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*RU-486
Endocrinology Vol. 143, No. 8 3071-3082
Copyright © 2002 by The Endocrine Society

ARTICLE

Identification and Characterization of Novel Estrogen Receptor-ß-Sparing Antiprogestins

Ganesan Sathya, Michelle S. Jansen, Susan C. Nagel, C. Edgar Cook and Donald P. MCDonnell

Department of Pharmacology and Cancer Biology (G.S., M.S.J., S.C.N., D.P.M.), Duke University Medical Center, Durham, North Carolina 27710; and Chemistry and Life Sciences (C.E.C.), Research Triangle Institute, Research Triangle Park, North Carolina 27709

Address all correspondence and requests for reprints to: Donald P. McDonnell, Department of Pharmacology and Cancer Biology, Duke University Medical Center, Box 3813, Durham, North Carolina 27710. E-mail: . donald.mcdonnell{at}duke.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The steroid hormones estrogen and progesterone together regulate the development and maintenance of the female reproductive system. The actions of these two hormones are mediated by their respective nuclear receptors located within overlapping cell populations in target organs. The molecular mechanism of action of these two hormones has been defined to a large extent using estrogen receptor (ER) and progesterone receptor (PR) antagonists. In the case of ER, the available antagonists are highly receptor selective. With respect to PR, however, the available antiprogestins also interact with the receptors for glucocorticoids, mineralocorticoids, and androgens. Whereas these cross-reactivities can usually be managed in studies of female reproductive function, it is the recent demonstration that RU486 is an effective antagonist of the ß-isoform of ER that suggested the need for more selective antiprogestins. In this study, we used cell-based transcriptional assays combined with screens using coactivator peptide analogs to identify two novel classes of antiprogestins that distinguish themselves from the antiprogestin RU486 in the manner they interact with PR. One class exhibits the characteristics of a pure antiprogestin in that its members bind to the receptor and induce a conformational change that prevents the presentation of two potential coactivator binding surfaces on the protein. The second class of compounds distinguish themselves from RU486 in that they are ERß sparing. When tested in vivo the ER-sparing antiprogestins were as effective as RU486 in suppressing superovulation. It is anticipated that the availability of these new antiprogestins will advance the studies of PR pharmacology in a manner similar to how the availability of selective ER modulators has helped the study of ER action.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE STEROID HORMONE progesterone is a key regulator of the cellular processes involved in development and maintenance of the female reproductive system. Produced in the ovaries and transported in the bloodstream, progesterone manifests its biological activities only in cells that contain a high affinity progesterone receptor (PR) (1, 2). In the absence of an activating ligand, this receptor resides in an inactive state in the nuclei of target cells associated with a multiprotein inhibitory complex comprised of heat shock and corepressor proteins. Upon binding a ligand, PR undergoes a conformational change that facilitates displacement of associated inhibitory proteins and permits the interaction of a receptor dimer with specific progesterone response elements (PREs) located within the regulatory regions of target genes. The magnitude of the positive or negative effects of the DNA bound receptor on transcription is subsequently determined by the structure of the ligand-receptor complex, promoter environment, and the expression level of receptor associated proteins such as coactivators and corepressors (3, 4, 5, 6, 7, 8). Thus, PR may not function in an equivalent manner in all cells.

The molecular pharmacology of PR is confounded by the existence of two functionally distinct receptor isoforms whose relative expression levels vary in different cell types. These receptor isoforms, human (h) PR-A and hPR-B, are derived from different transcripts initiating from two different promoters within a single gene. The larger receptor, hPR-B, differs from hPR-A by an additional 164 amino acids located at the amino terminus (2). When analyzed in PR-responsive transcription systems reconstituted in vitro, it has been demonstrated that hPR-B is the most active with respect to the positive regulation of progesterone responsive gene transcription and that with few exceptions hPR-A is inactive under the same conditions (3, 5). Indeed, it has now been demonstrated in several systems that the primary function of hPR-A appears to be as a negative regulator of hPR-B mediated gene expression in cells where both receptor isoforms are expressed. More interestingly, however, it has also been noted that agonist or antagonist activated hPR-A can inhibit the transcriptional activity of the estrogen receptor (ER) (2, 6) providing a potential explanation for the physiologically important antiestrogenic activities of progestins.

It has been demonstrated recently that RU486, at physiologically relevant concentrations, is an effective competitive antagonist of ERß activity, whereas it has no direct effect on ER{alpha} function (9). This surprising finding necessitated a reevaluation of studies that based their conclusions on the assumption that RU486-mediated inhibition of a particular response implicated PR. RU486 is also a potent antiglucocorticoid and a partial agonist of androgen receptor (AR) (10, 11), and in the presence of PR-A it inhibits the transcriptional activity mediated by all other steroid receptors (12, 13). Another well-studied antiprogestin, onapristone (ZK98,299), also interacts with and antagonizes glucocorticoid receptor (GR), albeit at high, nonphysiologically relevant concentrations. Interestingly, ZK98,299 exhibits weak ER binding affinity, although in cells expressing PR-A it too can inhibit ER transcriptional activity (12, 14, 15). The cross-talk between PR and other nuclear receptors and the promiscuity of the available antiprogestins have made it difficult to define processes that 1) result from direct transcriptional activities of PR; 2) reflect the interaction of ligand activated PR with other nuclear receptor mediated signaling pathways; or 3) represent the actions of ligands acting in a PR-independent manner through different receptors. Currently, there is no satisfactory way to distinguish between these possibilities.

The complexity of PR pharmacology also bears on the development and use of progestins and antiprogestins as contraceptives or as treatments for endocrinopathies. For conditions that require acute administration, the lack of receptor/pathway specificity may not be of much concern. However, it is likely that PR ligands with improved selectivity would be useful for the treatment of chronic conditions such as endometriosis, uterine fibroids, breast cancer, and brain meningiomas. Positive clinical data exist to support the use of RU486 in these conditions (16, 17, 18, 19, 20, 21). Thus, as tools to study PR biology and as potential novel therapeutics, there is a need to develop new PR ligands with improved receptor specificity. These reagents will help to link different activities of PR and its ligands with specific biological responses.

In this study, we have undertaken to address one aspect of the PR ligand specificity problem by screening a library of structurally diverse PR ligands for novel antiprogestins (1) that alter the shape of PR in a manner distinct from RU486 and (2) that do not directly inhibit ERß-mediated transcriptional activity. Previously, we have shown that a relationship exists between the overall structure of the PR-ligand complex and biological activity (4, 22). It is likely that different receptor-ligand conformations exhibit dissimilar cofactor binding preferences and that the expression level of PR coactivators and corepressors in target cells influences the pharmacological response to a ligand. We therefore devised an assay using coactivator peptide analogs to distinguish between different receptor-ligand conformations in an effort to identify novel antiprogestins that do not interact with ERß. Such ERß-sparing ligands could be used to distinguish between the antiestrogenic activities of antiprogestins that require PR from those that occur as a consequence of a direct interaction of the ligand with either of the two ER subtypes. We believe that studies of this nature will provide the tools necessary to more carefully dissect the biology of PR and may lead to the development of new clinically useful antiprogestins.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
17ß-estradiol, RU486 (mifepristone), dexamethasone, pregnant mare’s serum gonadotropin (PMSG), human chorionic gonadotropin (hCG) and hyaluronidase were obtained from Sigma (St. Louis, MO). The synthetic androgen R1881 and promegestone (R5020) were purchased from NEN Life Science Products (Boston, MA).

Test ligands
Table 1GoGo depicts the chemical structures of the 11ß-aryl compounds used in this study. The compounds fall into six categories based on their D-ring substitution pattern: 17ß-acetyl, 17ß-nitro, methylthio group at 17ß, spirothiolanes, cyclic ketones, and spironitrones. The prototype of these ligands is RU486, which has a 17ß-hydroxyl-17ß-propynyl D-ring substitution pattern (reviewed in Ref. 23). We reported previously that 17{alpha}-acetoxy combined with 17ß-acetyl substitution of the D-ring led to the development of RTI 3021-012, an antiprogestin with three times greater potency than RU486 (24, 25). Introduction of 16{alpha}-ethyl-17ß-acetyl substitution led to the development of RTI 3021-020, -021, and -022, a set of compounds that exhibited mixed agonist/antagonist activity on PR, with much weaker antiglucocorticoid activity than RU486 (4, 24, 25). These observations underscored the importance of D-ring substitutions in modifying the biological activities of 11ß-aryl steroids and led us to develop the analogs shown in Table 1GoGo. Synthetic procedures are to be published elsewhere (see also Refs. 26, 27, 28, 29, 30).


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  		Table 1. Structures of compounds evaluated

 

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  		Table 1A. Continued.

 
Cell culture
HepG2 cells were cultured in minimum essential medium (Invitrogen Corp., Carlsbad, CA) supplemented with 8% fetal calf serum (Hyclone Laboratories, Inc., Logan, UT), 1 mM sodium pyruvate and 0.1 mM nonessential amino acids (Invitrogen Corp.) and maintained in a humidified 37 C incubator with 5% CO2.

Plasmids
The mammalian expression vectors expressing full-length receptor proteins pBKC-hPR-B, pRST7 ER{alpha} and ERß, and the control vector to normalize for the amount of cytomegalovirus (CMV) promoter used (rev TUP1) have been described previously (31, 32, 33). The mammalian expression vector pRST7-GR was a gift from Dr. Jeff Miner, Ligand Pharmaceuticals, Inc. (San Diego, CA). The luciferase (luc) reporters 2XPRE-tk-luc containing two copies of the progesterone response element (PRE) upstream of a thymidine kinase (tk) promoter and 3XERE-TATA-luc containing three copies of the estrogen response element (ERE) upstream of a minimal TATA promoter, and the normalization vector pCMVß-gal have been reported elsewhere (6, 22, 33). 5XGal4-TATA-luc reporter was a gift from Dr. X. F. Wang, Duke University (Durham, NC). The mammalian two-hybrid plasmid VP16-PR was a gift from Dr. D. X. Wen, Ligand Pharmaceuticals, Inc. VP16-ER{alpha} and VP16-ERß vectors have been described previously (34). The Gal4DBD-peptide fusion constructs F6 and EBIP44 encode the nuclear receptor interaction motif LXXLL fused to the yeast Gal4 DNA binding domain. The isolation and characterization of the peptides F6 and EBIP44 have been reported elsewhere (34, 35). The Gal4DBD-peptide fusion of the {alpha}/ß V peptide was reconstructed into pM vector (CLONTECH Laboratories, Inc., Palo Alto, CA) between EcoRI and XbaI sites in frame with the Gal4DBD using oligonucleotides synthesized (Integrated DNA Technologies, Coralville, IA) based on the published amino acid sequence (36).

Transfections
Transfections were carried out in 100-mm dishes or T75 flasks (as required) using lipofectin reagent (Invitrogen Corp.) for 5 h. For mammalian two-hybrid assays, a total of 33 µg of DNA containing 10 µg each of the 5X-Gal4-TATA-luc reporter, VP16-receptor fusion, and pM-Gal4DBD-peptide fusion, and 3 µg of pCMV-ßgal plasmid was transfected into cells grown in 100-mm dishes for 5 h. The transfection medium was replaced with phenol red-free medium supplemented with 8% charcoal-dextran-stripped fetal calf serum. After 20 h, the cells were trypsinized using 0.25% phenol red-free trypsin and seeded into two 96-well plates. The cells were allowed to attach to the wells for 4 h, and the ligands dissolved in ethanol (or DMSO + ethanol in some cases), were added at the indicated concentrations in quadruplicate wells. Ethanol was used as a vehicle control. Luciferase and ß-gal assays were performed after 20 h, as described previously (37).

For transactivation assays, cells in T75 flasks or 100-mm dishes were transfected with a total of 45 µg or 30 µg, respectively. For T75 flasks, 22.5 µg of 2XPRE-TK-luc reporter (or 3XERE-TATA-luc for estrogen response), 750 ng of pBKC-hPR-B that expresses PRB (or 6 µg RST7ER{alpha} or ERß or RST7GR expression vectors), 1.5 µg of the pCMVßgal normalizing vector, and pBSIIKS vector (Stratagene, La Jolla, CA) to make up for the total amount of DNA, was used for transfection. After transfection, the cells were grown in phenol red-free medium for 20 h, at which point they were trypsinized and seeded into four 96-well plates for the addition of ligands at the indicated concentrations in quadruplicate wells.

In vivo ovulation experiments
C57BL/6 mice (21 d old) were purchased from Charles River Laboratories, Inc. (Raleigh, NC) and housed three per cage. All housing and procedures were approved by the Duke University Animal Care and Use Committee and were performed in accordance with federal, state, and local rules for the humane treatment of laboratory animals. Mice were housed in polystyrene cages with a 14-h light, 10-h dark cycle with food (Purina Mouse Chow 5001) and water ad libitum. Superovulation was induced by ip injection of 5.0 IU PMSG on the morning of postnatal day 25 followed 48 h later by ip injection of 5.0 IU hCG in 0.1 ml of sterile PBS. Control vehicle (corn oil), or various doses of RU486 (mifepristone, Sigma) or RTI 6413-49a dissolved in tocopherol-stripped corn oil (ICN Biomedicals, Inc., Aurora, OH) were administered by sc injection 6 h after hCG injection (d 27) in a volume of 0.1 ml per animal. The time point for administering the antiprogestins was based on a previous study with RU486 (38). The higher doses (40 and 60 mg/kg of body weight) of the compounds were administered as a suspension. On the morning of d 28, mice were euthanized by CO2 asphyxiation. The oviducts were excised and placed in a drop of culture medium (MEM) containing 25 mM HEPES and 0.01% hyaluronidase to digest the cumulus cells surrounding the oocytes. Oocytes were removed when the ampulla was gently punctured with a 28-gauge needle. The oocytes were dissociated from cumulus cells and counted under a dissecting microscope. If ampulla were not present, oviducts were punctured in two to three regions to verify the absence of oocytes. Uteri were also removed and weighed. Mice with a uterine weight (mg)/body weight (g) ratio less than 1.5 were not included in the study, as these mice failed to superovulate. The total number of oocytes per animal was compared among the treatment groups.

Statistics
ANOVA was conducted on data collected from the ovulation experiment using Stat View (SAS Institute, Inc., Cary, NC). Planned comparisons were conducted using Fisher’s planned least significant difference test when the overall ANOVA was statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of novel antiprogestins
The overall objective of these studies was to identify novel pure antiprogestins that do not directly inhibit the activity of either of the two ERs. The realization that nuclear receptor structure is influenced by the nature of the ligand with which it interacts (39), and the observation that cofactor recruitment is influenced by receptor conformation (40, 41) suggests that classical receptor binding studies are not the most efficient way of identifying compounds with the desired characteristics. As an alternative way of identifying novel antiprogestins, we have devised mechanism-based screens that incorporate elements of our current understanding of PR and ER action. The general flow chart for the studies is outlined in Fig. 1Go, A and B.



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  		Figure 1. Screening strategy used in these studies. A, Approach used to identify mechanistically distinct antiprogestins. B, Approach used to identify ER-sparing antiprogestins.

 
As a first step, we evaluated each member of a library of novel PR ligands (Table 1GoGo) for its ability to oppose agonist (R5020)-activated, PR-mediated gene transcription in transfected cells. In this assay, HepG2 cells were transfected with a hPR-B expression vector and a luciferase reporter gene containing a 2X-PRE-thymidine kinase promoter (2XPRE-TK-luc). The antagonist activity of each compound (at 1 µM) was evaluated in the presence of 10 nM R5020. For comparative purposes, we evaluated under the same conditions the antagonist activity of RU486 (a pure antagonist) and RTI3021-021 (a compound that can function as a partial agonist or an antagonist in a tissue/cell type-dependent manner). The results of this study indicated that most of the compounds from the selected series effectively inhibited agonist activation of hPR-B (Fig. 2AGo). The differences in efficacy noted (6413-045ox for example) reflected weaker affinity of the compounds or instability in this cell system as further suppression of PR antagonist activity was observed when these antagonists were tested at 1000-fold excess over R5020 (data not shown). None of these compounds exhibited agonist activity when tested in the agonist mode (Fig. 2BGo). Those compounds that did not effectively inhibit PR transcriptional activity were not brought forward for further analysis.



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  		Figure 2. Evaluation of selected compounds for antiprogestational activity. A, PR antagonist assay. HepG2 cells were transfected for 5 h with PBKC-hPR-B, 2XPRE-TK-luc reporter, pCMVßgal, and PBSIIKS to make up for the total amount of DNA for transfection. After 20 h, the transfected cells were seeded in 96-well plates and treated in quadruplicate with vehicle (NH), R5020 alone (10-8 M), or R5020 (10-8 M) plus each of the test compounds including partial agonist 3021-021 and antagonist RU486 at 10-6 M. Luciferase and ß-gal activities were measured after 20 h. B, PR agonist assay. HepG2 cells transfected as described in A were seeded in 96-well plates and treated in quadruplicate with vehicle (ethanol), R5020 (10-7 M), and all other ligands at 10-6 M final concentration for 20 h. A representative experiment is shown. Each data point represents the average of quadruplicate determinations of normalized luciferase activity with error bars indicating the SD.

 
One of the primary objectives of this project was to identify novel pure antiprogestins. Thus, it was important to eliminate compounds that had the potential to manifest partial agonist activity. The findings of the assays described above indicated that the antiprogestins evaluated do not manifest partial agonist activity in the selected cell and promoter background. It was possible, however, that these compounds could manifest agonist activity in other environments where different receptor associated proteins were present. This is a formidable problem as it has been difficult to develop in vitro assays that predict whether or not a compound will function as a partial agonist when assayed in vivo. The need to develop an assay with the potential to identify latent agonist activity of putative antiprogestins was strengthened by the fact that RTI 3021-021, which functions as an antagonist in standard transfection assays, has been shown to exhibit partial agonist activity in some cells manifesting 20–30% the efficacy of pure agonists (4).

To develop an appropriate assay, we took advantage of the fact that, although PR has two activation domains, activation function (AF)-1 in the amino terminus and AF-2 in the carboxyl terminus of the molecule, its transcriptional activity in most contexts requires an intact AF-2. The AF-2 domain is a complex structure that provides a docking surface for LXXLL motifs contained within the coactivators that endow upon this domain its ability to regulate target gene transcription (42). Therefore, because all the AF-2 interacting proteins contain an LXXLL motif and given the importance of this domain for transcriptional activity, we believed that the potential for agonist activity existed in any compound that facilitated the formation of an LXXLL binding pocket on PR. In a previous study, we identified an LXXLL-containing peptide (F6) that interacted with hPR-B in the presence of agonists (34). We reasoned, therefore, that we could screen for potential agonist activity by evaluating the ability of a compound to facilitate PR-F6 interactions. For this purpose, we developed a two-hybrid assay wherein the 19-amino-acid LXXLL- containing F6 peptide was fused to the heterologous Gal4DBD and the ability of this fusion to recruit a hPR-VP16 chimera to a Gal4-responsive promoter was assayed. The results of this assay are shown in Fig. 3AGo. As expected, R5020 addition facilitates a robust interaction between PR and the F6 peptide. Interestingly, the partial agonist RTI 3021-021 also promotes this association albeit to a lesser degree. This assay scored RTI 3021-021 as a partial agonist, in line with previous observations in PR-expressing T47D cells (4) and not a pure antagonist as indicated by the results of the cotransfection assay (Fig. 2Go). This further confirmed that the latter assay was unable to distinguish between antagonists and partial agonists. When this peptide-binding assay was used to evaluate the antiprogestins under investigation, we determined that, like RU486, none of the compounds tested were able to facilitate a PR-F6 interaction (Fig. 3AGo) and therefore were likely to function in vivo as pure antagonists.



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  		Figure 3. Evaluation of antiprogestin induced conformational changes in PR. A, Mammalian two-hybrid assay to detect coactivator binding pocket. The PR was fused to the VP16 activation domain and the F6 peptide (GHEPLTLLERLLMDDKQAV) was expressed as Gal4DBD-fusion protein. The peptide-receptor interaction was assayed using a luciferase reporter gene regulated by five copies of the Gal4 responsive element. HepG2 cells were transfected with VP16-PRB, Gal4DBD-F6, 5XGal4-TATA-luc, and pCMVßgal normalizing vector. After 20 h, the transfected cells were seeded in 96-well plates and treated in quadruplicate with vehicle (ethanol), R5020 (10-7 M), and all other ligands at 10-6 M for 20 h. Luciferase activities were normalized by ß-gal activities. B, Identification of mechanistically distinct antiprogestins. HepG2 cells were transfected with VP16 PRB, Gal4DBD-{alpha}/ßV, 5XGal4-TATA-luc, and pCMVßgal for 5 h. Amino acid sequence of {alpha}/ßV: SSPGSREWFKDMLSR. The transfected cells were seeded in 96-well plates after 20 h, and ligands were added as described in A. Average + SD of normalized luciferase values of a representative experiment is shown. *, Antiprogestins mechanistically distinct from RU486.

 
The AF-2 pocket is the only protein-protein interaction surface that has been demonstrated to be important for PR-cofactor interactions thus far. However, it is possible that other surfaces could be presented on PR in the presence of synthetic ligands that permit additional, possibly ectopic interactions between the receptor and coactivators. Indeed, the partial agonist activity of tamoxifen on ER{alpha} does not require a functional AF-2 domain but uses a surface for activity that is only presented in the presence of tamoxifen (33, 43). Previously, we have used phage display to identify peptides that had the potential to interact with additional surfaces on PR presented upon binding different ligands. In these studies, we determined that a peptide of the sequence -SSPGSREWFKDMLSR ({alpha}/ßV), originally identified as a peptide that binds tamoxifen-activated ER{alpha}, interacts with RU486-activated PR (36). As yet, we do not know the significance of the surface on PR implicated by this peptide. However, in our quest to obtain novel PR antagonists we reasoned that it would be advantageous to have compounds that did not facilitate the presentation of this potential cofactor interaction surface on PR. Consequently, we used a two-hybrid assay, wherein the {alpha}/ßV peptide was fused to the Gal4 DBD, to score for the presence of this potential protein-protein interaction surface on PR when occupied by the compounds under study. The results of this analysis are shown in Fig. 3BGo. Not surprisingly, R5020-activated PR was unable to bind the {alpha}/ßV peptide, whereas PR activated by the partial agonist RTI 3021-021 and the antagonist RU486 interacted well. Interestingly, this assay, unlike others performed thus far, separated the antiprogestins under study into two groups, those that allowed PR to interact with this peptide and those that did not. Most notable were the compounds RTI 6413-050b, 6413-051b, and 6413-058, which, unlike RU486, did not enable the presentation of an {alpha}/ßV binding surface on PR. Interestingly, the epimers of these compounds, RTI 6413-050a and RTI 6413-051a, respectively, did permit this interaction. Collectively, these assays have led to the identification of two groups of novel antiprogestins that, by the criteria of the assays available to us, are likely to function as pure antagonists in vivo. The next step was to evaluate the ability of these novel antiprogestins to modulate the transcriptional activity of ERß.

Identification of antiprogestins that do not interact directly with the human ERs
The second major objective of this project was to identify compounds that did not interact directly with either of the two ERs. The general approach used for these studies is outlined in Fig. 1BGo. The ability of each antiprogestin to modulate ERß activity was evaluated first. In the first assay, we examined the ability of each antiprogestin to inhibit estrogen-induced ERß-mediated transcriptional activity in HepG2 cells. The results of this assay are shown in Fig. 4AGo. As shown previously, RU486 effectively inhibited the activity of ERß as did some of the antiprogestins evaluated, most notably RTI 6413-001, 013, 016, 028, 031, 043, 043ox, 046a, 046b, 057, and 058. Clearly, there were compounds, RTI 6413-009a, 015, 039, 042, 045, 049a, 049b, 050b, 051a, 051b, 052, 054, and 055 that did not impact ER signaling under the conditions of this assay. Although this assay led to the identification of several ERß-sparing antiprogestins, we were concerned that the result could be influenced by promoter and cell context. In addition, because this is a classical inhibition assay, compounds with weak, though significant, inhibitory activity may be missed. For instance, it is difficult to determine the significance of the weak antagonist activity exhibited by some compounds such as 6413-002, 006, 050a, and 044.



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  		Figure 4. A subset of antiprogestins lack ERß antagonist activity. A, Transactivation assay to detect ERß antagonists. HepG2 cells were transfected for 5 h with pRST7-ERß expression vector, 3XERE-TATA-luc, pCMVßgal reporter constructs and pBSIIKS to make up for the total DNA for transfection. After 20 h, the cells were seeded in 96 well plates and treated in quadruplicate with ethanol vehicle (NH), estradiol alone (10-8 M) or estradiol 10-8 M plus each of the compounds at 10-6 M. Luciferase and ß-gal activities were measured after 20 h. B, Mammalian two-hybrid assay to detect ERß antagonists. HepG2 cells were transfected with VP16ERß, Gal4DBD-EBIP44, 5XGal4-TATA-luc and pCMVßgal reporter constructs for 5 h. After 20 h, the cells were seeded in 96-well plates and treated in quadruplicate with ethanol (NH), estradiol 10-8 M, or each of the compounds at 10-6 M. EBIP44: YGLKMSLLESLLREDISTV. Luciferase and ß-gal activities were measured after 20 h. *, Antiprogestins that lack ERß antagonist activity in transactivation assay (A) and mammalian two-hybrid assay (B).

 
To address this issue, we developed a new assay that performs in a context- independent manner and that may have utility as a screen for ER modulators. Previously, we identified an LXXLL-containing peptide (EBIP44: YGLKMSLLESLLREDISTV) whose ability to bind to the AF-2 pocket of ERß does not require ligand activation (35). It appears as if this peptide is able to lock the receptor into an active conformation and thus bypassing the need for a ligand induced change in receptor conformation (Jansen, M. S., and D. P. McDonnell, unpublished results). When incorporated into a two-hybrid assay, we were able to demonstrate that this LXXLL-peptide was able to tether an ERß-VP16 chimera to DNA in an agonist-independent manner. Importantly, agonist addition has no significant impact on this interaction. Compounds that display antagonist activity on the other hand, like RU486, alter the receptor structure in a manner that makes it incompatible with peptide binding. This assay has two major advantages over classical cotransfection assays. First, because the assay reports on a simple receptor-peptide interaction, it is less likely to be influenced by cell and promoter contexts. Second, this is a more sensitive assay for antagonist activity because an activating agonist is not required. We found that all of the compounds that score as antagonists in the classical transcription assay (Fig. 4AGo) also inhibited peptide binding (Fig. 4BGo). More importantly, however, we also determined that several compounds that had no or weak antagonist activity in the cotransfection assay did have potential ERß-antagonist activity. Most notable in this regard were RTI 6413-002, 003, 006, 018, 042, and 044. Following these experiments, we eliminated all antiprogestins that displayed ERß-antagonist activity and selected the eight promising compounds for further study. A comprehensive evaluation of the ability of different concentrations of the selected antiprogestins to inhibit the transcriptional activity of estradiol-activated ER{alpha} (Fig. 5AGo) or ERß (Fig. 5BGo) confirmed the lack of ER antagonist activity of the selected compounds. Thus, it has been possible to identify antiprogestins that do not directly inhibit ER transcriptional activity.



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  		Figure 5. Antiprogestins lacking ERß antagonist activity do not influence ER{alpha} transcriptional activity. HepG2 cells grown in 100-mm dishes were transfected for 5 h with 3XERE-TATA-luc, pCMVßgal, pBSIIKS to make up for the total amount of DNA and RST7ER{alpha} (A) or RST7ERß (B). After 20 h, the transfected cells were seeded in 96-well plates to treat with ethanol vehicle (NH), estradiol alone (10-9 M) or estradiol 10-9 M with increasing concentrations (10-8–10-6 M) of each of the tested ligands. Luciferase and ß-gal activities were measured after 20 h. Average + SD of quadruplicate determinations of normalized luciferase activity from a representative experiment is shown.

 
Before going into animals to evaluate the biological consequences of separating the antiprogestenic from the antiestrogenic activities of the compounds identified, we wished to select the compounds that would have minimal cross-reactivity with the GR. This was accomplished using an in vitro cotransfection assay where the ability of each of the selected compounds to inhibit GR-mediated transcriptional activity was compared with their antiprogestenic activities under the same conditions. The results of this analysis, shown in Fig. 6Go, A and B, indicate that all of the antiprogestins inhibit GR to some degree. However, from these analyses it appears that RTI 6413-49a or 49b gave the widest separation between PR and GR antagonism. We note, however, that these two compounds effectively antagonize GR at 1 µM. We chose 6413-49a for studies in vivo as this compound also failed to inhibit AR transcriptional activity, a property common to most of the known antiprogestins (data not shown).



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  		Figure 6. Evaluation of the antiprogestin RTI 6413-49a for receptor cross-reactivity. A, Determination of relative potency of antiprogestin activity. HepG2 cells in a 100-mm dish were transfected with pBKC-hPRB expression vector, 2XPRE-TK-luc reporter, pCMVßgal, and pBSIIKS to make up for the total amount of DNA. After 20 h, the cells were seeded in 96-well plates and treated in quadruplicate with ethanol (NH), R5020 alone (10-9 M), or R5020 (10-9 M) with increasing concentration (10-8-10-6 M) of each ligand. Luciferase and ß-gal activities were measured after 20 h. B, Determination of relative potency of antiglucocorticoid activity. HepG2 cells grown in a 100 mm dish were transfected for 5 h with RST7-GR expression plasmid, 2XPRE-TK-luc reporter, pCMVßgal, and pBSIIKS to make up for the total amount of DNA. After 20 h, the cells were seeded in 96-well plates and treated in quadruplicate with ethanol (NH), dexamethasone alone (10-9 M), or dexamethasone (10-9 M) with increasing concentration (10-8-10-6 M) of each ligand. Luciferase and ß- galactosidase activities were measured after 20 h. Average + SD of normalized luciferase activity from a representative experiment is shown.

 
The ER-sparing antiprogestin RTI 6413-49a is biologically active in vivo
Ovulation is a precisely timed multistep process culminating in the release of a mature oocyte from the ovary. Evidence for a direct role of PR in ovarian function has been supported by the demonstration that 1) the antiprogestin RU486 inhibits ovulation (38, 44); 2) LH transiently up- regulates PR mRNA in preovulatory follicles (45); and perhaps most importantly, 3) PRKO mice fail to ovulate even after exposure to superovulatory levels of gonadotropins (1, 46, 47, 48). We used ovulation as a classical endpoint to test the bioavailability and efficacy of the identified ER-sparing antiprogestin. From the studies above, 6413-049a was chosen as a potent ER-sparing antiprogestin. Following superovulation, RU486 or 6413-49a was administered 6 h post hCG injection and the number of oocytes in the oviducts was recorded for each treatment. As shown in Fig. 7Go, compared with the control, animals receiving higher doses of RU486 or 6413-49a (40 mg/kg) showed significant reduction in the number of oocytes. An independent experiment using 60 mg/kg of RU486 or 6413-49a inhibited superovulation in a similar manner (data not shown).



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  		Figure 7. The ER-sparing antiprogestin 6413-49a inhibits superovulation in C57BL/6 mice. Immature female C57BL/6 mice were stimulated with gonadotropins 5 IU PMSG (on d 25) and 5 IU hCG (on d 27). Six hours post hCG stimulation, sc injections of corn oil (control, n = 5), RU486 (8 mg/kg, n = 6; 40 mg/kg, n = 5) or 6413-49a (8 mg/kg, n = 5; 40 mg/kg, n = 5) were performed. The oviducts were collected 20 h post hCG injection, and the total number of oocytes per animal was determined. The average + SEM are shown. P < 0.05 for RU486 (40 mg/kg) and 6413-49a (40 mg/kg).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Until recently, it has been considered that the role of PR agonists was that of a switch, which upon binding to the receptor facilitated its conversion from an inactive state to an active state that was capable of regulating target gene transcription (49). Conversely, it was believed that antagonists functioned by competitively inhibiting agonist binding to the receptor. However, as the mechanisms by which progestins manifest their biological activity in target tissues are unraveled, it has become clear that the terms agonist and antagonist do not adequately describe the complex pharmacology of ligands for this receptor. Indeed, it has been demonstrated that the same PR-ligand complex is not recognized in the same manner in all cells and the same compound can function as an agonist in one environment and an antagonist in another (4, 50).

Although a complete understanding of the molecular basis for the observed cell-selective actions of PR ligands is likely some years off, there are three major factors that have been found to determine how a cell recognizes and distinguishes between different PR-ligand complexes. The first was the observation that cells can contain either or both forms of the PR, hPR-A and hPR-B (2, 6, 47). These receptor isoforms are functionally distinct: hPR-B is primarily involved in mediating the positive transcriptional responses to ligands, whereas hPR-A serves to modulate hPR-B function in most cells. The extent of these different activities has recently been demonstrated in studies using breast cancer cell lines engineered to express either PR-B or PR-A. Specifically, it was shown that around 65 genes are uniquely regulated by PR-B, 25 regulated by both PR-A and PR-B, and only 4 genes that are regulated uniquely by PR-A (51). Adding to this complexity is the finding that hPR-A is a negative regulator of hER transcriptional activity, providing a mechanism to explain how progestins exhibit antiestrogenic activity in vivo (5, 13). The second major observation is that the structure of PR is influenced by the nature of the ligand with which it interacts. Specifically, we have shown previously that agonists, antagonists, and partial agonists each induce distinct conformational changes in receptor structure and more importantly that the biological activity of an uncharacterized compound can be predicted based on the conformation it induces in PR (4). These latter observations suggested that there existed a link between PR structure and its ability to positively or negatively regulate target gene transcription. Third, the discovery of receptor associated proteins (coactivators that enhance and corepressors which repress PR transcriptional activity) (52, 53, 54) and the observation that their ability to interact with PR is regulated by receptor conformation reveals why receptor structure is linked to function. In addition, it suggests that alterations in the relative expression level of coactivators and corepressors may enable cells to distinguish between agonists and antagonists.

In addition to the complexity outlined above is the observation that some PR ligands can interact with other nuclear receptors. RU486 for instance, used clinically as an antiprogestin, is able to interact with and inhibit the transcriptional activity of GR, AR, as well as ERß; and in the presence of PR-A, RU486 inhibits the activity of PR-B, GR, AR, ER, and mineralocorticoid receptor via an indirect PR-A mediated transrepression mechanism (9, 10, 11, 12, 13). The documented promiscuity of the existing antiprogestins has made it difficult to use these agents to assess the involvement of PR in specific biological processes. For these reasons, we feel that the antiprogestins identified in this study may have clinical utility and will be useful for preclinical studies of PR biology.

In this study, we screened for compounds that permit PR to adopt a conformational state that is distinct from that which is observed in the presence of RU486. Previously, we used phage display to identify peptides that interacted with ER{alpha} in the presence of different ligands (36, 39). The peptides identified in these screens were subsequently used to map potential protein-protein interaction surfaces on ER{alpha} and to demonstrate that different ligands induced different conformational changes in the receptor and that the biological activity of a specific ligand could be attributed to the presentation of specific binding surface(s). The importance of this finding was highlighted by the observation that peptides that bound specifically to the tamoxifen-activated ER{alpha} could inhibit the agonist activity manifest by this compound but not estradiol when expressed in target cells. Indeed, we have been able to demonstrate that all of the surfaces on ER identified using this peptide mapping approach are functionally important. This was an important observation as it suggested that even if the identity of the coactivators that bound to these surfaces were not known, it was reasonable to expect that compounds that presented different surfaces on the receptor would not be functionally equivalent. It is not surprising, given the high degree of sequence homology between members of the nuclear receptor superfamily, that some of the peptides that bound to ER{alpha} in the presence of agonists or antagonists were also found to interact with PR. In particular, we observed that a peptide of the sequence SSPGSREWFKDMLSR ({alpha}/ßV) was able to interact with both tamoxifen-activated ER and RU486-activated PR. Because the {alpha}/ßV pocket was required for tamoxifen partial agonist activity, we reasoned that it may enable RU486 to manifest agonist activity under certain circumstances and that it would be useful to screen for antiprogestins that did not permit this surface to be presented. Using peptide binding as a discriminator, we were able to identify three novel antiprogestins 6413-50b, 51b, and 58 that do not present the {alpha}/ßV surface and thus by virtue of their unique actions on PR structure are mechanistically distinct from RU486. As yet, we do not know if these antiprogestins are functionally distinct from RU486, although we are currently addressing this question using cDNA array technologies.

Most studies that have examined the pharmacology of PR have used the antiprogestin RU486 to demonstrate that a particular progestin-induced response was actually occurring through PR. While it is known that this antiprogestin is also capable of inhibiting mineralocorticoid receptor, AR, and GR, it has been possible to eliminate the confounding influence of these cross-reactivities in most experiments. However, it was the recent discovery that RU486 is an efficient antagonist of ERß that has begged a reevaluation of conclusions of studies that used this compound to implicate PR. In HepG2 cells, RU486 exhibited significant antagonist activity on ERß (IC50 ~ 100 nM), albeit at a lower potency than its antiprogestin activity (IC50 ~ 1 nM) (data not shown). Because of the importance of determining whether the observed activities of RU486 are due to inhibition of PR or ERß, we made the identification of ERß sparing antiprogestins a primary goal of this study. This goal was realized with the identification of RTI 6413-49a, an antiprogestin exhibiting similar efficacy and potency as RU486 but lacking ERß antagonist activity under the conditions of our assays. The biological activity and efficacy of RTI6413-049a as an antiprogestin in vivo was demonstrated in the superovulation model.

Some initial structure-activity correlations may be drawn from the data. Clearly, the presence of a 17{alpha}-propynyl group exerts a powerful influence on interaction of these compounds with ERß, as all of the most potent inhibitors of ERß transcription contained this moiety (6413-001, 013, 016, 028, 043 and 043ox in Table 1GoGo and Fig. 4AGo). The presence of a 17{alpha}-propanol or propenol tended to result in less potent inhibition, as detected by the peptide binding assay, but this effect was modified by other substituents on the molecule. The eight ERß sparing compounds (Fig. 5Go, A and B) were all characterized by highly polar substituents (nitro, nitrone, sulfoxide) in the 17ß-position.

The 11ß-aryl substituent plays a significant role in progestin receptor binding. Thus, it is of interest that the three compounds (RTI 6413-050b, 6413-051b, and 6413-058) which, unlike RU486, did not enable the presentation of an {alpha}/ßV binding surface on PR, do not have the p-N,N-dimethylamino moiety of RU486. These compounds have either a p-methylthio or p-acetyl substituent. However, the 11ß- substituent does not act alone in this regard, because the sulfoxide 20-epimers of two of these compounds, RTI 6413-050a and RTI 6413-051a, respectively, did permit the interaction with the {alpha}/ßV peptide. This finding suggests that rather subtle differences in structure can alter the mode of interaction of these types of compounds. A more complete characterization of the structure-activity relationship and stereochemistry of these compounds will be published elsewhere.

In conclusion, we have identified two new classes of antiprogestins. One class competitively inhibits the actions of agonists but differs from RU486 in that they induce a unique conformational change in PR that prevents the presentation of at least two potential coactivator binding surfaces on the receptor. Members of the second class of antiprogestins differ from RU486 in that they do not directly inhibit ERß. The potential clinical utility of these two new classes of antihormones remains to be determined. However, they are likely to be as useful for the study of PR as selective ER modulators have been in the study of ER and thus independent of whether they are clinically useful these new antiprogestins should provide investigators new tools to probe the mechanism of action of PR.


   Acknowledgments
 
We would like to thank Dr X. F. Wang at Duke University (Durham, NC) and Drs. Jeff Miner and D. X. Wen of Ligand Pharmaceuticals, Inc. (San Diego, CA) for gifts of plasmid constructs used in this study. We thank Dr. Patricia Saling at Duke University (Durham, NC) for help with the ovulation studies. We thank J. A. Kepler, J. M. O’Reilly, G. S. Bartley, R. S. Shetty, P. Raje, and D. Y.-W. Lee (all from Research Triangle Institute, Durham, NC) for synthetic work described in Refs. 24 25 26 27 28 . We thank the members of our laboratory for critical reading of the manuscript and Ms. Trena Martelon for editorial assistance.


   Footnotes
 
These studies were supported by NIH Grants DK-50495 (to D.P.M.), CA-92984 (to M.J.), and DK-07012-23 (to S.C.N.) and by Department of Defense Breast Cancer Research Program postdoctoral fellowship DAMD17-01-1-0233 (to G.S.).

Abbreviations: AF, Activation function; AR, androgen receptor; CMV, cytomegalovirus; ER, estrogen receptor; gal, galactosidase; GR, glucocorticoid receptor; h, human; hCG, human chorionic gonadotropin; luc, luciferase; PMSG, pregnant mare’s serum gonadotropin; PR, progesterone receptor; PRE, progesterone response element; TK, thymidine kinase; ZK98,299, onapristone.

Received January 9, 2002.

Accepted for publication April 10, 2002.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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