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Endocrinology Vol. 143, No. 10 3822-3829
Copyright © 2002 by The Endocrine Society

ARTICLE

Identification and Characterization of a Selective, Nonpeptide Follicle-Stimulating Hormone Receptor Antagonist

Brian J. Arey1, Darlene C. Deecher, Emily S. Shen, Panayiotis E. Stevis, Edwin H. Meade, Jr, Jay Wrobel, Donald E. Frail2 and Francisco J. López3

Women’s Health Research Institute and Medicinal Chemistry, Department of Chemical Sciences, Wyeth Research, Collegeville, Pennsylvania 19680

Address all correspondence and requests for reprints to: Darlene C. Deecher, Women’s Health Research Institute, Wyeth Research, 500 Arcola Road, Collegeville, Pennsylvania 19680. E-mail: deeched{at}wyeth.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The glycoprotein hormones (LH, FSH, and TSH) are critical to the maintenance of physiological homeostasis and control of reproduction. However, despite an obvious utility for synthetic pharmacological agents, there are few reports of selective, nonpeptide agonists or antagonists to receptors for these hormones. We have identified and characterized a novel synthetic molecule capable of inhibiting the action of FSH. This compound, 7-{4-[Bis-(2-carbamoyl-ethyl)-amino]-6-chloro-(1,3,5)-triazin-2-ylamino)-4-hydroxy-3-(4-methoxy-phenylazo)-naphthalene}-2-sulfonic acid, sodium salt (compound 1), is a selective, noncompetitive inhibitor of the human (h) and rat (r) FSH receptors (FSHRs). Compound 1 selectively inhibited binding of [125I]hFSH with an IC50 value of 5.4 ± 2.3 µM. Radioligand-binding assays were performed using the baculovirus expressed extracellular domain of hFSHR (BV-tFSHR) to demonstrate site-specific interaction. Compound 1 competed for [125I]hFSH binding to BV-tFSHR with an IC50 value of 10 ± 2.8 µM. Functionally, compound 1 inhibited hFSH-induced cAMP accumulation and steroidogenesis in vitro with an IC50 value of 3 ± 0.6 µM. Competition of compound 1 for binding to other glycoprotein hormone receptors and other G protein-coupled receptors demonstrated select activity for FHSRs. Compound 1 inhibited ovulation in immature and cycling adult rats. These data provide proof of concept that selective, small molecule antagonists can be designed for glycoprotein hormone receptors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLYCOPROTEIN HORMONES ARE evolutionarily conserved across a wide range of phyla. Homologous glycoprotein hormone proteins have been identified in species ranging from worms to mammals (1). Despite having a rich ancestry, however, only four members of this family have been identified in mammals. Homologs of this ancient family of proteins are represented in mammals by chorionic gonadotropin and the pituitary hormones, LH, TSH, and FSH (2). These hormones are known to play an important role in the regulation of crucial functions, including metabolic rate and reproduction. LH, TSH, and FSH are secreted into the circulation by cells within the anterior pituitary gland in which they are synthesized as heterodimeric proteins. The family members share a common {alpha}-subunit but contain hormone-specific ß-subunits that confer receptor selectivity.

Similar to their ligands, receptors for these hormones also share structural similarity. The glycoprotein hormone receptors are members of the superfamily of G protein-coupled receptors (GPCRs). These receptors are characterized by the presence of seven putative transmembrane domains and activation of G protein-coupled signaling molecules upon hormone binding (3, 4, 5). Glycoprotein hormone receptors, however, are somewhat unique among GPCRs by the presence of an extraordinarily long NH2-terminal extracellular domain (~350 aa) containing several leucine-rich repeats. This domain of the receptor is known to be an important site of hormone binding and subsequent signal transduction.

FSH plays a major role in both female and male reproduction. In the female, FSH induces follicular maturation and concomitant estradiol production that is required for ovulation and proper preparation of the uterus for implantation. In the male, FSH regulates the function of testicular Sertoli cells, which are important for the proper progression of spermatogenesis and production of testicular peptide hormones. Clinical data demonstrate the importance of FSH for reproductive success because mutations to either FSH or its receptor leads to decreased fertility or infertility in both sexes (6, 7, 8). Therefore, these observations have provided a suitable proof of concept for the development of nonsteroid hormonal contraceptives that may be useful for males and females. These drugs would target membrane [e.g. the FSH receptor (FSHR)] rather than intracellular receptors as current steroidal contraceptives do. Currently, a scarcity of pharmacological agents exists to study the mechanisms of FSH interaction with its receptor (9, 10, 11, 12). In the studies described here, we report the identification and pharmacological characterization of the first known selective, small molecule antagonist of the FSHR. These studies provide a theoretical basis for the development of selective small molecule glycoprotein hormone receptor antagonists for potential use as novel contraceptive agents.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Affinity-purified human (h) pituitary FSH as well as the other glycoprotein hormones (hLH, hTSH, hCG) were purchased from Dr. Patrick Sluss (Reproductive Endocrine Unit, Massachusetts General Hospital, Charlestown, MA) or Cortex Biochem, Inc. (San Leandro, CA). Stock solutions of hormones (100 µM) were prepared in binding buffer (10 mM Tris HCl, 1 mM MgCl2, 1 mM CaCl2, 0.1% bovine albumin, 0.025% NaN3), aliquoted and stored at -80 C until the day of the assay. Iodinated hFSH was purchased from NEN Life Science Products (Boston, MA).

Compound 1 Identification
7-{4-[Bis-(2-carbamoyl-ethyl)-amino]-6-chloro-(1, 3, 5)-triazin-2-ylamino)-4-hydroxy-3-(4-methoxy-phenylazo)-napthalene-2-sulfonic acid, sodium salt (compound 1) and analogs were synthesized using methods previously described in a U.S. patent (Wrobel J, Rogers JF, and Kao W, 2001, Aryl Sufonic Acids as FSH Antagonists, US 6,200,963).

Cell lines
A Chinese hamster ovary cell line (3D2 cells) stably expressing the human FSHR was kindly provided by Dr. Kerry Koller (Affymax Inc., Palo Alto, CA). Cells were maintained at 37 C in 1:1 DMEM/F12 medium that was supplemented with 10% fetal bovine serum (FBS) (Life Technologies, Inc., Grand Island, NY), 146 µg/ml L-glutamine and 100 U/ml penicillin/10 µg/ml streptomycin) as previously described (13). Chinese hamster ovarian (CHO) cells that stably express the hTSH receptor (hTSHR) gene (CHO-25) were kindly provided by Dr. Leonard D. Kohn (Metabolic Diseases Branch of the NIDDK). CHO-25 cells were grown in F-12 nutrient mixture medium (Life Technologies, Inc.) supplemented with 10% (vol/vol) heat-inactivated fetal calf serum (Life Technologies, Inc.), 2 mM GlutaMax-I (Life Technologies, Inc.), penicillin G sodium (100 U/ml), and streptomycin sulfate (100 µg/ml). Cells were maintained at 37 C in an atmosphere saturated with water and containing 95% air, 5% CO2.

An adrenocortical tumor cell line (Y1 cells) purchased from American Type Culture Collection (Manassas, VA) was engineered to stably express the full-length hFSHR cDNA (provided by Dr. Kerry Koller). The hFSHR cDNA was inserted into the Bluescript II KS+ vector (Stratagene, La Jolla, CA) using XhoI and KpnI restriction enzyme sites at the 5' and 3' ends, respectively. The hFSHR cDNA was then subcloned in the correct orientation into the pcDNA3 expression vector (Invitrogen, San Diego, CA) using KpnI and NotI restriction enzyme sites at the 5' and 3' ends, respectively. This plasmid was used to introduce the hFSHR gene into the Y1 cell genome. A clonal cell line (Y1/5E5/s3) was isolated and cultured in growth medium consisting of F-10 nutrient mixture (Life Technologies, Inc.), 2 mM GlutaMax I (Life Technologies, Inc.), 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate (Life Technologies, Inc.), 12.5% horse serum (heat inactivated; BioWhittaker, Inc.), 2.5% FBS (heat inactivated; BioWhittaker, Inc.), and 75 µg G418/ml (Life Technologies, Inc.).

Radioligand-binding assays
FSH binding.
Binding assays were performed using 3D2 cell membrane preparations. Cells were grown to 90% confluency on 150-mm tissue culture plates. The medium was removed, cells were harvested from the plates into FSH-binding buffer (10 mM Tris-HCl, 1 mM MgCl2, 1 mM CaCl2, 0.1% BSA, and 0.025% sodium azide, pH 7.2) and homogenized by polytron (VirTis, Gariner, NY). The homogenate was centrifuged at 15,000 x g for 15 min, and the pellet was resuspended in binding buffer, homogenized, and centrifuged again. The supernatant was discarded and the pellet resuspended to approximately 100 µg/ml protein in FSH-binding buffer for assay. Membranes were dispensed into 96-well microtiter plates at 100 µl/well (~10 µg protein). Test compounds at varying concentrations or the nonspecific determinant (1 µM hFSH) or buffer (total binding determinant) were added to the wells in 50-µl aliquots. Reactions were initiated by the addition of 50 µl [125I]hFSH (50 pM, 55,000 cpm/well for competition assays or varying radioligand concentrations for saturation experiments, specific activity: 90–200 µCi/µg (3500–4500 Ci/mmol; NEN Life Science Products) for a total reaction volume of 200 µl. Plates were incubated on an orbital shaker for 2 h at room temperature for the reaction to reach steady state.

The binding assay was terminated by harvesting the cell membranes onto glass fiber filters (Skatron Blue mat 11740) presoaked (50 mM Tris/1% BSA, pH 7.2, 30 min) using a 96-well vacuum harvester (Skatron Instruments, Sterling, VA). Unbound ligand was removed from the glass fiber filter mats with five cycles of 3.5 ml wash buffer (50 mM Tris-HCl, pH 7.2, 4 C). Filters were individually divided using an automatic filter punch and the remaining bound radioactivity (<10% of total) determined by counting single filter disks in a {gamma} counter (ICN Biomedical, Costa Mesa, CA) for 1 min.

The equilibrium radioligand-binding assays to characterize compound 1 interaction were performed by preincubating membranes with either 10 µM compound 1 or vehicle for 1 h then adding varying concentrations of radioligand to initiate the binding reaction. Reactions were incubated for an additional 2 h at room temperature to ensure steady state of the radioligand.

The methods for using the baculovirus-produced extracellular domain of the FSHR (BV-tFSHR) and the solid phase radioligand binding were previously described (14).

Rat LH binding.
Receptor membrane preparation.
Rat testes were purchased from Pel-Freez Biologicals (Rogers, AR). The tunica albuginea was removed from each testis and the tissue was weighed. Two testes were minced in binding buffer and homogenized using a polytron homogenizer (model PT 1200C, Brinkmann Instruments, Inc., Westbury, NY). The homogenates were spun at 500 x g for 10 min, and the supernatants were pooled. A second centrifugation at 17,000 x g for 12 min was conducted, and the supernatants were discarded and the pellets collected, frozen, and stored at -80 C until use.

Receptor-binding assay.
Frozen pellets were weighed and resuspended in binding buffer to a concentration of 60 mg/ml (approximately 0.73 mg protein/reaction). Binding reactions were set up according to the hFSHR-binding assay described above with human LH (50 µl, 4 µM solution). The binding reactions were initiated by the addition to each well of 50 µl [125I]hLH (250 pM final concentration, specific activity: 90–200 µCi/µg, NEN Life Science Products). Plates were incubated for 2 h at room temperature on an orbital shaker, and the reactions were terminated as described for the hFSH-R binding assay.

cAMP accumulation assays
Compounds were screened for functional activity by monitoring their ability to inhibit FSH-induced cAMP accumulation in 3D2 cells. The cells were cultured in 96-well plates at 30,000 cells/well for 24 h before assay. Compounds were prepared as 1 mM stock solutions in dimethylsulfoxide (DMSO) and serially diluted for assay. On the day of the assay, cells were washed twice with assay media [Optimem, Life Technologies, Inc., 0.1% (wt/vol) BSA]. Following the second wash, cells were preincubated in 100 µl assay media for 30 min at 37 C. The medium was removed from the wells, and triplicate wells containing cells were challenged with 1 ng/ml hFSH in assay media containing varying concentrations of test compound for 30 min at 37 C. The final DMSO concentration of the challenge medium did not exceed 0.1% (vol/vol) and was the same among all the experimental groups. Experiments were terminated by the addition of 100 µl 0.2 N HCl. Determination of cAMP concentrations was conducted by RIA using a commercially available RIA kit (13). The sensitivity of the assay was 2 fmol/tube. The intra- and interassay variability (percent coefficient of variation at approximately the IC50 value) for this assay were 6.7% and 10.8%, respectively. Data were expressed in terms of nmol cAMP/ml assay medium.

The cAMP accumulation assays using CHO-25 (hTSHR) cells were performed as described above for the CHO-3D2 cells with the following exceptions. CHO-25 cells were plated at a density of 20,000 cells/well. All test compounds were evaluated in a concentration-response paradigm ranging from 0.01 to 30 µM. Controls and test compounds were evaluated in quadruplicate. Cells were treated with vehicle, the EC20 concentration of hTSH (5 nM, hTSH >98% pure, Cortex Biochem, Inc.) or the test compounds in the presence or absence of the 5 nM hTSH. The ability of the compounds to inhibit the cAMP accumulation induced by hTSH was evaluated by RIA as stated above.

Granulosa cell culture and aromatase bioassay
All procedures using animals were approved by the Wyeth Research Animal Care and Use Committee (Collegeville, PA). Twenty-one-day-old female Sprague Dawley rats were housed at 25 C under a 12-h light, 12-h dark cycle with food and water provided ad libitum. Before the harvesting of granulosa cells, animals were treated with daily injections of 100 µg/kg diethylstilbestrol in corn oil for 3 d. On the fourth day, animals were euthanized and ovaries collected. The ovaries were washed three times in 50 ml sterile HEPES-buffered saline containing glucose. The ovaries were then incubated in a hypertonic medium consisting of McCoy’s 5A medium supplemented with 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml sodium selenite (ITS, Sigma, St. Louis, MO), 146 µg/ml L-glutamine, 100 nM testosterone, 100 nM diethylstilbestrol, 100 U/ml penicillin/10 mg/ml streptomycin/250 ng/ml Amphotericin B (antibiotic/antimycotic, Life Technologies, Inc.), 0.5 M sucrose, and 0.1 mM EGTA for 45 min at 37 C in a humidified incubator (95% air/5% CO2). Following this incubation, the ovaries were washed in isotonic medium (using the above medium without sucrose and EGTA) and then incubated for an additional 45-min period at 37 C in the isotonic medium.

Granulosa cells were harvested by extrusion from swollen follicles by placing the ovaries between two sterile glass slides and applying pressure. Following extrusion, the ovaries were triturated by pipetting three times in 30 ml isotonic medium. The medium containing the extruded granulosa cells and the ovarian washes were combined and centrifuged for 5 min (700 x g at room temperature). Pelleted cells were resuspended in isotonic medium. Cell number was determined using a hemocytometer, and viability was assessed by trypan blue exclusion. Cells were plated into 24-well tissue culture plates (Nunc, Naperville, IL) at 100,000 viable cells/well.

The aromatase bioassay was performed as previously described (13). In brief, cells were challenged with hFSH in isotonic medium supplemented with 0.1% (wt/vol) BSA in a total incubation volume of 500 µl. The cells were incubated with hFSH and test compounds for 72 h at 37 C and estradiol in the media measured by RIA using the Coat-a-Count kit (Diagnostic Products Corp., Los Angeles, CA). The sensitivity of this assay is 0.25 pg/tube and the intra- and interassay variability (percent coefficient of variation at approximately the IC50 value) were 4.3% and 6.8%, respectively.

Y1/5E5/s3 steroidogenesis assay
Y1/5E5/s3 cells stably expressing the hFSHR were plated 2 d before treatment into 24-well plates at a density of 200,000 cells/well in growth medium. On the day of treatment, each well of cells was washed twice with 1 ml prewarmed (37 C) assay medium ({alpha}MEM, Life Technologies, Inc.), 2 mM GlutaMax I, 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate, 15% horse serum, and 2.5% FBS (heat inactivated, BioWhittaker, Inc.). After removing the last medium wash, an additional 1 ml assay medium was added to each well, and the cells were incubated for 30 min at 37 C in a humidified incubator with 5% CO2/95% air. Cells were treated with different concentrations of test compounds in 0.5 ml assay medium containing 0.1% (vol/vol) DMSO or vehicle/DMSO alone for 20–24 h at 37 C. Each treatment condition was tested in quadruplicate. For studying antagonist activity, each compound was tested at four different concentrations (four wells/treatment) in a concentration-response paradigm vs. a concentration of hFSH (EC50 value 25 ng/ml, Cortex Biochem). In addition, vehicle alone and FSH alone (25 ng/ml final concentration) were tested as negative and positive controls, respectively. Along with the test compounds, hFSH was tested in a dose response (1, 10, 30, 300, 1000 ng/ml) paradigm as an additional assay control. At the end of the incubation period, the medium was removed from each well and assayed for progesterone concentration by RIA using the Coat-a-Count progesterone kit (Diagnostic Products Corp.) according to the recommended specifications. The sensitivity of this assay was 2.5 pg/tube and the intra- and interassay variability (percent coefficient of variation at approximately the IC50 value) were 6.6% and 12.3%, respectively.

Ovulation inhibition assay
Adult random-cycling female Sprague Dawley rats (200–225 g body weight) were purchased from Charles River (Raleigh, NC) and housed at 25 C under a 12-h light, 12-h dark cycle. Rats were provided with a casein diet and water ad libitum. The estrous cycles of the rats were synchronized by a sc injection of [D-trp6, DesGly10]-LHRH ethylamide (Peninsula Laboratories, Inc., Belmont, CA) [0.1 ml/rat; 20 µg/ml 0.1% (wt/vol) BSA in PBS] at 0900 h and again at 1400 h (15). The estrous cycle was monitored for each rat over two cycles by cytological evaluation of vaginal smears. All rats were in estrus on the first day of treatment. Body weights were recorded on the first day of treatment and the morning after treatment was completed. Rats were treated twice a day (0900 h and 1400 h) with ip injections of vehicle [50% (vol/vol) DMSO in buffered saline] or compound 1 (30 or 100 mg/kg body weight) for four consecutive days. Animals were euthanized by CO2 asphyxiation on the morning following the last day of treatment (estrus). The ampullary region of the oviduct was removed from each animal and the number of ova contained within each ampulla counted to assess ovulation. The uterus and spleen were removed from each animal and weighed. The ovaries were removed from each animal and fixed in 10% neutral buffered formalin. Growth rates were calculated by subtracting the body weights of the animals at the end of treatment from the body weights at the start treatment and dividing this difference by the number of days of treatment. The experiment was performed twice.

Histological evaluation
Ovary samples were sent to the Investigative Pathology Group at Wyeth Research (Andover, MA) for preparation of tissue sections. The ovarian samples were embedded in paraffin wax medium, processed, sectioned at 5 µm, and stained with hematoxylin and eosin for microscopic examination and morphometrical measurement. Ovarian inflammation was graded according to the following scoring system: grade 0, no abnormal findings; 1, slight or minimal inflammation; 2, mild inflammation; 3, moderate inflammation; 4, marked inflammation; and 5, severe inflammation. Ovaries were evaluated for the presence or number of corpora lutea, Graafian follicles, all stages of follicle development, and general histological features of the ovaries. Morphometry measurements were made using Image Pro Plus (version 3.0, Media Cybernetics, Inc., Silver Spring, MD) and included the number and the surface of the corpora lutea. A 1-mm bar, embedded in each micrograph, was used to calibrate the instrument for all measurements. For each rat ovary, all corpora lutea were traced and the areas were recorded.

Statistical analyses
Statistical analyses of bioassay data were performed using EXCEL scripts that preanalyzed data for normality and homogeneity of variances. Differences among treatment groups were analyzed by ANOVA in conjunction with Huber weighting and paired differences determined using Dunnett’s test or least significant difference test. In those cases in which data were not normally distributed or had heterogeneous variance, a logarithmic transformation of the data was performed before ANOVA. Differences between treatment groups were considered significant if P was less than 0.05. EC50 and IC50 values were estimated using EXCEL scripts using a four-parameter logistic equation. For the morphometry results, the mean corpora lutea area was calculated per ovary, and this value was then used to calculate the mean and SEM per treatment group.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The primary goal in these studies was to identify selective small molecule modulators of the FSHR. Therefore, we screened a large chemical library for compounds capable of competing for the binding of [125I]hFSH to the hFSHR expressed in 3D2 cells. In this assay, hFSH competes for the binding of [125I]hFSH to 3D2 cell membrane preparations with an IC50 value of 411 ± 97 pM (Fig. 1BGo, open circles). A known, nonselective FSHR antagonist, suramin, also competed for binding of [125I]hFSH to the hFSHR with an IC50 value of 70 ± 10.2 µM (Fig. 1BGo, inverted triangles) (16, 17). Using this screening paradigm, we identified, a diazonapthylsulfonic acid, sodium salt (Fig. 1AGo, compound 1) capable of inhibiting the binding of [125I]hFSH. This compound competed for the binding of [125I]hFSH to hFSHR expressed in 3D2 cells with an IC50 value of 5.4 ± 2.3 µM (Fig. 1BGo, solid circles).



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  		Figure 1. Chemical structure and competition of compound 1 for the binding of [125I]hFSH to the hFSHR. A, Chemical structure of compound 1. B, Competition of compound 1 for [125I]hFSH using membranes prepared from 3D2 cells. Values depicted in the graph are the means ± SEM of triplicate determinations of percent specific bound at each concentration of compound tested. Control binding (100%) was measured in the presence of buffer only. The competition curve for hFSH depict an IC50 value of 411 ± 97 pM (open circles). Competition curves using hFSHR from 3D2 cell membranes yielded IC50 ± SE values of 5.8 ± 2.3 µM for compound 1 (solid circles) and 70 + 10.2 µM for suramin (open triangles). C, Compound 1 interacts with the extracellular domain of the hFSHR (BV-tFSHR). A truncated form of the hFSHR was expressed and used in a solid-phase binding radioligand assay to determine the competition of compound 1 for hFSH binding. Compound 1 inhibited the binding of [125I]hFSH to the BV-tFSHR with an IC50 value of 10 ± 2.8 µM (solid circles), whereas hFSH competed for [125I]hFSH binding with an IC50 value of 370 ± 21 pM (open circles).

 
Numerous reports have documented the importance of the extracellular domain of the hFSHR for binding of its native ligand (14, 18, 19, 20, 21, 22). To study whether compound 1 could inhibit binding of hFSH via direct interaction with this domain, we employed a solid-phase radioligand-binding assay using the extracellular domain of the hFSHR captured in individual wells of 96-well plates as described earlier (14). Compound 1 inhibited the binding of [125I]hFSH to the extracellular domain of the FSHR with an IC50 value of 10 ± 2.8 µM (Fig. 1CGo, solid circles). As expected, hFSH competed for [125I]hFSH binding with an IC50 value of 370 ± 21 pM (Fig. 1CGo, open circles).

To further analyze the nature of the binding of compound 1 to the hFSHR, equilibrium binding experiments were performed using 3D2 cell membranes. As is shown in Fig. 2Go, association of compound 1 with the hFSHR appeared to be noncompetitive in nature, compared with the native ligand, hFSH. Preincubation of compound 1 (10 µM) with 3D2 membranes significantly reduced the maximal binding density (Bmax) values. The reported KD values for vehicle, compared with membranes preincubated with compound 1 were 385 ± 70.9 and 350 ± 71.9 pM, whereas the Bmax values were 245 ± 22.4 and 82 ± 9.9 fmol/mg protein, respectively. Therefore, the radioligand binding studies suggest that compound 1 associates with the hFSHR via the extracellular domain of the receptor and that this interaction is noncompetitive. However, these data do not provide information concerning the functional properties of the compound (i.e. agonist or antagonist).



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  		Figure 2. Compound 1 noncompetitively inhibits binding of FSH with the FSHR. The graph illustrates data from equilibrium-binding assays using the hFSHR as described in Materials and Methods. Values from parallel Scatchard analyses reveal that preincubation of compound 1 (10 µM, open circles) with 3D2 cell membranes statistically decreases the Bmax values for the hFSHR.

 
To assess the functional activity of this molecule, we evaluated the biological response of the FSH receptor in various in vitro models. The CHO-3D2 cell line was used to study the ability of compound 1 to affect cAMP accumulation through the hFSHR. Compound 1 had no effect on cAMP accumulation when incubated with 3D2 cells alone (data not shown). However, when incubated in the presence of 1 ng/ml hFSH (EC20) compound 1 inhibited FSH-induced cAMP accumulation in a concentration-dependent manner with 100% efficacy (Fig. 3Go). The IC50 value of compound 1 in this assay was 3.3 ± 0.6 µM. In addition, compound 1 also inhibited steroid production in two separate models of steroidogenesis. In Y1/5E5/s3 cells, compound 1 inhibited hFSH-induced progesterone secretion with an IC50 value of 14 ± 5 µM (Fig. 4AGo) and an efficacy of approximately 100%. However, using primary cultures of granulosa cells, which represent a more physiologically relevant model, the compound demonstrated a 5-fold greater potency (3 ± 0.8 µM) to inhibit FSH-induced estradiol production (Fig. 4BGo).



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  		Figure 3. Compound 1 inhibits FSH-induced cAMP accumulation in a concentration-dependent manner. The 3D2 cells were incubated in 96-well plates with medium containing DMSO alone (white bar), 1 ng/ml hFSH/DMSO (black bar), or increasing concentrations of compound 1 plus 1 ng/ml hFSH (circles). Compound 1 inhibited FSH-induced cAMP accumulation in a concentration-dependent manner with an apparent IC50 value of approximately 3.3 ± 0.6 µM. Asterisks denote significant differences, compared with the positive control, hFSH (P < 0.05).

 


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  		Figure 4. Compound 1 dose-dependently inhibits FSH-induced steroidogenesis in vitro. A, Compound 1 inhibited hFSH-induced progesterone secretion in a mouse adrenal cell line, Y1/5E5/s3 with an IC50 value of 14 ± 5 µM. B, Compound 1 inhibited estradiol production in granulosa cells in a concentration-dependent manner with an IC50 value of 3.0 ± 0.8 µM. Asterisks denote significant differences from the positive control (hFSH/DMSO, P < 0.05). Granulosa cells were isolated and then plated in 24-well plates in serum-free isotonic medium containing DMSO alone (0.1%, white bar), 0.5 ng/ml hFSH/DMSO (black bar), or increasing concentrations of compound 1 plus hFSH (open circles).

 
Selectivity of compound 1 was studied by evaluating the ability of the compound to bind or activate signaling through related glycoprotein hormone receptors. Binding of compound 1 to the LH receptor was tested using bovine testis membrane preparations. In addition, the ability of the compound to inhibit cAMP accumulation by the bound TSHR was performed in CHO-25 cells. Compound 1 inhibited binding and activation of signaling through the FSHR in the low micromolar range. However, compound 1 did not significantly interact with the LH and TSH receptors (up to 100 µM) (data not shown). These data conservatively suggest a minimal 20- to 30-fold separation in affinity of compound 1 for the FSHR, compared with the other glycoprotein hormone receptors. Competition of compound 1 for binding to a variety of nonglycoprotein hormone GPCRs as well as the estrogen receptor was also performed, and no significant interaction at 10 µM was noted (data not shown). Taken together, these data strongly suggest that compound 1 exhibits a high degree of selectivity for the FSHR.

To discern structure/activity relationships for the antagonistic properties of the compound at the FSHR, a number of analogs of compound 1 were synthesized and analyzed in vitro. Evaluation of activity of these analogs revealed certain key features to the bioactivity of the molecule: esterification of the sulfonic acid moiety, methylation of the phenolic group, removal of the diazo substituent or the triazine ring all led to drastic reductions in activity (data not shown). Similarly, replacing the bis (2-carbamoylethyl)amino or the chlorine substituents on the pendant triazine nucleus also led to reductions in potency. One alternative triazine substituent that was successful in retaining activity was the bis (2-hydroxyethyl)amino group. Replacement of the triazine nucleus with a dimethoxybezoyl group also produced an active compound without much reduction in potency. However, none of the analogs prepared were more potent than compound 1.

In preliminary experiments using an immature rat model (23), decreases in uterine, ovarian, and body weight were detected after ip administration of compound 1 (data not shown). Consequently, we sought to determine the effect of compound 1 on ovulation in an adult cycling rat. The effect of compound 1 on ovulation was determined after ip administration of vehicle or compound 1 at either 30 or 100 mg/kg body weight. The results from one of the replicate studies are summarized in Table 1Go. Administration of compound 1 at 30 mg/kg inhibited ovulation in two of eight rats but did not affect the number of ova produced in those animals that did ovulate. The highest dose of compound 1 studied (100 mg/kg) inhibited ovulation in all of the animals in that treatment group. Under the conditions of this study, ip injection of compound 1 was associated with mild chronic inflammation with brightly eosinophilic foreign material seen at the peritoneal surface (also called the surface epithelium) of the ovary from rats dosed at 100 mg/kg body weight (Table 1Go). No microscopic differences were seen in the other ovarian structures as well as in the total number of follicles (including all stages of the follicle development), de Graaf follicles, and corpora lutea of the rats injected with the vehicle control and compound 1 at 30 or 100 mg/kg (Table 1Go). In addition, there was no difference in the number, size, and microscopic aspect of the corpora lutea in the control and compound 1-treated rats (data not shown). Administration of compound 1 affected growth rate with the highest dose producing a loss in body weight as indicated by the negative growth rate (Table 1Go).


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  		Table 1. Effects of compound 1 on ovulation, follicular development, organ weight, and growth rate after ip administration

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The goal of these series of experiments was to identify and characterize small molecular weight compounds capable of interacting with the hFSHR. A radioligand-binding assay was employed to search a large chemical library for such compounds. We identified one compound that has been described here. Compound 1 inhibited binding of [125I]hFSH to the hFSH full-length receptor expressed in CHO cell membranes but also to the truncated extracellular domain of the receptor. The potencies of the compound against the two receptor preparations was similar, suggesting that the membrane spanning portion of the FSHR has little interaction with compound 1. These data support previous work, suggesting the critical role of the extracellular amino-terminal domain of the FSHR in ligand binding. Interestingly, however, the Scatchard analysis of the binding of hFSH in the presence of compound 1 revealed that the competition for binding with the receptor is noncompetitive in nature. Additionally, the functional consequence of this interaction was studied to determine the effects of compound 1 on signaling by the hFSHR. Initial studies in 3D2 cells demonstrated that the compound did not induce cAMP production in the absence of hFSH. However, when incubated in the presence of hFSH, compound 1 inhibited FSH-induced production of cAMP in a concentration-dependent manner. Further studies confirmed the antagonistic nature of compound 1 to inhibit steroidogenesis induced by FSH in both a steroidogenic adrenal cell line expressing the hFSHR and primary granulosa cells in culture. In addition, when tested in vivo the compound blocked ovulation at the highest dose tested.

FSH is known to contact numerous sites in the extracellular region of the FSHR to promote signaling (14, 18, 19, 20, 21, 22). In addition, other regions near the extracellular surface of the receptor also have been shown to be important in binding to FSH (24) including the extracellular loops formed by the membrane spanning regions of the receptor. These associated regions are thought to play a more structural role by stabilizing the correct three-dimensional structure of the amino-terminal domain. The observations outlined above suggest that compound 1 does not bind to the same site on the receptor as FSH. Functional antagonism of compound 1 at the FSHR may arise from a partial overlap of binding sites for compound 1 and hFSH. Alternatively, compound 1 may block FSH binding by allosteric modulation of the correct three-dimensional structure of the extracellular domain required for high affinity association between the receptor and its ligand. Unfortunately, the studies presented here cannot distinguish between these two possibilities. This will require further experiments studying the amino acids of the FSHR involved in binding compound 1. The noncompetitive nature of the antagonistic effects of compound 1 at the FSHR, however, suggests that it acts to decrease the ability of the native ligand to interact with its receptor.

Because the glycoprotein hormone receptors are structurally similar, selectivity of compound 1 for the FSHR was evaluated. Studies performed with closely related glycoprotein hormone receptors or other unrelated GPCRs demonstrated no effect of compound 1 on binding or functional activation of these receptors at the active concentration used for FSHR. These data demonstrate that compound 1 is a selective, noncompetitive functional antagonist of the FSHR.

In our characterization of compound 1, we found that the potency of the antagonist to inhibit FSH functional activity was similar to that reported for another small molecule, suramin (Fig. 1Go and Ref. 14). However, compound 1 was approximately 7- to 10-fold more potent in binding assays than suramin. Clinically, suramin is used as both an antiparasitic and cytostatic therapy, but it has been shown to have numerous other activities, including blockade of glycoprotein hormone action (16). One of the clear differences between suramin and compound 1 is that suramin does not display selectivity for the glycoprotein hormone receptors. It has been reported to associate with a number of different receptors including GPCRs (16, 17), growth factors (16), and kinase receptors (25). The mechanism of the interaction of suramin with this diverse list of receptors is not currently known but has been hypothesized to be due to its polyionic structure (3). If this is the case, the portion of suramin interacting with different classes of receptor is likely to be somehow unique to each class. In contrast, analysis of the similarities between suramin and compound 1 revealed the presence of sulfonic acid residues as an interesting commonality between the two molecules. The appearance of this moiety in both compounds may be a critical feature for the association with the extracellular domain of the FSHR (14). This hypothesis was supported through the synthesis of analogs of compound 1 in which the sulfonic acid moiety was masked as its isopropyl ester. These compounds were completely devoid of activity. Furthermore, other analogs of compound 1, in which the triazine group was modified, highlighted the importance of this portion of the compound for biological activity. Similarly, analogs in which the molecule was truncated at either the triazine or diazo moieties also had drastically reduced activity (data not shown).

One major physiological action of FSH is to stimulate steroid production in the ovarian follicle. The subsequent result of this effect is to induce maturation of the uterine lining in preparation for implantation of an embryo as well as to induce maturation and recruitment of ovarian follicles and in males proper progression of spermatogenesis (26). We tested the ability of compound 1 to block FSH-induced steroidogenesis using a mouse adrenal Y1 cell line that stably expresses the hFSHR gene as well as primary cultures of rat granulosa cells. Compound 1 blocked FSH-induced steroidogenesis using both the human and rat FSHR systems. In addition, we used the ovarian response in the primary rat granulosa cells to assess the bioactivity of compound 1 (27). Compound 1 inhibited FSH-induced aromatase activity in granulosa cells in a concentration-dependent manner with a similar potency to that which blocked cAMP accumulation. These data clearly show that compound 1 is capable of inhibiting FSH-induced functional steroidogenic responses in vitro.

The utility of blocking FSH action as a contraceptive strategy is borne out in the literature through reports of naturally occurring mutations in either the hormone or its receptor (26). Similarly, knockout models have confirmed the importance of FSH in fertility. Inactivating mutations in either the ligand or receptor leads to decreased fertility or infertility (6, 7, 8). The effect of these mutations to render women completely infertile is well documented. However, studies of men with such mutations have led to conflicting reports. It is clear that disruption of biological action of the FSH system will at the very least lead to reduced fertility in such subjects, but in some cases only reduced spermatogenesis or partial effects on sperm quality were found (8, 28, 29, 30). Thus, a potential compensatory mechanism for gonadotropin action on spermatogenesis may exist. This has led reproductive biologists to question the critical nature of FSH in male fertility. However, knockout mouse models and research in primates in some cases have led to drastic effects on male fertility (31, 32, 33, 34, 35, 36). Therefore, resolution of this issue awaits further research. Compound 1 may provide a means to further research the role of FSH in spermatogenesis. Administration of compound 1 completely inhibited ovulation at the highest dose tested in rats. The ability of compound 1 to block FSHR activation in vivo could not be ascertained in our studies, although we have demonstrated that the compound has the ability to block FSH binding to the FSHR in vitro. The inhibition of ovulation does not appear to be related to alterations in the number of or microscopic appearance of ovarian follicles or corpora lutea. Because histological evidence is lacking on the affect of compound 1 on folliculogenesis, nonreceptor effects of compound 1 (such as periovarian inflammation or decreased growth rate) may have indirectly contributed to the ovulation inhibition. However, studies of the in vitro efficacy of compound 1, taken together with the in vivo data, point to the ability of this compound to inhibit ovulation through the FSHR.

In summary, we have identified a small-molecule, selective FSHR antagonist. Identification of a selective, nonpeptide antagonist such as compound 1 represents the first step in the development of nonsteroid hormone-based contraceptive compounds and provides a pharmacological tool to further study the role of FSH and its receptor in the control of reproductive processes.


   Acknowledgments
 
The authors would like to thank the following contributors to this body of work: Drs. Andre Jean Lambert for evaluation of tissue samples from in vivo experiments; M. Claudia Perez for evaluation of compound 1 in TSH bioassay; and Wenling Kao and John Rogers for scale-up chemistry of compound 1 for testing in vivo.


   Footnotes
 
1 Current address: Bristol-Myers Squibb Co., P.O. Box 4000, Hopewell, New Jersey 08543. Back

2 Current address: Pharmacia Corp., 310 Henrietta Street, Kalamazoo, Michigan 49007. Back

3 Current address: Ligand Pharmaceuticals, Inc., 10275 Science Center Drive, San Diego, California 92121. Back

Abbreviations: Bmax, Maximal binding density; BV-tFSHR, baculovirus expressed extracellular domain of human FSH receptor; CHO, Chinese hamster ovary; DMSO, dimethylsulfoxide; FBS, fetal bovine serum; FSHR, FSH receptor; GPCR, G protein-coupled receptor; h, human; TSHR, TSH receptor.

Received April 3, 2002.

Accepted for publication June 11, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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