using [3H]E2 as tracer and hydroxylapatite to adsorb ligand-receptor complex (25). Receptor preparations were human ER and ERß (53-kDa form), expressed in baculovirus and purified (PanVera, Madison, WI). Values given represent the mean of two or three repeat determinations that have a coefficient of variation of 0.3.
Cell culture and transient transfections
Human endometrial cancer (HEC-1) cells were maintained in culture as previously described (14). Transfection of HEC-1 cells in 24-well plates used a mixture of 0.35 ml serum-free improved MEM medium and 0.15 ml HBSS containing 5 µl lipofectin (Life Technologies, Inc., Gaithersburg, MD), 1.6 µg transferrin (Sigma, St. Louis, MO), 0.5 µg pCMVß-galactosidase as internal control, 1 or 2 µg of the reporter gene plasmid, 100 or 250 ng ER expression vector, and carrier DNA to a total of 3 µg DNA/well. The expression levels of ER and ERß differ by no more than 2-fold based on FLAG epitope-tagged ER and ERß in Western blots (19). The cells were incubated at 37 C in a 5% CO2-containing incubator for 6 h. The medium was then replaced with fresh medium containing the desired concentrations of ligands. Reporter gene activity was assayed 24 h after ligand addition. CAT or luciferase activity, normalized for the internal control ß-galactosidase activity, was assayed as previously described (14). All transfections used the long ERß form except the NHE-RF/EBP50-Luc and TGFß3-CAT assays, in which the short form of ERß was used because it gave slightly more robust stimulation by E2.
MCF-7 cells were maintained in culture as previously described (20, 26). About 0.3 x 106 cells were seeded into each well of a six-well plate, and after 3 d, fresh medium containing the desired concentration of ligands was added to each well. Twenty-four hours later the cells were harvested for RNA extraction.
Real-time PCR analysis
The total RNA from MCF-7 cells was isolated using TRIzol reagent (Life Technologies, Inc.) according to the manufacturer’s protocol. One microgram of total RNA was reverse transcribed using the GeneAmp RNA PCR kit (PE Applied Biosystems, Foster City, CA). Part (1/100th) of each cDNA product was subjected to real-time PCR analysis on an ABI PRISM 7700 sequence detection system using TaqMan universal PCR master mix (PE Applied Biosystems). The pS2 cDNA (27, 28) was amplified with the primers pS2f (5'-GCGCCCTGGTCCTGGTGTCCAT-3') and pS2r (5'-GAAAACCACAATTCTGTCTTTCAC-3') and was detected by the probe 6-carboxyfluorescein-CCCAGACAGAGACGTGTACAGTGGCCC-TAMRA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was used as the internal control, was amplified with the primers GAPDH forward (5'-GAAGGTGAAGGTCGGAGTC-3') and GAPDH reverse (5'-GAAGATGGTGATGGGATTTC-3'). The signal was detected by the probe 6-carboxyfluorescein-CAAGCTTCCCGTTCTCAGCC-6-carboxytetramethylrhodamine. The comparative threshold cycle method was used to determine the relative expression level of pS2 mRNA.
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Results |
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and ERß binding selectivity of BSC-pyrazoles
(see Fig. 1 for a simple nomenclature for these pyrazoles). From earlier work, we identified a single position on 1,3,5-triaryl-4-alkyl-pyrazoles where the BSC substitution is well tolerated by the ERs, namely, the C5 phenol group (24), and we developed a regio-selective synthesis to prepare these compounds efficiently (23). Compounds(I-(A-G)-Et) exemplify various BSC placed on our initially discovered C4-ethyl-triaryl pyrazole I that is an ER-selective agonist ligand (11, 12, 14). Once we had identified the most promising basic side chains (A, C, and E; see below), we explored other C4 substituents on this core system [methyl (A, C, and E) and in one case propyl (A)].
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and ERß, determined in a radiometric competitive binding assay (25), are listed in Fig. 1
. The affinities are expressed as relative binding affinity (RBA) values, where the RBA of E2 is 100, and also as Ki values calculated from the RBA values. All of the compounds investigated had ER binding affinity selectivity, with some showing ER/ERß selectivity ratios of 100–400. It is instructive to compare the ER binding affinity and selectivity of these BSC-pyrazoles to those of the parent triaryl pyrazoles (i.e. R = H instead of the BSC). The parent triaryl pyrazoles (Cpd. No. I, Fig. 1) have ER RBA values of 5.4–51 and ER/ERß ratios of 80–390; interestingly, the BSC analogs (Cpd. No. I-A through I-G) can reach similar levels of ER selectivity (ER/ERß ratio of 220), and they show similar binding affinity for ER.
Although we have not explored these series exhaustively, where comparisons can be made, the ER/ERß affinity ratio reaches a maximum with a smaller C4 substituent in the BSC analogs than in the triphenol series (compare pyrazole triols I, where the propyl analog PPT has the highest ratio, with BSC-pyrazoles I-A, where the methyl analog has the highest ER/ERß affinity ratio). This may reflect differences in the way in which triphenolic and BSC-pyrazoles are oriented in the ligand binding pockets of the ERs (24, 29) (see Discussion). All of the BSC-pyrazoles had ER/ERß affinity ratios much higher than the ratios seen with the well known antiestrogens trans-hydroxytamoxifen, raloxifene, and ICI 182,780 (Fig. 1, bottom).
ER and ERß transcriptional selectivity of BSC-pyrazoles
To evaluate which BSC were effective in converting the pyrazoles into ER antagonists, we first screened the activity of the seven BSC-pyrazoles with C4-ethyl substituents as agonists or as antagonists of E2-induced transcription through ER and ERß by a cotransfection assay, using a single concentration regimen. Expression plasmids for ER and ERß (pCMV5-ER and pCMV5-ERß) together with an estrogen-responsive reporter gene plasmid (3ERE-pS2-CAT) containing three consensus estrogen response elements (EREs) and the promoter from the pS2 gene and a control plasmid (pCMV5-ßgal) were transfected into HEC-1 cells. The results of these assays are summarized in Fig. 2.
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or ERß; all failed to evoke significant transcriptional activation of ER or ERß. Several of them showed considerable ER subtype selectivity as transcriptional antagonists, being more potent antagonists of E2 stimulation on ER than on ERß. We selected three of these [piperidinyl (A), pyrrolidino (C), and diethylamino (E)] for more extensive dose-response studies together with their analogs that had different C4-alkyl substituents. These data are shown in Figs. 3 and 4.
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, A and B, respectively. Comparison of the C4-ethyl series (Fig. 3A) with the C4-methyl series (Fig. 3B) shows, in each case, that the methyl series is more selective. Within the C4-methyl series (Fig. 3B), the piperidinyl analog (A) appears to be both a more potent and a more complete antagonist with ER selectivity than the two other analogs. Thus, of these six compounds, I-A-Me appears to be most selective as an ER antagonist (cf. Fig. 3B). It is of note that the BSC in this compound is the same as that found in the antiestrogen raloxifene.
An additional evaluation of piperidinyl congeners (A) with three different C4 substituents, methyl, ethyl, and propyl (Fig. 4A), demonstrated again that in terms of both antagonist potency and ER selectivity, the best compound is I-A-Me. Overall, there is a good correlation between the selectivity of these BSC pyrazoles for ER in terms of their binding affinity and their potency as antagonists.
The selectivity of MPP in inhibiting ER is in noted contrast to that of the two well known antiestrogens, raloxifene and ICI 182,780 (Fig. 4B). Raloxifene showed a similar dose response for suppression of both ER and ERß; ICI 182,780 was somewhat more potent in suppressing ERß than ER. However, in contrast to MPP, both of these antiestrogens fully suppressed ER and ERß stimulation by E2.
Characterization of the most ER-selective antagonist, MPP
Based on the above gene transcriptional assays, the pyrazole I-A-Me, which we have termed MPP, was found to be the most ER-selective antagonist of the various compounds examined. We therefore evaluated its ER antagonist selectivity further in a different estrogen-responsive gene construct, NHERF/EBP50-Luc (containing the 3.5-kb 5'-flanking and promoter region of the sodium-hydrogen exchanger regulatory factor/ezrin-radixin-moesin binding protein 50) (21, 22) (Ediger, T. R., S.-E. Park, and B. S. Katzenellenbogen, manuscript submitted) (Fig. 5). This promoter contains no palindromic full EREs, and its estrogen responsiveness is, instead, attributable to multiple half EREs (22). Even at high (1 µM) concentrations, MPP had no stimulatory activity on ER or ERß, and it fully inhibited ER activity by E2 in a concentration-dependent manner while having no suppressive activity on ERß stimulation by E2 (Fig. 5).
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), where ER is thought to act not by direct DNA binding, but by being tethered to the DNA by other proteins (30). MPP again behaved as an ER-selective antagonist: It evoked little or no stimulation through ER or ERß, and it was able to suppress E2 stimulation through ER, but had no effect on E2 stimulation of the TGFß3 promoter via ERß. In contrast, the synthetic selective ER modulators trans- hydroxytamoxifen and ICI 182,780, which also alone did not stimulate activity through ER or ERß, suppressed E2 stimulation through both ER and ERß (Fig. 6).
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stimulation of an endogenous gene, we monitored its effectiveness in blocking E2 induction of pS2 mRNA production in MCF-7 breast cancer cells, which contain high levels of ER and minimal, if any, ERß. As shown in Fig. 7, MPP fully suppressed pS2 mRNA induction by E2, consistent with the fact that these cells contain almost exclusively ER. ICI 182,780 also fully suppressed pS2 mRNA induction by E2, as expected.
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Discussion |
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and ERß (6, 7, 19), and show little selectivity in their potency toward these receptors. We have been able to develop estrogen antagonists that are ER selective by starting with 1,2,5-triaryl-4-alkyl-pyrazoles, a class of ER agonists that has up to a 400-fold affinity preference for ER (11, 12). By introducing BSC substituents, similar to those found in antiestrogens, on the C5-phenol of these pyrazoles, we have obtained BSC-pyrazole compounds that are antagonists, but still retain this ER binding selectivity (up to 200-fold, as in MPP). These findings confirm that although the ligand binding pockets of ER and ERß are highly conserved, there is sufficient structural difference in the overall ligand-binding domains (amino acid identity only 59%) (1, 2, 5, 6) that ER subtype-selective antagonists as well as agonists can be developed.
Within the BSC pyrazole series, we observed a good correlation between ER selectivity in terms of their binding affinity and their antagonist potency. What is interesting in this comparison is that the C4-alkyl substituent that gives the highest selectivity for the BSC ER antagonists, namely a methyl group as in MPP, is smaller than the C4-propyl group needed to obtain the highest ER/ERß selectivity in the triarylpyrazole agonist series. Based on molecular modeling and structure-binding affinity relationships, we believe that this is due to differences in how these two classes of ligands are oriented in the ligand binding pocket of ER (11, 24, 29), so that the methyl substituent of the BSC pyrazole and the propyl substituent of the pyrazole project into different regions of the pocket. Because of these different orientations, groups of different sizes are required to obtain the highest selectivity in binding in the agonist vs. antagonist ligand structures.
ERs are known to work at several different types of gene regulatory sites (9, 10, 22, 30, 31). These sites include consensus and nonconsensus EREs (such as vitellogenin and pS2, respectively) (27, 28), sites comprised of multiple half-EREs (such as NHERF/EBP50) (22), and sites at which ER works indirectly by tethering to other DNA-bound proteins (such as TGFß3) (30). MPP showed ER-selective antagonism at all of these types of gene regulatory sites, suggesting that it can induce an antagonist conformation of ER that is not able to activate gene transcription through diverse regulatory sites.
It is becoming increasingly well documented that different estrogen target tissues and cells contain varying amounts of one or both of the ER subtypes (3, 4, 6, 32). An important aspect still to be fully elucidated is which estrogen-responsive genes and biological responses are under regulation by ER, ERß, or both. The availability of a compound such as the ER-selective antagonist MPP provides a new tool for investigating the biological functions of ER and ERß. It can be used by itself to selectively block responses that might be mediated through ER, or it could be used together with an ER subtype-nonselective agonist, such as E2, to achieve stimulation of only ERß. The use of an ER-selective antagonist such as MPP should therefore allow investigation of the roles of ER and ERß in diverse cells and tissues in which these receptors are expressed as well as attribution of estrogen activities to ER in cells expressing both ER and ERß through the selective suppression of ER-mediated responses.
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Acknowledgments |
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Footnotes |
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Abbreviations: BSC-pyrazoles, Basic side-chain pyrazoles; CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; ERE, estrogen response element; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HEC, human endometrial cancer; MPP, methyl-piperidino-pyrazole; PPT, propylpyrazole triol; RBA, relative binding affinity.
Received October 12, 2001.
Accepted for publication November 28, 2001.
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T. L. Ramsey, K. E. Risinger, S. C. Jernigan, K. A. Mattingly, and C. M. Klinge Estrogen Receptor {beta} Isoforms Exhibit Differences in Ligand-Activated Transcriptional Activity in an Estrogen Response Element Sequence-Dependent Manner Endocrinology, January 1, 2004; 145(1): 149 - 160. [Abstract] [Full Text] [PDF]
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J. Lassurguere, G. Livera, R. Habert, and B. Jegou Time- and Dose-Related Effects of Estradiol and Diethylstilbestrol on the Morphology and Function of the Fetal Rat Testis in Culture Toxicol. Sci., May 1, 2003; 73(1): 160 - 169. [Abstract] [Full Text] [PDF]
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J. Sun, J. Baudry, J. A. Katzenellenbogen, and B. S. Katzenellenbogen Molecular Basis for the Subtype Discrimination of the Estrogen Receptor-{beta}-Selective Ligand, Diarylpropionitrile Mol. Endocrinol., February 1, 2003; 17(2): 247 - 258. [Abstract] [Full Text] [PDF]
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 A. S. Clark, M. C. Kelton, and A. C. Whitney Chronic Administration of Anabolic Steroids Disrupts Pubertal Onset and Estrous Cyclicity in Rats Biol Reprod, February 1, 2003; 68(2): 465 - 471. [Abstract] [Full Text] [PDF]
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H. A. Harris, J. A. Katzenellenbogen, and B. S. Katzenellenbogen Characterization of the Biological Roles of the Estrogen Receptors, ER{alpha} and ER{beta}, in Estrogen Target Tissues in Vivo through the Use of an ER{alpha}-Selective Ligand Endocrinology, November 1, 2002; 143(11): 4172 - 4177. [Abstract] [Full Text] [PDF]
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) or ERß (
) and an (ERE)3-pS2-CAT reporter gene and were treated in the absence or presence of E2, BSC-pyrazole compound, or BSC-pyrazole compound plus E2 for 24 h at the concentrations indicated. CAT activity was normalized for ß-galactosidase activity from an internal control plasmid. The maximal activity with E2 was set at 100. Values are the mean ± SD from three or more separate experiments.
