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A Structural and in Vitro Characterization of Asoprisnil: A Selective Progesterone Receptor Modulator

Departments of Computational, Analytical and Structural Sciences (K.P.M., E.L.S., S.P.W.), and Gene Expression and Protein Biochemistry (S.-J. D., T.B.S., S.A.S.) GlaxoSmithKline Discovery Research, Research Triangle Park, North Carolina 27709; and Departments of Urogenital Biology (E.T.G., A.C.S., C.W., N.J.L., J.D.B.) and Medicinal Chemistry (S.K.T.), GlaxoSmithKline Cardiovascular and Urogenital Center for Excellence in Drug Discovery, King of Prussia, Pennsylvania 19406

Address all correspondence and requests for reprints to: Jeffrey D. Bray, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, King of Prussia, Pennsylvania 19406. E-mail: jeffrey.d.bray{at}gsk.com.

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Selective progesterone receptor modulators (SPRMs) have been suggested as therapeutic agents for treatment of gynecological disorders. One such SPRM, asoprisnil, was recently in clinical trials for treatment of uterine fibroids and endometriosis. We present the crystal structures of progesterone receptor (PR) ligand binding domain complexed with asoprisnil and the corepressors nuclear receptor corepressor (NCoR) and SMRT. This is the first report of steroid nuclear receptor crystal structures with ligand and corepressors. These structures show PR in a different conformation than PR complexed with progesterone (P₄). We profiled asoprisnil in PR-dependent assays to understand further the PR-mediated mechanism of action. We confirmed previous findings that asoprisnil demonstrated antagonism, but not agonism, in a PR-B transfection assay and the T47D breast cancer cell alkaline phosphatase activity assay. Asoprisnil, but not RU486, weakly recruited the coactivators SRC-1 and AIB1. However, asoprisnil strongly recruited the corepressor NCoR in a manner similar to RU486. Unlike RU486, NCoR binding to asoprisnil-bound PR could be displaced with equal affinity by NCoR or TIF2 peptides. We further showed that it weakly activated T47D cell gene expression of Sgk-1 and PPL and antagonized P₄-induced expression of both genes. In rat leiomyoma ELT3 cells, asoprisnil demonstrated partial P₄-like inhibition of cyclooxygenase (COX) enzymatic activity and COX-2 gene expression. In the rat uterotrophic assay, asoprisnil demonstrated no P₄-like ability to oppose estrogen. Our data suggest that asoprisnil differentially recruits coactivators and corepressors compared to RU486 or P₄, and this specific cofactor interaction profile is apparently insufficient to oppose estrogenic activity in rat uterus.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
PROGESTERONE (P₄) IS A steroid hormone essential for coordinating normal mammalian female reproductive physiology (1, 2, 3). The physiological actions of P₄ are mediated by interaction with the progesterone receptor (PR), a member of the nuclear hormone superfamily of ligand-activated transcription factors (4, 5). Ligand-occupied PR binds directly to DNA at P₄ response elements (6) and recruits coregulatory proteins that activate or repress transcription via interactions with the general transcription apparatus (7, 8, 9, 10). PR-dependent transcriptional specificity depends on the availability of PR isoforms and coregulatory factors in a target cell (11, 12, 13, 14). PRs can also interact with other transcription factors or signaling pathways to modulate transcriptional activity, such as AP-1 and the RelA(p65) subunit of nuclear factor-B (15, 16, 17). They can also interact with Src kinase to activate MAPK signaling (18, 19) and compete for binding of general transcriptional machinery components, thus preventing access of other transcriptional activators in a process known as "squelching" (20, 21). Therefore, synthetic PR ligands could differentially affect transcription of PR target genes through selective recruitment of factors resulting in the differential regulation of gene expression in the various P₄ target tissues.

There are two predominant receptor isoforms, designated PR-A and PR-B, transcribed from the same gene by two distinct promoters, with the only difference being that human PR-B is larger by an additional 164 amino acids at the amino terminus (22, 23, 24). As a result, PR-A and PR-B have differing transcriptional activities (11, 12, 25). Both isoforms have two activation function domains (AFs): the constitutive AF-1 proximal to the DNA-binding domain, and the ligand-dependent AF-2 domain in the C terminus (26, 27). There is a unique third AF, designated AF-3, in the amino terminus of PR-B that may contribute to its differential transactivational properties (28). Studies in mice with selective ablation of a PR isoform revealed that PR-A is necessary for ovulation and modulates the antiproliferative effects of P₄ in the uterus, and PR-B is required for normal mammary gland development and function (29, 30). Recent evidence has confirmed the existence of a functional third isoform lacking both AF-2 and AF-3 domains, designated PR-C, that appears to be most critical for the onset of parturition (31). The presence of multiple PR isoforms potentially increases the specificity and versatility of hormone action in any given target tissue.

Synthetic progestins interact with PR to activate or repress gene expression in target cells in a manner similar to P₄. Progestins are used for contraception, hormone therapy, and treatment of some gynecological disorders. The clinical profile of each progestin differs, but all have the ability to repress estrogen- induced endometrial proliferation in vivo (32). The ability of PR to repress estrogen receptor (ER) function has been demonstrated in vitro as well (11, 12, 13, 33). Often the inclusion of a progestin in contraception and hormone therapy is for its ability to repress the endometrial mitogenic effects of estrogens. PR antagonists oppose the action of P₄, and the only clinically relevant therapeutic agent in this class is mifepristone (RU486). RU486 is used primarily for pregnancy termination in combination with the prostaglandin misoprostol, but it has usages for treatment of certain PR-mediated or gynecological conditions (34, 35). A third class of PR ligands now being developed are the selective PR modulators (SPRMs) (36, 37).

Asoprisnil (Fig. 1) is a steroidal SPRM in late-stage clinical development that exhibits partial agonist/antagonist properties and tissue selective effects in animals and humans. The partial agonist effects of asoprisnil, unlike RU486, can be demonstrated by changes in the morphology of the rabbit uterine epithelium (McPhail test) and on the guinea pig uterus and vagina (36). However, the agonist effects of asoprisnil in the rabbit uterus never reached the level of maximum epithelial stimulation of a full agonist. Asoprisnil has at least one metabolite, designated J912 (Fig. 1), that differs by a single hydrogen and also has activity on PR, albeit with reported reduced affinity and efficacy (38). In addition, asoprisnil had a unique endometrial effect based on histological examination of healthy cycling women when taken for 30 d (39). The preclinical and clinical data suggest that asoprisnil is a SPRM.

View larger version (11K): [in this window] [in a new window]   Fig. 1. Chemical Structures of PR Ligands Discussed in this Study The Ki of these ligands used for human PR are (in nanomoles ± SE): P4, 4.3 ± 1.0; RU486, 0.82 ± 0.01; and asoprisnil, 0.85 ± 0.01 (obtained from Ref. 55 ).

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Crystal Structures of PR-LBD Bound to Asoprisnil/SMRT (2.1 Å) and Asoprisnil/Nuclear Receptor Corepressor (NCoR) (2.6 Å) Show the Antagonist Conformation of PR-LBD with Direct Ligand/Corepressor Interaction
The structure of the PR/asoprisnil/corepressor complex demonstrated that the functional changes associated with compound binding are matched by structural changes in the LBD of PR (PR-LBD) compared with the standard agonist position seen in the P₄/PR-LBD crystal structure (pdb code: 1A28) (40). Surprisingly, the PR-F domain (residues 920–933) maintained the same conformation in the agonist (P₄) and antagonist (asoprisnil) complexes (Fig. 2). Together with helix 11, the F domain defined two fixed points that limited AF-2 conformational changes to the flexible linker connecting helix 11 with the AF-2. Constrained by the fixed F domain and helix 11, the PR-LBD AF-2 helix shifted from the agonist conformation to pack antiparallel to helix 11 (Fig. 2). This movement had two consequences: it made room for the longer corepressor helix, and it displaced Glu911, a residue observed to be critical for coactivator binding (41).

View larger version (52K): [in this window] [in a new window]   Fig. 2. Overlay of PR Agonist Conformation with PR Antagonist Conformation A, Overlay of PR agonist conformation with PR antagonist conformation. Cyan is PR from the asoprisnil/SMRT structure, yellow is the corepressor peptide SMRT, and green is PR from the P4 structure. Note that the peptide clashes with the AF-2 of the P4 structure. B, Same overlay as panel A, but the view is rotated 180°.

View larger version (27K): [in this window] [in a new window]   Fig. 3. Overlay of PR/Asoprisnil Complexed with SMRT or NCoR Corepressor Structures A, Asoprisnil/SMRT structure (yellow) and SMRT (purple), overlaid with asoprisnil/NCoR structure (blue) and NCoR (blue). Note the asoprisnil interaction with the leucine in the corepressor peptides. The leucine in the SMRT structure is 3.16 Å from the ligand asoprisnil. B, Asoprisnil/SMRT structure (yellow) and the P4 structure (green) binding pockets overlaid with the ligands in bold. Note that the 11ß extension of asoprisnil collides with Met909 of the P4 binding pocket.

Mammalian Two-Hybrid and Fluorescence Polarization Interaction Assays with NCoR Confirmed that Asoprisnil Can Induce an Antagonist-Like Conformation of the PR-LBD
To confirm the crystallographic observations, we used a mammalian two-hybrid assay to examine the effect of asoprisnil on the PR-LBD. This assay measures the ability of certain PR antagonists to promote an interaction between the PR-LBD and the C-terminal domain of NCoR. The PR antagonist RU486 had an EC₅₀ of 0.04 nM, and asoprisnil similarly induced a concentration-dependent (EC₅₀ = 0.02 nM) interaction with 73% efficacy compared with RU486 (Fig. 4). In addition, fluorescence polarization showed that the relative potency of asoprisnil-induced NCoR peptide recruitment was less than RU486 but more that P₄ (Fig. 5A) and is consistent with the observed interaction of asoprisnil with the corepressor peptides in the solved crystal. The ability of transcription intermediary factor 2 (TIF2) coactivator or NCoR corepressor peptides to compete for the binding of fluorescent NCoR peptide to asoprisnil or RU486 bound to glutathione-S-transferase (GST)-PR was investigated. Unlabeled NCoR and TIF2 peptides successfully competed for binding with RU486 as the ligand; however, NCoR displayed approximately 10-fold more affinity than TIF2 (Fig. 5B). In contrast, both TIF2 and NCoR displayed similar affinities when asoprisnil was the ligand (Fig. 5C). These data confirmed that asoprisnil induced an antagonist-like conformational change to the PR-LBD, consistent with the structural analyses; however, asoprisnil produced a unique coactivator and corepressor peptide interaction profile compared with RU486.

View larger version (9K): [in this window] [in a new window]   Fig. 4. Asoprisnil and RU486 Induced a Functional Interaction between PR-LBD and NCoR C-Terminal Domain in a Mammalian Two-Hybrid Assay Plasmids expressing the chimeric proteins VP16/PR-LBD and GAL4/NCoR, and a GAL4-luciferase reporter plasmid (pGL5-luc) were transiently transfected into COS-7 cells. At 24 h after transfection, cells were treated with increasing concentrations (10–15 to 10–7 M) of P4, asoprisnil, or RU486 for another 24 h, and then luciferase activity was measured as described in Materials and Methods. The data for asoprisnil (73%), RU486 (100%), and P4 (3%) are from four independent experiments and represented as mean relative luciferase units ± SEM normalized to maximal RU486 activity equal to 100%.

View larger version (16K): [in this window] [in a new window]   Fig. 5. Affinities of Coactivator and Corepressor Peptides for GST-PR-LBDs Purified with Ligands A, The apparent affinities of GST-PR-LBDs purified with RU486 (circles), asoprisnil (triangles), or P4 (squares) were determined by fluorescence polarization using a fluorescein-labeled NCoR peptide. Each data point is represented as mean ± SD. Apparent affinities from four independent measurements are 0.75 ± 0.2 µM (RU486), 1.5 ± 0.2 µM (asoprisnil), and 7.0 ± 0.4 µM (P4). B and C, Inhibition of GST-PR-LBD (2 µM) purified with asoprisnil (B) or RU486 (C) binding to 5 nM fluorescein NCoR peptide by dimethylsulfoxide (circles), NCoR (triangles), and TIF2 (squares). Apparent affinities for the NCoR peptide are 2.4 ± 0.3 µM with asoprisnil and 1.5 ± 0.4 µM with RU486. Apparent affinities for the TIF2 peptide are 1.9 ± 0.2 µM with asoprisnil and 14 ± 2 µM with RU486.

View larger version (27K): [in this window] [in a new window]   Fig. 6. Asoprisnil Demonstrated Only Antagonism in Human or Rat PR-B Ligand-Dependent MMTV-Luciferase Activity in CV-1 Cells Plasmids expressing either full-length human or rat PR-B and a MMTV-luciferase reporter plasmid were transiently transfected into CV-1 cells. At 24 h after transfection, cells were treated with increasing concentrations (10–14 to 10–5 M) of P4, asoprisnil, or RU486 for another 24 h, and then luciferase activity was measured as described in Materials and Methods. A, Asoprisnil was unable to stimulate luciferase activity through human or rat PR-B in a ligand-dependent manner. B, Asoprisnil and RU486 demonstrated full antagonism as assayed by the ability to oppose 10 nM P4. The data are from three independent experiments and are represented as mean relative luciferase units ± SEM normalized to maximal P4 activity equal to 100%.

View larger version (22K): [in this window] [in a new window]   Fig. 7. Asoprisnil Demonstrated the Ability to Weakly Recruit Coactivators via the PR-LBD in COS-7 Cells Plasmids expressing the chimeric protein GAL4/PR-LBD and either full-length coactivators and a GAL4-luciferase reporter plasmid (pGL5-luc) were transiently transfected into COS-7 cells. At 24 h after transfection, cells were treated with increasing concentrations (10–15 to 10–5 M) of P4, asoprisnil, or RU486 for another 24 h, and then luciferase activity was measured as described in Materials and Methods. A, Asoprisnil (23%) weakly induced an interaction between GAL4/PR-LBD and SRC-1. B, Asoprisnil (21%) weakly induced an interaction between GAL4/PR-LBD and AIB1. C, Asoprisnil partially antagonized SRC-1 by 49% and AIB1 by 43%, whereas RU486 fully antagonized 10 nM P4-stimulated luciferase activity with either coactivator. The data are from four independent experiments and represented as mean relative luciferase units (RLU) ± SEM normalized to maximal P4 activity equal to 100%.

There is a significant amount of literature describing PR-mediated gene expression in T47D breast cancer cells (14, 47). To investigate the effects of asoprisnil on gene expression, we selected two genes that have been demonstrated to be PR-regulated with large and consistent fold-change expression by agonists: Sgk-1 (48) and PPL (Ref. 49 ; and Grygielko, E. T., and J. D. Bray, unpublished data). Asoprisnil demonstrated weak, but significant agonism on PPL gene expression with a maximal induction of 2.5-fold (15% of P₄), whereas RU486 failed to stimulate PPL expression (Fig. 8A). Asoprisnil significantly antagonized by 80% and RU486 fully antagonized P₄-induced PPL expression (Fig. 8B). Similar results were obtained for P₄-stimulated Sgk-1 expression with a maximal 3.9-fold increase (5% of P₄), with RU486 having no detectable agonism (Fig. 8C). Asoprisnil and RU486 inhibited P₄-stimulated Sgk-1 expression by 95 and 99%, respectively (Fig. 8D). Using sensitive P₄-responsive endpoints, we showed that asoprisnil demonstrated weak partial agonism on selected T47D cell gene expression.

View larger version (22K): [in this window] [in a new window]   Fig. 8. Asoprisnil Demonstrated Weak Partial Agonist Activity on Endogenous PPL and Sgk-1 Gene Expression in T47D Human Breast Cancer Cells After 24 h of serum starvation, T47D cells were treated with increasing concentrations (10–14 to 10–6 M) of P4, asoprisnil, or RU486 for 18 h, then RNA was extracted and subjected to quantitative TaqMan RT-PCR analysis as described in Materials and Methods. A, Concentration response showed that asoprisnil had weak agonism (15%) on PPL gene expression, whereas RU46 was inactive. B, Asoprisnil (100 nM) demonstrated 80% partial antagonism compared with RU486 (100 nM) on 10 nM P4-stimulated PPL gene expression. C, Concentration response showed that asoprisnil had weak agonism (5%) on Sgk-1 gene expression, whereas RU46 was inactive. D, Asoprisnil (100 nM) demonstrated reduced antagonism compared with RU486 (100 nM) on 10 nM P4-stimulated Sgk-1 gene expression (95% vs. 99%, respectively). The data are from three independent experiments and represented as fold induction ± SEM normalized with RPL32 gene expression. *, P < 0.05; **, P < 0.001.

View larger version (22K): [in this window] [in a new window]   Fig. 9. Asoprisnil and RU486 Antagonized P4-Mediated Inhibition of COX Enzymatic Activity in ELT3 Cells After 24 h of serum starvation, ELT3 cells were treated with increasing concentrations of E2, P4, asoprisnil, or RU486, or E2 (10 nM) plus increasing concentrations of P4, asoprisnil, or RU486 for 24 h. A and B, Arachidonic acid (10–5 M) was added, the supernatant was removed, and PGE2 production was measured by EIA as described in Materials and Methods. C–E, RNA was extracted from the cells and subjected to quantitative TaqMan RT-PCR analysis as described in Materials and Methods.

View larger version (14K): [in this window] [in a new window]   Fig. 10. Asoprisnil Did Not Decrease E2-Stimulated Uterine Wet Weight and Gene Expression Activities and Antagonized the Antiestrogenic Activities of P4 on the Rat Uterus Ovariectomized Sprague-Dawley rats were treated daily with vehicle, E2 (0.08 mg/kg), P4 (10 mg/kg), asoprisnil (30 mg/kg), E2 plus P4, E2 plus asoprisnil, or E2 plus P4 plus asoprisnil for 2 d. The animals were killed, and the whole uterus was carefully excised, trimmed, blotted, and weighed. Tissue was snap-frozen in liquid nitrogen for later RNA extraction. RNA was extracted from the tissue and subjected to quantitative TaqMan RT-PCR analysis as described in Materials and Methods. P4 can reverse these endpoints, but asoprisnil does not. Furthermore, asoprisnil blocked the effects of P4 (n = 5 or 6). a, P < 0.05; b, P < 0.01, compared with vehicle; c, P < 0.05; d, P < 0.01 compared with E2 only.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

The PR/asoprisnil complex crystal structure was solved in the presence of peptides from the corepressors SMRT (pdb code: 2OVH) and NCoR (pdb code: 2OVM). These structures revealed how specific interactions with the compound produced a dramatic rearrangement of the AF-2, favoring corepressor binding. A direct contact between the compound and a side chain on the corepressor peptide was observed, which may account for the decreased affinity of corepressor for PR/asoprisnil relative to PR/RU486. This direct contact also suggests the possibility that ligands can be designed to influence specific cofactor binding. The asoprisnil/SMRT structure is overlaid with the PPAR/SMRT structure (pdb code: 1KQQ) in Fig. 11 (54). The ligand in the PPAR structure (GW6471) does not interact with the SMRT peptide, and the AF-2 helix is in a different conformation from the PR/asoprisnil structure. The different AF-2 helix conformations show that for antagonist conformations in NRs, it is more important that the AF-2 moves to disrupt the coactivator binding groove and make room for the corepressor than where the AF-2 is moved.

View larger version (32K): [in this window] [in a new window]   Fig. 11. Overlay of PR/Asoprisnil/SMRT with PPAR/GW4671/SMRT A, The PR protein is in cyan, the PPAR protein is in magenta, the PR/SMRT and asoprisnil is in yellow, and the PPAR/SMRT and ligand is in orange. Note that the corepressors have the same general position and orientation, but that the PPAR/SMRT is closer to the protein. B, The proteins have been dropped, and the ligands and SMRT peptides remain. Note that the PPAR ligand does not interact with the PPAR SMRT.

Interaction of the corepressor NCoR with asoprisnil-bound PR was consistently observed in our studies. NCoR is an essential gene product (57), but the relevance of corepressor interaction with steroid NRs is poorly understood. Although antagonist-bound receptors interact with corepressors (reviewed in Ref. 58), to our knowledge, no endogenous ligand for this class demonstrates physiological activities with corepressors. Because these experiments presented are not exhaustive, it may be hypothesized that the most significant PR agonism demonstrated by asoprisnil will be found in cells, tissues, or species that have an excess of coactivator proteins, a specific coactivator, or a ratio favoring coactivators compared with corepressor proteins. It has been shown that altering the ratios or levels of coactivator proteins results in enhanced agonism for RU486 (59). However, due to the large number of bona fide cofactors already identified, establishing the relevant cofactors for a given ligand will be difficult.

The coactivators SRC-1 and AIB1 are physiologically relevant for PR based on genetic manipulation studies in mice performed in the O’Malley laboratory. Targeted disruption of SRC-1 resulted in a severe reproductive phenotype, especially in the uterus (60), whereas AIB1 knockout animals showed impaired mammary gland development and function (61). Recently, use of a PRAI transgene on the background of mice lacking SRC-1 or AIB1 confirmed the contribution of PR to the phenotypes; suggesting that SRC-1 is the predominant PR-used coactivator in the uterus, and PR predominantly recruits AIB1 in the mammary gland (62, 63). Pharmacological evaluation of SPRMs in these animals may provide a way to improve the therapeutic index by demonstrating tissue-selective activation of the transgene. Again, it is possible that asoprisnil may recruit other coactivators for PR, thus explaining a potential discrepancy among assays and species.

We observed that asoprisnil had assay-dependent effects, appearing as either a pure antagonist or a weak partial agonist. In both CV-1 cells transfected with PR-B and the T47D alkaline phosphatase assay, asoprisnil was a pure antagonist. Asoprisnil, like RU486, exhibited no P₄-like inhibition of COX enzymatic activity in ELT3 cells. But unlike the complete reversal of P₄ suppression of COX activity produced by RU486, asoprisnil could only partially reverse the P₄ suppression. Examining COX-2 gene expression in these PR-responsive cells, asoprisnil demonstrated partial P₄-like agonism inversely correlating to its degree of antagonism. Despite these suggestions of agonist activity, asoprisnil was never observed to have agonism efficacy comparable to P₄ in any assay.

To increase our ability to detect weak agonism, we chose genes with large fold induction after P₄ stimulation in T47D cells. These genes demonstrate no PR isoform selectivity and represent both direct (Sgk-1) and indirect (PPL) regulation (Ref. 14 ; and Bray, J. D., K. B. Horwitz, and C. R. Lyttle, unpublished data). Examination of genes with less robust induction (>10-fold) failed to demonstrate asoprisnil-induced activation (data not shown). In general, the observed gene expression changes were more sensitive for identifying activities than transfection-based or functional assays but, in turn, suffer from the inability to control for certain variables, such as the presence of other NRs and cofactors or the concentrations and ratios of such.

Ultimately, the weak partial agonistic character exhibited by asoprisnil in some cell-based assays did not translate into PR-mediated antiestrogenic uterine activities in the rat. Indeed, asoprisnil was previously reported to be relatively inactive in the rat uterotrophic assay and could induce labor in rodents, consistent with a high degree of PR antagonism (45). However, in the highly sensitive McPhail test of estrogen-primed immature rabbit epithelium, asoprisnil exhibits both significant agonism and antagonism, albeit reduced compared with P₄ and RU486, respectively (36). It may be that because the rabbit has only the PR-B isoform that asoprisnil has increased agonism in that model due to the lack of potential PR-A mediated inhibition of PR-B (64). Additionally, asoprisnil demonstrated PR agonism in guinea pig luteolysis inhibition and lack of labor-inducing activity (36). We also confirmed these findings in guinea pigs (Sulpizio, A. C., unpublished data). Preliminary in vitro experiments with primary guinea and rat endometrial stromal cells do not show any clear asoprisnil-mediated agonistic differences (Grygielko, E. T., and J. D. Bray, unpublished data). These preclinical effects clearly reflect the mixed agonism/antagonism of asoprisnil and may be suggestive of species-selective activity. Asoprisnil is metabolized to a less efficacious SPRM, J912, as evaluated by the rabbit epithelial transformation assay (45), although this appears not to matter in the rabbits because asoprisnil is more efficacious than J912. Because it has been reported that asoprisnil undergoes similar metabolism to J912 in a variety of species including rats, it may explain the lack of detectable efficacy in our uterotrophic model. However, confirmation would require additional pharmacokinetic/pharmacodynamic evaluations comparing both asoprisnil and J912. Because asoprisnil is the most advanced SPRM in terms of clinical development, the predictive value of the animal model is less well understood compared with full PR agonists.

Receptor modulators are distinct from traditional ligands of NRs in that they possess both tissue-dependent antagonist and agonist activity. The concept of NR modulation has been successfully demonstrated for the ER, and the discovery of tamoxifen, the first selective ER modulator (SERM) has provided an effective therapy for estrogen-dependent breast cancer (65). However, tamoxifen demonstrated weak partial agonism on the uterus. Second-generation representatives of these agents such as raloxifene are able to fully antagonize estrogen activity in the uterus while maintaining sufficient agonist activity on bone ERs (66). For PR ligands, SPRMs, as represented by asoprisnil, exhibit partial agonist/antagonist and/or tissue selective effects. Agents of this type may provide improved therapies for the treatment of uterine proliferative diseases such as endometriosis and leiomyoma. Because it is possible that SPRMs are less transcriptionally active than progestins and less transcriptionally repressive than PR antagonists, they may provide the benefits of each class, without the undesired adverse events. For progestins, these include weight gain, breast tenderness, acne, breakthrough bleeding, loss of libido, and mood effects; and for antiprogestins (defined by RU486), these include abortifaciency, endometrial thickening, and potential antagonism of other NRs. At this time, it is not understood where the balance of therapeutic efficacy and reduced adverse effects lies. However, it is likely that the weak agonist/strong antagonist character of asoprisnil will oppose long-term progestogenic actions on the uterus in humans, thus limiting its therapeutic potential to conditions that do not rely on PR agonism for efficacy, or for a reduced duration of treatment.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
PR-LBD (residues 678–933) was expressed and purified as described previously (67) with the following modifications. NCoR or SMRT corepressor peptide (SMRT 2337–2353: TNMGLEAIIRKALMGKY; NCoR 2051–2075: GHSFADPASNLGLEDIIRKALMGSF) was added to GST-PR fusion protein (GST-PR 1:2 peptide).

The protein was concentrated to 4 mg/ml. The PR/NCoR/asoprisnil complex crystallized spontaneously in the fraction collection tube simply requiring the collection of crystals. The collection was accomplished by pipetting the protein solution directly from the fraction collection tube onto a cover slide and harvesting the largest crystals. The crystals were frozen by quick dunk in 25% glycerol. The PR/SMRT/asoprisnil crystals came from 10% PEG 3350, 0.4 M NaCl, 0.1 M Tris (pH 8.0), and were frozen by slow exchange in 25% glycerol. The crystal structures were solved using the molecular replacement program Amore (PR-LBD/P₄ structure as a model pdb code 1A28), and built and refined using Quanta/CNX. The data sets were collected at IMCA-17ID at the Advanced Photon Source.

The fluorescence polarization assay was conducted using varied concentrations of GST-PR-LBDs purified in the presence of ligand and incubated with 10 nM of fluorescein-labeled NCoR peptide [NCoR2 2051–2075 (GHSFADPASNLGLEDIIRKALMGSF)] in 10 mM HEPES (pH 7.4), 0.15 M NaCl, 3 mM EDTA, 0.005% polysorbate-20, and 5 mM dithiothreitol in a final volume of 40 µl in black 384-well plates for 2 h at room temperature. Plates were read using the Fusion FP plate reader using 485 nm excitation and 520 nm emission filters. Binding curves were plotted, and apparent affinities were determined by nonlinear least squares fit of the data to a simple 1:1 interaction model. Competition assays were done by competing the binding of 2 µM GST-PR-LBDs to 5 nM of the fluorescein-labeled peptide in the presence of 20 µM ligand by either the NCoR (GHSFADPASNLGLEDIIRKALMGSF) or TIF2 732 to 756 (QEPVSPKKKENALLRYLLDKDDTKD) peptides.

Chemicals and Reagents
P₄, RU486, and 17ß-estradiol (E₂) were purchased from Sigma Chemical Co. (St. Louis, MO). Asoprisnil was synthesized for GlaxoSmithKline CVU CEDD Medicinal Chemistry (according to the method described in Ref. 68). These compounds were prepared in 100% ethanol for in vitro assays. P₄ and E₂ were diluted into a vehicle of 10% ethanol/90% corn oil, and asoprisnil was prepared in a vehicle consisting of 3% dimethylsulfoxide/6% Cavitron (pH 3.5) before administration to the animals. Tissue culture medium reagents were purchased from Invitrogen (Carlsbad, CA), except for fetal bovine serum (FBS), which was purchased from HyClone (Logan, UT).

Cell Culture
T47D human breast carcinoma cells and ELT-3 rat leiomyoma cells (obtained from Cheryl Walker, University of Texas MD Anderson Cancer Center) were maintained in DMEM/F12 supplemented with 10% FBS, 2 mM glutamine, and penicillin/streptomycin (Invitrogen) at 37 C in a humidified incubator with 5% CO₂. CV-1 African green monkey cells and COS-7 African green monkey cells were maintained in DMEM (high glucose) supplemented with 10% FBS, 2 mM glutamine, and penicillin/streptomycin (Invitrogen) at 37 C in a humidified incubator with 5% CO₂.

Plasmids
The cDNA encoding amino acids 634–933 of human PR were subcloned in-frame into the pM and the pVP16 plasmids (Invitrogen) to generate GAL4 DNA binding domain and VP16 activation domain fusions, respectively. The cDNA encoding amino acids 1944–2453 of human NCoR were subcloned in-frame into pM plasmid to generate a GAL4 DNA binding domain fusion. Plasmid pG5luc is a reporter construct with firefly luciferase under control of the yeast GAL4 promoter (Promega, Madison, WI). Full-length rat PR-B cDNA was subcloned into pcDNA3.1 expression vector (Invitrogen). Full-length human PR-A and PR-B were subcloned into pFastBac1 (Invitrogen) as previously described (69). Plasmid pFBM-MMTV-luc had MMTV promoter from pLM253 inserted into pFastBac1 to replace the CMV promoter to control expression of firefly luciferase as previously described (70). The cDNAs encoding full-length rat PR-B and human SRC-1 and AIB1 were subcloned into pcDNA3.1 expression vector (Stratagene, La Jolla, CA). All plasmids were confirmed by restriction digestion and DNA sequencing, with expression in cultured cells confirmed by Western blot, when applicable.

Transfections
Briefly, 30,000 COS-7 or CV-1 cells per well were plated into 96-well plates in DMEM containing 5% charcoal-stripped FBS overnight. Transfection of cells was performed using Lipofectamine Plus following manufacturer’s specifications (Invitrogen). The medium was removed, and 50 µl of OptiMEM (Invitrogen) was added to each well. The transfection mix (10 µl) was added, containing appropriate plasmid DNAs detailed separately below for each experiment. After 3 h, 40 µl of DMEM containing 5% FBS was added, and cells were permitted to recover overnight. Medium was removed and the cells were treated for 24 h with P₄, asoprisnil, or RU486 alone, or 10 nM P₄ plus increasing concentrations of asoprisnil or RU486. Each plasmid was tested individually and in combination, demonstrating no ligand-independent reporter transactivation, and ligand-dependent signaling required all plasmids. Plasmid pM3VP16 was cotransfected with pG5luc as a positive control for transfection and luciferase quantification. A modified full-length coactivator recruitment assay was performed (71). The amount of plasmid DNA per well was 0.02 µg of GAL4/PR-LBD, 0.04 µg of coactivator, and 0.04 µg of pG5luc in COS-7 cells. The mammalian two-hybrid assay used 0.02 µg of GAL4/NCoR, 0.04 µg of VP16/PR-LBD, and 0.04 µg of pG5luc plasmid DNA per well in COS-7 cells. The PR-MMTV assay used 0.025 µg of pFBM-MMTV-luc and 0.025 µg of either human PR-B or human PR-A, or 0.01 µg of rat PR-B DNAs in CV-1 cells.

Luciferase Assays
At the end of DNA transfection assays, 100 µl of Steady-Glo reagent (Promega) was added to each well. Plates were incubated for 15 min to ensure complete cell lysis and luciferase reaction and read in a Wallac 1420 Victor2 Multilabel (PerkinElmer, Waltham, MA), and the data were analyzed to obtain EC₅₀ and IC₅₀ values using GraphPad Prism 4 (GraphPad Software, San Diego, CA).

Alkaline Phosphatase Assay
A modified alkaline phosphatase assay was performed (72, 73, 74). Briefly, 40,000 T47D cells per well were plated in 96-well plates in DMEM/F12 containing 2% charcoal-stripped FBS overnight. The next day, the cells were treated overnight with P₄, asoprisnil, or RU486 alone, or 10 nM P₄ plus increasing concentrations of asoprisnil or RU486. The medium was removed, cells were washed with PBS, and then 50 µl of 0.1 M Tris-HCl (pH 9.8) containing 0.2% Triton X-100 was added to each well with 15 min of shaking. After this incubation, 150 µl of 0.1 M Tris-HCl (pH 9.8) containing 4 mM p-nitrophenyl phosphate was added. V_max (rate of reaction; the velocity at maximal concentrations of substrate) measurements were taken at 5-min intervals for 30 min at 405 nM using a SpectraMAX PLUS spectrophotometer (Molecular Devices, Sunnyvale, CA), and the data were analyzed to obtain EC₅₀ and IC₅₀ values using GraphPad Prism 4.

T47D and ELT3 Gene Expression
T47D cells were plated as above. Cells were treated for 18 h and then lysed with RLT buffer containing guanidine thiocyanate (QIAGEN, Valencia, CA). Total RNA is isolated from the lysed cells in the Biorobot Universal System using RNeasy technology (QIAGEN). Briefly, the RNA isolation methodology combines the selective binding properties of a silica gel-based membrane with the speed of vacuum technology. A Rnase-free Dnase step was added to the isolation method to remove genomic DNA. Approximately 500 ng of RNA from each sample was used to generate cDNA using Multiscribe enzyme (Applied Biosystems, Foster City, CA). For quantitative PCR (qPCR), 1x Fast Universal PCR mastermix was combined with approximately 50 ng of sample and specific probe and primer sets and processed under accelerated PCR conditions for 40 cycles. The following genes were quantified: human Sgk-1, probe, 6FAM-TCCACCTTCTGTGGCAC-GCCG-TAMRA; forward primer, TGCAAGGAGAACATTGAACACAA; reverse primer-GCACCTCAGGTGCGAGATACT; and human PPL, probe, 6FAM-CAGGCCAAGCATTTT-ATACATACCCTCGCT-TAMRA; forward primer, GACGCAGTGACCTCCTTGGT; and reverse primer, TGTGGGCGGGACTCTGA. A relative quantification methodology was used for fold change analysis using the housekeeper gene human ribosomal protein L32 (RPL32) for normalization: probe, VIC-CGCTCACAATGTTTCCTCCA-TAMRA; forward primer, TGA-CTCTGATGGCCAGTTGG; and reverse primer, CGCAA-AGCCATCGTGGAAAGAGCT.

The ELT3 cells were processed using the same above-mentioned methods used for T47d cell RNA isolation and qPCR. The gene for rat COX-2 was quantified in ELT-3 cells with the following probe and primers: probe, 6FAM-CTCTGCGCTTGCCCTGGCCTC-TAMRA; forward primer, CCACCTCTGCGATGCTCTTC; and reverse primer, CATTCACCACGGTTTTGACATG. A relative quantification methodology was used for fold change analysis using the housekeeper gene rat RPL32 for normalization: probe, VIC-AGGCATCGACAACAGGGTGCGG-TAMRA; forward primer, GAAACTGGCGGAAACCCA; and reverse primer, GGATCTGGCCCTTGAATCTTC.

COX Assay
ELT3 cells were plated at 10,000 cells per well in 96-well plates in DMEM 0.5% FBS the day before the experiment. Next day, the cells were washed two times with PBS, and DMEM phenol red free and 5% charcoal-stripped FBS medium were added. Cells then were treated with different concentrations of estrogen, P₄, RU486, asoprisnil alone, or 1 nM estrogen plus P₄, RU486, or asoprisnil in 200 µl total volume and incubated for 24 h. The following day, arachidonic acid was added to a final concentration of 10 µM and further incubated for 10 min. The reaction was stopped on ice, and 50 µl of supernatant was transferred to the enzyme-linked immunoassay (EIA) plate for prostaglandin E2 (PGE2) measurement (Cayman 514040 PGE2 EIA kit; Cayman Chemical Co., Ann Arbor, MI). Measurements were taken at 405 nm using a SpectraMAX PLUS spectrophotometer (Molecular Devices), and the data were analyzed to obtain EC₅₀ and IC₅₀ values using GraphPad Prism 4.

Ovariectomized Rat Uterotrophic Model
The procedures involving the use of rats in these experiments were reviewed and approved by the Institutional Animal Care and Use Committee in accordance with National Institutes of Health guidelines (NIH Publication No. 85-23). The animals were housed singly with food and water ad libitum on a 12-h light, 12-h dark cycle.

Ovariectomized female, 10-wk-old female Sprague-Dawley rats (Charles River Laboratories, Inc., Wilmington, MA) weighing between 250 and 300 g were used. The ovariectomies were performed by the supplier with a minimum of 10 d before treatment. The animals were randomized into groups of five or six and treated once daily for 3 d, orally by gavage (by mouth) in a volume of 3 ml with either asoprisnil (30 mg/kg) or vehicle, and/or sc injection in the hindquarter with P₄ (10 mg/kg) in a volume of 0.2 ml. On the second and third days of treatment, the animals, except the untreated control and P₄-only groups, were also treated with E₂ (0.08 mg/kg) sc. Approximately 24 h after the final treatment, the uteri were surgically removed, stripped of remaining fat and mesentery, weighed, and snap-frozen on dry ice. The animals were then euthanized by exsanguination.

Rat Uterus Gene Expression
A subsample of frozen rat uterus was homogenized in 750 µl of Qiazol containing phenol using a bead mill (QIAGEN). The homogenized samples were chloroform-extracted, and the resulting aqueous phase was separated for total RNA isolation in the Biorobot Universal System (QIAGEN). The methodology is similar to the aforementioned cell isolation protocol. The isolated total RNA was checked for quality and quantity using spectrophotometric OD in the M2 plate reader (Molecular Devices). Approximately, 1 µg of RNA from each sample was used to generate cDNA using Multiscribe enzyme (Applied Biosystems). For qPCR, 1x Fast Universal PCR mastermix was combined with approximately 50 ng of sample and specific probe and primer sets and processed under accelerated PCR conditions for 40 cycles. The following genes were quantified: rat v-fos FBJ murine osteosarcoma viral oncogene homolog (c-fos), probe, 6FAM-AGC CAA GTG CCG GAA TCG GAG G-TAMRA; forward primer, TCC GAA GGG AAA GGA ATA AGA TG; reverse primer, CGC TTG GAG CGT ATC TGT CA; and complement 3 (C3), probe, 6FAM-AGCATTCCATCGTCCTTCTCCGGATG; forward primer, GGTCGGTCAAGGTCTACTCCTACTA; reverseprimer, CACAGCGGCACATTTCATTG. A relative quantification methodology was used for fold change analysis using the housekeeper gene rat RPL32 for normalization: probe, VIC-AGGCATCGACAACAGGGTGCGG-TAMRA; forward primer, GAAACTGGCGGAAACCCA; and reverse primer, GGATCTGGCCCTTGAATCTTC.

	ACKNOWLEDGMENTS

 
The authors acknowledge Juan-Li Gu and Natalie Burrows for their excellent technical assistance; Roy Katso for providing the full-length PR containing plasmids and pFBM-MMTV-luc; and Bruce Wisely for providing the SRC-1 and AIB1 expression plasmids. Use of the IMCA-CAT beamline 17-ID at the Advanced Photon Source was supported by the companies of the Industrial Macromolecular Crystallography Association through a contract with the Center for Advanced Radiation Sources at the University of Chicago.

	FOOTNOTES

 
Disclosure Statement: The authors have nothing to disclose.

First Published Online March 13, 2007

Abbreviations: AF, Activation function domain; AIB1, amplified in breast cancer 1; COX, cyclooxygenase; E₂, 17ß-estradiol; EIA, enzyme-linked immunoassay; ER, estrogen receptor; FBS, fetal bovine serum; GAL4, galactosidase-4; GST, glutathione-S-transferase; LBD, ligand binding domain; MMTV, mouse mammary tumor virus; NCoR, nuclear receptor corepressor; NR, nuclear receptor; P₄, progesterone; PGE2, prostaglandin E2; PPAR, peroxisome proliferator-activated receptor; PPL, periplakin; PR, progesterone receptor; qPCR, quantitative PCR; RPL32, ribosomal protein L32; Sgk-1, serum glucocorticoid kinase-1; SPRM, selective PR modulator; SRC, steroid receptor coactivator; TIF2, transcription intermediary factor 2.

Received for publication December 6, 2006. Accepted for publication March 5, 2007.

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