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J. Biol. Chem., Vol. 280, Issue 31, 28468-28475, August 5, 2005

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Molecular and Pharmacological Properties of a Potent and Selective Novel Nonsteroidal Progesterone Receptor Agonist Tanaproget^*

Zhiming Zhang, Andrea M. Olland¶||, Yuan Zhu, Jeff Cohen, Tom Berrodin, Susan Chippari, Chandrasekaran Appavu**, Shen Li**, James Wilhem¶||, Raj Chopra¶||, Andrew Fensome¶, Puwen Zhang¶, Jay Wrobel¶, Rayomand J. Unwalla¶, C. Richard Lyttle, and Richard C. Winneker

From the Women's Health Research Institute and the Departments of ^¶Chemical and Screening Sciences and ^**Drug Safety and Metabolism, Wyeth Research, Collegeville, Pennsylvania 19426 and ^||Wyeth Research, Cambridge, Massachusetts 02140

Received for publication, April 15, 2005 , and in revised form, May 27, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Progesterone receptor (PR) agonists have several important applications in women's health, such as in oral contraception and post-menopausal hormone therapy. Currently, all PR agonists used clinically are steroids. Because of their interactions with other steroid receptors, steroid-metabolizing enzymes, or other steroid-signaling pathways, these drugs can pose significant side effects in some women. Efforts to discover novel nonsteroidal PR agonists with improved biological properties led to the discovery of tanaproget (TNPR). TNPR binds to the PR from various species with a higher relative affinity than reference steroidal progestins. In T47D cells, TNPR induces alkaline phosphatase activity with an EC₅₀ value of 0.1 nM, comparable with potent steroidal progestins such as medroxyprogesterone acetate (MPA) and trimegestone (TMG), albeit with a reduced efficacy (60%). In a mammalian two-hybrid assay to measure PR agonist-induced interaction between steroid receptor co-activator-1 and PR, TNPR showed similar potency (EC₅₀ value of 0.02 nM) and efficacy to MPA and TMG. Importantly, in key animal models such as the rat ovulation inhibition assay, TNPR demonstrates full efficacy and an enhanced progestational potency (30-fold) when compared with MPA and TMG. Furthermore, TNPR has relatively weak interactions with other steroid receptors and binding proteins and little effect on cytochrome P450 metabolic pathways. Finally, the three-dimensional crystal structure of the PR ligand binding domain with TNPR has been delineated to demonstrate how this nonsteroidal ligand achieves its high binding affinity. Therefore, TNPR is a structurally novel and very selective PR agonist with an improved preclinical pharmacological profile.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Progesterone (P₄)¹ plays a pivotal role in female reproduction. It is involved in the regulation of uterine development and differentiation, implantation, ovulation, and mammary gland development (1–3). Progesterone exerts its physiological roles primarily through the progesterone receptor (PR), a member of the nuclear receptor superfamily of transcription factors (4, 5). The PR consists of two isoforms, PR-A and PR-B, which are derived from a single gene through alternative usage of promoters and translation initiation sites (6, 7). Divergent as well as overlapping functions of the two PR isoforms have been identified both in vitro and in vivo (2, 8–14).

The molecular mechanism of PR actions has been extensively studied in the last decade. It is now generally accepted that binding of P₄ causes the PR to undergo conformational changes, phosphorylation, dimerization, and interaction with its target genes (15–19). The conformational change induced by P₄ binding also facilitates the interactions between the PR, coregulators, and basal transcriptional factors, eventually leading to altered gene expression (19, 20). It is hypothesized that PR conformational changes conferred by different PR ligands will lead to various biological responses. In addition to its genomic action, PR has been shown to elicit biological effects through other signaling pathways. It has been demonstrated that PR can interact with the Src/Ras mitogen-activated protein kinase pathway to modulate cellular activities in a ligand-dependent manner (21–23). Furthermore, ligand-independent PR actions involving other signaling cascades have been shown in vitro and in vivo (24–28). Recently, G protein-coupled membrane progesterone receptors have been identified (29, 30). Furthermore, P₄ and/or steroidal progestin metabolites have been shown to interact with a variety of non-PR pathways such as the -aminobutyric acid type A receptor (31–33). Therefore, PR ligands have the potential to elicit a wide variety of biological and pharmacological responses.

PR agonists, i.e. progestins, have many therapeutic uses in women's health, such as in oral contraception and hormone therapies and for the treatment of certain reproductive disorders. In the past few decades many progestins have been developed. Most of them and all of those in clinical usage are steroidal compounds. Even though these compounds are effective in achieving their intended efficacy endpoints, such as inhibition of ovulation in contraception and suppression of estrogen-induced uterine epithelial proliferation in hormone therapy, they also carry unwanted side effects due to interactions with other closely related steroid receptors, steroid-metabolizing enzymes, or other signaling pathways. As a first step to circumvent some of these drawbacks, we set out to identify novel nonsteroidal structures of PR ligands that would maintain the required biological activity while reducing the side effects often associated with steroidal progestins. In this report we describe the biological and molecular characterization of a novel PR agonist, tanaproget (TNPR; Fig. 1), which demonstrates improved pharmacological properties as compared with those of traditional steroidal progestins. We also have delineated the first crystallographic structure of a PR ligand binding domain (PR-LBD) with a nonsteroidal ligand.

View larger version (14K): [in this window] [in a new window]   FIG. 1. Chemical structure of tanaproget.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Cell Culture and in Vitro Assays—The human breast carcinoma cell lines T47D and MCF-7, the human lung carcinoma cell line A549, the mouse skin fibroblast cell line L929, and the monkey kidney cell line COS7 were obtained from American Type Culture Collection (Manassas, VA). T47D and MCF-7 cells were maintained in DMEM/F12, A549 cells in DEF/NPM, L929, and COS7 cells in DMEM, all with 10% FBS. Cells were passed 2–3 times every week.

T47D Alkaline Phosphatase Assay—The effect of TNPR and reference steroids on alkaline phosphatase activity in T47D cells was determined as described (34, 35). Briefly, cells were plated in 96-well plates at 50,000 cells/well in DMEM/F12 with 10% FBS. After overnight culture, the medium was changed to phenol red-free DMEM/F12 containing 2% charcoal-stripped FBS (experimental medium). The next day, cells were treated with a test compound in the absence (agonist mode) or presence (antagonist mode) of 1 nM progesterone in the experimental medium. Twenty-four hours after treatment, cellular alkaline phosphatase activity was measured using p-nitrophenyl phosphate as a substrate. Optical density measurements were taken at 5-min intervals for 30 min at a test wavelength of 405 nm.

PR Binding Assay—Uterine tissues from the rat, monkey, and rabbit were homogenized in 10 mM Tris buffer (1.5 nM EDTA, pH 7.4) with 20 mM sodium molybdate and 1 mM dithiothreitol, whereas T47D cells were homogenized in 20 mM HEPES buffer (1 mM EDTA, pH 7.6) with protease inhibitors (0.5 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.7 µg/ml pepstatin A, and 0.5 mM phenylmethylsulfonyl fluoride). After centrifugation at 100,000 x g for 1 h (4 °C), the supernatant containing PR was collected and measured for protein concentration. The PR competition binding assay was done with 100 µg of cytosolic protein, 3 nM [³H]R5020, and increasing concentrations of test compounds. Following overnight incubation at 4 °C, free [³H]R5020 and bound [³H]R5020 were separated by using 0.5% charcoal and 0.05% dextran (molecular weight 69,000) in Tris-EDTA buffer (pH 7.4). Total [³H]R5020 and bound [³H]R5020 were counted in a Beckman LS6500 scintillation counter (Beckman Instruments, Inc.).

Protease Digestion Assay—The protease digestion analysis was performed essentially as described (15, 37) with minor modifications. The plasmid pT7BPRB, kindly provided by Dr. B. W. O'Malley (Baylor College of Medicine, Houston, TX), was used to generate ³⁵S-radiolabeled PR-B using a TNT T7 quick coupled transcription/translation system according to the manufacturer's protocol (Promega, Madison, WI). After the translation reaction, an aliquot (30 µl) of the lysate was incubated for 10 min in the absence or presence of ligands at a final concentration of 100 nM. Aliquots (5 µl) of the ligand-treated receptor mixture were then incubated with a trypsin solution (Worthington Biochemicals) giving various final concentrations of the enzyme (0, 25, 50, and 75 µg/ml). After incubation at room temperature for 10 min, the digestion reaction was terminated with the addition of 20 µl of gel-denaturing buffer and boiling for 5 min. The digestion products were separated on a 4–12% Bis-Tris NuPAGE gel (Bio-Rad). After electrophoresis, the gel was treated with a 50% (v/v) methanol and 10% acetic acid (v/v) solution for 30 min and immersed in Amplify (Amersham Biosciences) for 30 min. The gel was then dried under vacuum, and the radiolabeled products were visualized by autoradiography.

PR/SRC-1 Mammalian Two-hybrid Assay—COS7 cells were transfected with PR-LBD in the GAL4 DNA binding domain plasmid pM (Clontech), full-length SRC-1 or SRC-3 in the VP16 activation domain plasmid pVP16 (Clontech), and a GAL4-responsive luciferase reporter (5x GALuas) using Lipofectamine 2,000 (Invitrogen) according to the manufacturer's instructions. Cells were treated with test compounds for 24 h, and luciferase activity was measured on a Victor2 luminometer (PerkinElmer Life Sciences) using a luciferase reporter assay kit (Promega).

Adenovirous Hormone Response Element-Luciferase Reporter Assays—Luciferase reporter assays using cell lines expressing endogenous steroid receptors (MCF-7 cells for the estrogen receptor (ER), A549 cells for the glucocorticoid receptor (GR), and L929 cells for the androgen receptor (AR)) were used as described previously (34) to examine potential cross-interactions between TNPR and these steroid receptors.

SHBG Competition Binding Assay—An SHBG competition binding assay was run based upon published methods (38, 39) with modifications. Briefly, human blood was collected in Vacutainer SST tubes (BD Biosciences) and kept at room temperature for 30 min before centrifugation at 1,000 x g for 10 min (4 °C). The serum was aliquoted and stored at -76 °C. Human serum was diluted 20–60 times with TG buffer (10 mM Tris and 10% glycerol, pH 7.4, at room temperature), depending on the source of the serum, for a total [³H]dihydrotestosterone ([³H]DHT) binding of 3,000 cpm. Seven hundred µl of dextran-coated charcoal (0.5% charcoal and 0.05% dextran (molecular weight 69,000) in Tris-EDTA buffer, pH 7.4) was added to every 1,000 µl of diluted serum and incubated on ice for 1 h, followed by centrifugation at 2,000 x g for 20 min. The SHBG competition binding assay was performed with 100 µl of the supernatant, 8 nM [³H]DHT, and increasing concentrations of test compounds. Following overnight incubation at 4 °C, free [³H]DHT and bound [³H]DHT were separated by using 0.5% dextran-coated charcoal. Bound [³H]DHT was counted in a Beckman LS6500 scintillation counter (Beckman Instruments, Inc.).

Cytochrome P450 3A4 Induction Assay in DPX2 Cells—The induction potential of the CYP3A4 enzyme by TNPR was evaluated in a DPX2 cell line by Puracyp, Inc. (Carlsbad, CA). The DPX2 cell line is a stably transformed tumor cell line. The enhancer of CYP3A4 (PXRE) and human PXR are stably integrated into the tumor cells. The incubations were performed in 96-well microtiter plates in a high throughput manner. TNPR and 3-KDG were incubated at 1, 5, 10, and 25 µM concentrations in DPX2 cell line. Rifampicin, a known inducer of CYP3A4, was used as positive control. Induction of CYP3A4 was assessed by monitoring reporter gene activity and comparing the results to analogous cells treated with solvent.

Rat Ovulation Inhibition Model—All animal studies were conducted under approved Wyeth Research Animal Care and Use Committee protocols. Animals were housed under a 12-hour light/dark cycle and fed casein-based Purina laboratory rodent diet 5K96 and water ad libitum unless otherwise indicated.

Ovulation inhibition experiments were run as described (40). Briefly, random cycling mature female Sprague-Dawley rats (200 g) were obtained from Charles River Laboratory (Boston, MA). Rats were synchronized for estrus with 2 µg of luteinizing hormone-releasing hormone (in phosphate-buffered saline containing 0.1% bovine serum albumin) administered subcutaneously per rat at 0900 h and again at 1600 h. Animals were allowed to rest for 8 days before the administration of test compounds. Animals were then grouped, with 8–9 rats per treatment group. The morning of the 9th day following luteinizing hormone-releasing hormone treatment the rats were treated with test compounds once daily, by gavage, for 4 consecutive days. The animals were euthanized the morning following the last treatment. Oviducts were removed, placed between two glass slides, and viewed through a dissecting microscope to count ova. The number of animals presenting ova in the oviduct from each treatment group and the number of ova in the oviduct of each animal were recorded.

TNPR·PR-LBD Crystal Structure Analysis
Gene Construct for Expression of the PR-LBD—PR-LBD was expressed in E. coli from a plasmid carrying the LBD coding sequence (residues 675–933, GS... KK) as a fusion protein with glutathione S-transferase (GST). A thrombin cleavage site separates the GST and LBD regions. The construct is essentially the same as that described by Williams and Sigler (41).

Cell Growth and Induction of Expression for PR-LBD—Growth and expression were performed in a B. Braun Biotech Biostat C 10-liter fermenter. A 100-ml preculture was used to inoculate 10 liters of fermenter salts, glucose, ampicillin, trace metals, and yeast extract media. Cells were grown overnight at 25 °C. Fifteen minutes prior to induction, the vessel temperature was dropped to 15 °C, and 5 ml of 66 mM P₄ was added. Cells were induced with 2.4 g of isopropyl 1-thio--D-galactopyranoside (1.0 mM final concentration) at an A₆₀₀ value of 5.4. Five ml of 66 mM P₄ was added at induction and every 15 min after induction throughout the entire expression (4 h total; 660 µM final concentration of P₄). After 4 h of induction, a final A₆₀₀ value of 8 had been obtained. The expression yielded 160 g of wet cell weight, and the protein of interest was approximated at 5–7% of total cell protein by SDS-PAGE.

Purification of PR-LBD (Complexed with TNPR) Cell Lysis and Isolation of Soluble Protein—Twenty grams of frozen cells were suspended in 300 ml of 50 mM HEPES (pH 7.3), 150 mM NaCl, 5 mM EDTA, 10% glycerol, 5 mM dithiothreitol with 0.33 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride (a protease inhibitor), 0.3 ml of protease inhibitor mixture (Sigma catalog number 8849), and 5 µM P₄. P₄ or TNPR was added to solutions from stocks of 50 mM dimethyl sulfoxide. Cells were broken by passage through a Microfluidizer^TM from Microfluidics (Newton, MA). Cell debris and aggregated GST·PR-LBD were removed by centrifugation (Sorvall RC5B centrifuge, SS34 rotor) at 5 °C for 2.5 h at 18,500 rpm (40,000 relative centrifugal force), CHAPS was added to 1.5%, and the solution was filtered (0.45-µm cellulose nitrate filter) and stored overnight.

Isolation of GST·PR-LBD—The solution was passed over two 5-ml columns (in tandem) of GSTrap FF at 1 ml/min. Resin was washed with 50 ml of 50 mM HEPES (pH 7.3), 150 mM NaCl, 5 mM EDTA, and 10% glycerol and then with 50 ml of the same solution containing 50 µM TNPR. GST·PR-LBD was eluted with 12 mM reduced glutathione in 50 mM HEPES (pH 7.3), 100 mM NaCl, 10% glycerol, 0.1% octyl--glucoside, and 50 µM TNPR. Fractions of 5 ml were collected and pooled after location of the GST·PR-LBD by SDS-PAGE (generally a pool of 30–40 ml). Thrombin was added to 25,000 NIH units/ml, and the solution was stored overnight.

Isolation of PR-LBD—The solution was diluted with 4 volumes of 10 mM HEPES (pH 7.3), 10% glycerol, 5 mM dithiothreitol, 0.1% octyl--glucoside, and 50 µM TNPR. The solution was passed over 1-ml column of SP FF at 1 ml/min. The column was washed with 5 ml of 10 mM HEPES (pH 7.3), 20 mM NaCl, 10% glycerol, 0.1% octyl--glucoside, and 1 µM TNPR. PR-LBD was eluted from the column with a 15-ml gradient of sodium chloride running from 20 to 220 mM (other components are described directly above). Fractions of 1 ml were collected and PR-LBD was located by SDS-PAGE, and those fractions containing PR-LBD at 1–2 mg/ml were used directly for crystallization.

Crystallization—Crystals were grown by hanging drop vapor diffusion at 18 °C in drops containing 2.0 µl of protein stock solution (5 mg/ml protein, 10 mM HEPES, pH 7.3, 10% glycerol, 5 mM dithiothreitol, 100 mM NaCl, 0.1% octyl--glucoside, and 1 µM TNPR) mixed with 1 µl of well solution (8% polyethylene glycol 3350, 300 mM MgSO₄, 50 mM PIPES, pH 6.5, and 10% glycerol) and 0.5 µl of 40% 1,3-propanediol (v/v) and equilibrated against 1 ml of well solution. Diamond-shaped crystals grew in 2–6 weeks, measuring 50 µm across.

Data Collection and Processing—Crystals belong to the space group P2₁ (unit cell parameters: a = 57.52 Å; b = 64.50 Å; c = 70.41 Å; and = 95.76°) and contain two molecules of PR-LBD in the asymmetric unit, resulting in a solvent content of 44%. Crystals were drawn through a solution of 20% ethylene glycol and 80% well solution and cooled rapidly in liquid nitrogen. Diffraction data were collected using a Rigaku rotating anode x-ray generator and recorded on a Raxis4 detector. Intensities were integrated and scaled using the programs Denzo and Scalepack (42).

Phasing, Model Building, and Refinement—The structure was determined by molecular replacement using the PR-LBD/P structure (41) as the search model. After several iterative cycles of refinement using CNX (43) and Refmac5 (44) and model improvement building using QUANTA (Molecular Simulations, Inc.) and Coot (45), TNPR was placed and refined. Final R_work and R_free values of 17.88 and 22.81% were obtained (Table I). Figs. 8 and 9 were prepared using PyMol, and Fig. 10 was prepared using Coot (44, 45).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
PR Activity of TNPR in T47D Cells—The in vitro PR activity of TNPR was evaluated in the T47D alkaline phosphatase assay. The compound was tested along with reference steroidal progestins MPA and TMG. As shown in Fig. 2, TNPR showed potent PR agonist activity with an EC₅₀ value of 0.15 ± 0.01 nM (n = 3), similar to MPA (EC₅₀ = 0.12 ± 0.01 nM, n = 3) and TMG (0.09 ± 0.02 nM, n = 3). TNPR had a reduced efficacy (60%) as compared with the steroid compounds in this assay. However, as shown later, TNPR is fully efficacious in the rat ovulation inhibition model.

TNPR Promotes PR and SRC-1 Interaction in Mammalian Cells—In a mammalian two-hybrid assay to determine the interaction between SRC-1 and PR induced by PR agonists, TNPR showed similar potency and efficacy to MPA, TMG, and LNG and was 50-fold more potent than P₄ (Fig. 3). Similar results were obtained when SRC-3 was used in the two-hybrid assay (data not shown).

TNPR Binds to PR with High Affinity—In the competition binding assay, TNPR displaced R5020 (a synthetic steroidal progestin) binding to the human PR with high relative affinity. The IC₅₀ values (50% inhibition of 3 nM [³H]R5020 binding to the human PR) for TNPR were 1.7 compared with 11.2 and 7.8 nM for MPA and TMG, respectively (Fig. 4). TNPR also showed a 6–30-fold higher affinity to the monkey, rat, and rabbit PR than MPA and TMG (Table II).

View larger version (18K): [in this window] [in a new window]   FIG. 2. Representative dose-response curves in a T47D alkaline phosphatase assay. T47 cells were plated at 50,000 cells/well in 96-well plates in DMEM/F12 with 10% FBS. The medium was changed to DMEM/F12 (phenol red-free) with 2% charcoal-stripped FBS the next day, and cells were treated with compounds overnight. Cellular alkaline phosphatase activity was measured as described under "Materials and Methods."

TNPR Does Not Induce Cytochrome P450 3A4 in DPX2 Cells—In the DPX2 cells TNPR did not increase luciferase activity at concentrations up to 25 µM, whereas the synthetic progestin 3-KDG showed a dose-response induction on CYP3A4 (Fig. 6). The fold induction over the control by 3-KDG was 1.7, 3.7, 8.3, and 21.3 at 1, 5, 10, and 25 µM, respectively. Under the same conditions, the positive control rifampicin showed 10, 21.6, 29.5, and 25-fold CYP3A4 induction at 1, 5, 10, and 25 µM, respectively. These data indicate that 3-KDG induces CYP3A4, whereas TNPR does not. Therefore, it is less likely that TNPR will interact with drugs that are metabolized by CYP3A4.

Partial Proteolytic Analysis of TNPR-bound PR—Partial protease digestion as described by Allan et al. (15) was carried out to determine the gross conformational changes of PR upon TNPR binding. As shown in Fig. 7, a TNPR-bound PR provided a trypsin digestion pattern that was similar to a PR bound with P₄ or TMG but distinct from that of a RU-486-bound PR. Partial digestion with chymotrypsin also demonstrated similar peptide patterns among TNPR-, P₄-, and TMG-bound PRs that were different from that of RU-486-bound PR (data not shown).

View larger version (18K): [in this window] [in a new window]   FIG. 3. Effects of TNPR, MPA, TMG, and P4 on PR-LBD/SRC-1 interaction in a mammalian two-hybrid assay. COS7 cells were transfected with PR-LBD in the GAL4 DNA binding domain plasmid pM, full-length SRC-1 in the VP16 activation domain plasmid pVP16, and a GAL4-responsive luciferase reporter (5x GALuas) using Lipofectamine 2000. Cells were treated with test compounds for 24 h, and luciferase activity was measured as described under "Materials and Methods." RLU, relative light units.

View larger version (18K): [in this window] [in a new window]   FIG. 4. Representative PR competition binding curves for TNPR, MPA, and TMG using PR isolated from human T47D cells. The PR competition binding assay was done with 100 µg of T47D cell cytosolic protein, 3 nM [3H]R5020, and increasing concentrations of test compounds. Following overnight incubation at 4 °C, free [3H]R5020 and bound [3H]R5020 were separated and measured as described under "Materials and Methods."

View larger version (18K): [in this window] [in a new window]   FIG. 5. Representative SHBG competition binding curves for TNPR, LNG, and norethindrone. The SHBG competition binding assay was performed with 100 µl of diluted human serum, 8 nM [3H]DHT, and increasing concentrations of test compounds. Following overnight incubation at 4 °C, free [3H]DHT and bound [3H]DHT were separated by using 0.5% dextran-coated charcoal. Bound [3H]DHT was counted as described under "Materials and Methods."

View larger version (25K): [in this window] [in a new window]   FIG. 6. TNPR does not induce cytochrome P450 3A4 in DPX2 cells. DPX2 cells stably transformed with the enhancer of CYP3A4 (PXRE) and human PXR were treated with TNPR and 3-KDG at 1, 5, 10, and 25 µM. Rifampicin, a known inducer of CYP3A4, was used as positive control. Induction of CYP3A4 was assessed by monitoring reporter gene activity and comparing results to analogous cells treated with vehicle control. DMSO, Me2SO.

View larger version (70K): [in this window] [in a new window]   FIG. 7. Protease digestion patterns of TNPR, P4, TMG, and RU486-bound PR. In vitro translated and 35S-radiolabeled PR-B was incubated for 10 min in the absence or presence of ligands at a final concentration of 100 nM. Aliquots of the ligand-treated receptor mixture were then incubated with a trypsin solution (0, 25, 50, and 75 µg/ml). After incubation at room temperature for 10 min, the digestion products were separated on a 4–12% Bis-Tris NuPAGE gel. The gel was then tried under vacuum, and the radiolabeled products were visualized by autoradiography. Veh, vehicle.

View larger version (45K): [in this window] [in a new window]   FIG. 8. The dimer of the PR-LBD/TNPR that is present in the crystallographic asymmetric unit. Single molecules of the PR-LBD are shown in spectral colors, with the N terminus in blue and the C terminus in red.

View larger version (58K): [in this window] [in a new window]   FIG. 9. Superposition of TNPR and progesterone-bound PR-LBD structures based on all C backbone positions. The PR-LBD/TNPR structure is shown in gold, and the PR-LBD/P structure is shown in blue. Side chains of residues participating in hydrogen-bonding with TNPR are shown.

TNPR is accommodated by the PR ligand binding pocket better than the ligand binding pockets of the other closely related receptors such as GR and AR (48–50). In the PR, residue Phe⁷⁹⁴ (helix 7) is substituted by methionine in the GR and AR, whereas Leu⁷⁹⁷ (helix 7) takes the place of the polar Gln of the GR and AR. Together, these two residues contribute to the creation of a hydrophobic environment that can better accommodate the two methyl substituents of the benzoxazine ring. Both the GR and AR have several other conservative differences with respect to the PR among the hydrophobic residues that line the ligand binding pocket, possibly contributing to the selectivity of TNPR for PR.

View larger version (116K): [in this window] [in a new window]   FIG. 10. Electron density for TNPR and the surrounding residues in a 2Fo - 1Fc map contoured at 1.3 .

View larger version (16K): [in this window] [in a new window]   FIG. 11. Effects of TNPR, MPA, and TMG on ovulation in the rat. Estrus-synchronized rats (n = 8 or 9 animals/group) were treated with test compounds for 4 days. Ova in the oviduct were counted the morning after the last treatment. Data were presented as the percentage of animals that ovulated in each dosage group. For each compound, experiments were repeated at least in two separate occasions with the same ED100 values.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Although several nonsteroidal tissue-selective ER modulators with an improved pharmacological profile and enhanced clinical utility have been developed over the past few decades, to our knowledge no nonsteroidal PR ligands have been in clinical development to date. In this report, we describe the identification and molecular and pharmacological characterization of a novel nonsteroidal PR agonist, TNPR. TNPR was initially identified as a PR agonist through a medicinal chemistry effort based upon its in vitro activity in T47D cells. It showed similar potency to potent steroidal compounds such as TMG and MPA in the alkaline phosphatase assay in T47D cells. However, TNPR had reduced efficacy in this in vitro model (Fig. 2) (34). The biological relevance of this reduced efficacy in the breast cancer line is unclear and is currently under investigation using in vivo models of mammary gland development. In an artificial system using a mammalian two-hybrid system to evaluate the interaction between PR and coactivators such as SRC-1 and SRC-3, TNPR showed similar potency and efficacy as the two steroidal progestins but was much more potent than P₄ or norethindrone (Fig. 3 and data not shown).

TNPR showed higher relative binding affinity as compared with the tested reference steroid progestins in various species. It was 4–6-fold more potent than TMG and MPA on human PR and up to 100-fold more potent than MPA on rat PR in the competition binding assays. The increased binding affinity may be explained by the additional hydrogen bond between TNPR and PR as observed in the crystal structure discussed below. Importantly, TNPR demonstrated potent in vivo progestational activity. In the ovulation inhibition model in the rat it was fully efficacious at 0.03 mg/kg orally, 30-fold more potent that MPA and TMG (40). This enhanced progestational activity has been shown in other animal models.²

TNPR demonstrated >250-fold selectivity for the PR versus other closely related steroid receptors. It interacted only weakly with GR in vitro, and this activity was not detected in vivo at 10 mg/kg, a dose that is 300-fold higher than the efficacious dose (0.03 mg/kg in the rat ovulation inhibition model; Fig. 11 and data not shown). Some of the side effects (e.g. metabolic changes) of the clinically used steroid progestins may be attributed to their interactions with other steroid receptors. For example, progestins with androgenic activity, such as LNG and MPA, have been shown to alter the plasma lipid profile (51, 52). The improved selectivity profile of TNPR will likely reduce these side effects.

SHBG is a serum sex steroid binding protein produced by the liver that regulates the availability of androgens and estrogens to target tissues. It is well known that some synthetic steroids bind to and/or regulate the production of SHBG, thus affecting endogenous sex steroid bioavailability (53–55). For example, LNG competes for SHBG binding with an IC₅₀ value of 16 nM (Fig. 5). It also dramatically reduces serum SHBG levels when given to primates (data not shown). Unlike some steroidal progestins, TNPR did not compete for SHBG binding at concentrations up to 10,000 nM, and a dose that was 100-fold its efficacious dose did not change serum SHBG levels in primates.² Therefore, TNPR offers advantages over steroidal progestins in that it will not affect blood SHBG levels and sex hormone availability to the target tissues.

Failures of contraceptives containing some steroidal progestins, although rare, are known to result from interactions with other drugs. Likewise, some oral contraceptives/progestins are also known to affect the efficacy of other drugs (56–60). These drug-drug interactions are caused by modulation of drug-metabolizing enzymes (61, 62). Steroidal progestins such are desogestrel are metabolized primarily by CYP3A4. CYP3A4 is known to be induced by several drugs that are sometimes co-administered with oral contraceptives. Elevated CYP3A4 concentrations can lead to lower efficacy of contraceptives. Results from recent metabolism studies clearly demonstrate that TNPR is superior to currently marketed steroidal progestins. For example, glucuronidation is the major metabolic pathway for TNPR, and TNPR is not metabolized by CYP3A4 (data not shown). TNPR does not have the potential to induce CYP3A4, whereas 3-ketodesogestrel, a metabolite of desogestrel, has the capacity to induce this isozyme. TNPR does not inhibit any of the major P450 enzymes (data not shown), whereas steroidal progestins have the potential to inhibit CYP2C19, an isozyme that is responsible for the metabolism of proton pump inhibitors such as omeprazole (36). These characteristics of TNPR clearly demonstrate that, unlike other steroidal progestins, the efficacy of TNPR will likely not be affected by any concomitant drugs and that TNPR should not interfere with the efficacy of other drugs.

The crystal structure of the PR/TNPR complex is the first reported PR-LBD structure with a non-steroidal ligand. This structure shows that TNPR retains many of the key interactions of P₄ with the protein, albeit from an unrelated chemical scaffold. As it is a fundamentally different compound, the common features of ligand binding between TNPR and P₄ delineate general features required for a potent PR agonist. Furthermore, TNPR interacts with the protein in novel ways. TNPR takes advantage of the ligand binding pocket even more effectively than P₄, with a novel hydrogen bond as the basis for its greater affinity for PR (41). Comparisons with the GR and AR help in understanding the subtle differences that make the PR ligand binding pocket unique from those of other closely related steroid receptors and show why TNPR takes advantage of it to achieve >250-fold selectivity for the PR (48–50).

In summary, TNPR is a structurally novel nonsteroidal PR agonist. It demonstrated potent PR agonist activity in vitro and in vivo. TNPR has an excellent receptor selectivity profile and improved pharmacological properties. Because of its novel chemical structure, TNPR may offer other unique biological properties. In fact recent evidence shows that TNPR possesses some tissue selective properties.² The PR isoform-selective effect of TNPR is currently under investigation. Finally, the crystal structure of PR-LBD-TNPR has been resolved, which supports the potent activity of this nonsteroidal compound.

	FOOTNOTES

 
The atomic coordinates and structure factors (code 1ZUC) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Division of Endocrinology and Reproductive Disorders, Women's Health Research Inst., Wyeth Research, 500 Arcola Rd., Collegeville, PA 19355. Tel.: 484-865-5627; Fax: 484-865-9389; E-mail: zhangz{at}wyeth.com.

¹ The abbreviations used are: P₄, progesterone; AR, androgen receptor; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; CYP, cytochrome P450; DHT, dihydrotestosterone; DMEM, Dulbecco's modified Eagle's medium; ER, estrogen receptor; FBS, fetal bovine serum; GR, glucocorticoid receptor; GST, glutathione S-transferase; 3-KDG, 3-ketodesogestrel; LBD, ligand binding domain; LNG, levonorgestrel; MPA, medroxyprogesterone acetate; PIPES, 1,4-piperazinediethanesulfonic acid; PR, progesterone receptor; SHBG, sex hormone binding globulin; SRC, steroid receptor coactivator; TMG, trimegestone; TNPR, tanaproget.

² Z. Zhang, Y. Zhu, S. Lundeen, S. Chippari, and R. C. Winneker, manuscript in preparation.

	ACKNOWLEDGMENTS

 
We thank L. Suhodolnik, S. Cosmi, L. Seestaler-Wehr, A. K. Malakian, S. A. Wolfrom, and the Tissue Culture Core staff at the Women's Health Research Institute for excellent technical support and Jennifer Deibert for assistance with the preparation of the manuscript.

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