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 [(125)I]hFSH with an IC(50) value of 5.4 +/- 2.3 micro M. Radioligand-binding assays were performed using the baculovirus expressed extracellular domain of hFSHR (BV-tFSHR) to demonstrate site-specific interaction. Compound 1 competed for [(125)I]hFSH binding to BV-tFSHR with an IC(50) value of 10 +/- 2.8 micro M. Functionally, compound 1 inhibited hFSH-induced cAMP accumulation and steroidogenesis in vitro with an IC(50) value of 3 +/- 0.6 micro M. 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.

The pituitary glycoprotein hormones, luteinizing hormone and follicle-stimulating hormone (FSH), act through their cognate receptors to initiate a series of coordinated physiological events that results in germ cell maturation. Given the importance of FSH in regulating folliculogenesis and fertility, the development of FSH mimetics has been sought to treat infertility. Currently, purified and recombinant human FSH are the only FSH receptor (FSH-R) agonists available for infertility treatment. By screening unbiased combinatorial chemistry libraries, using a cAMP-responsive luciferase reporter assay, we discovered thiazolidinone agonists (EC 50's ‫؍‬ 20 M) of the human FSH-R. Subsequent analog library screening and parallel synthesis optimization resulted in the identification of a potent agonist (EC 50 ‫؍‬ 2 nM) with full efficacy compared with FSH that was FSH-R-selective and -dependent. The compound mediated progesterone production in Y1 cells transfected with the human FSH-R (EC 50 ‫؍‬ 980 nM) and estradiol production from primary rat ovarian granulosa cells (EC 50 ‫؍‬ 10.5 nM). This and related compounds did not compete with FSH for binding to the FSH-R. Use of human FSH/thyroid-stimulating hormone (TSH) receptor chimeras suggested a novel mechanism for receptor activation through a binding site independent of the natural hormone binding site. This study is the first report of a high affinity small molecule agonist that activates a glycoprotein hormone receptor through an allosteric mechanism. The small molecule FSH receptor agonists described here could lead to an oral alternative to the current parenteral FSH treatments used clinically to induce ovarian stimulation for both in vivo and in vitro fertilization therapy. Follicle-stimulating hormone (FSH)2 is a glycoprotein hormone produced by the anterior pituitary that plays a key role in stimulating ovulation and spermatogenesis. Like other members of the glycoprotein hormone family (luteinizing hormone, chorionic gonadotropin, and thyroid-stimulating hormone (TSH)), FSH is a heterodimeric protein of ϳ30,000 Da that consists of a common ␣-subunit joined noncovalently to a hormone-specific ␤-subunit. FSH activity is mediated through binding to the FSH receptor (FSH-R), which belongs to family I of the large 7-transmembrane-spanning, G protein-coupled receptor (GPCR) superfamily. The glycoprotein hormone receptors are unique among the members of the GPCR family, because they recognize protein hormones and are dominated by a large N-terminal extracellular region (366 amino acids in the case of the FSH-R) that is the predominant site of hormone binding (1, 2) and is required for signal transduction.The binding of FSH to its receptor results in the activation of adenylyl cyclase through heterotrimeric G proteins. Interaction of the activated FSH-R with G s initiates the cAMP signaling cascade (3). The ability of the FSH-R to activate adenylyl cyclase was exploited to generate a Chinese hamster ovary (CHO) FSH-R reporter cell line (4 -6). Screening of a collection...

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To investigate the role of leptin in bone formation, the skeleton of the obese female leptin receptor-deficient Zucker rat was examined using pQCT, CT, and histomorphometry. A trend toward decreasing structural and bone formation parameters in these rats as they age suggest that leptin has a small positive effect on bone.Introduction: Evidence in the literature has suggested the possible role of leptin in bone formation. Leptin deficiency or leptin receptor deficiency results in higher bone mass. In an attempt to further investigate leptin's role in bone formation, we examined the skeleton of obese leptin receptor-deficient Zucker rats. Methods: Female leptin receptor-deficient Zucker (fa/fa) rats and their homozygous (Fa/Fa) and heterozygous (Fa/fa) lean controls were used at 9 and 15 weeks of age (n ϭ 5). Bone mineral density of the proximal tibia was measured by peripheral quantitative computed tomography (pQCT). Microcomputed tomography (CT) was used for the analysis of trabecular architecture in the proximal tibia metaphysis and cortical bone at the tibia-fibula junction. Static and dynamic parameters of bone resorption and formation were quantitated by histomorphometry. Statistical analysis was performed by Dunnett's one-way ANOVA. Results: Analysis of the proximal tibia by pQCT show no significant differences in the bone mineral density of obese rats compared with their corresponding lean controls in either age group. Trabecular architecture measured by CT indicate a trends toward decreasing bone volume (BV/TV) in the obese animals, evident by a decrease in trabecular number and thickness with an increase in trabecular separation. Histomorphometric evaluation further shows significant increases in osteoclast surface in the obese rats at both 9 and 15 weeks without a change in osteoclast number. Osteoid surface in the obese animals was also found to be decreased by 15 weeks of age. Fluorescent-based measurements of bone formation were not significantly different. Differences in the cortical compartment were not observed at either age. Conclusion: Based on the observed skeletal phenotype of the Zucker (fa/fa) rat, it is suggested that leptin exerts a positive effect on bone.

Circulating calcium (Ca(2+)) is a primary regulator of bone homeostasis through its action on PTH secretion. Extracellular Ca(2+) modulates PTH secretion through a cell surface G protein-coupled receptor, the calcium-sensing receptor (CaR). The expression of the CaR suggests a critical role in cellular regulation by calcium in various organs, including parathyroid gland, bone, and kidney. Despite an obvious pharmacological utility for CaR antagonists in the treatment of disease, only a limited number of such classes of compounds exist. We have identified a novel class of small molecules with specific activity at the CaR. This class of compounds is represented by compound 1. It possesses potent antagonist activity at the human CaR with IC(50) values of 64 nm and 230 nm in inhibiting intracellular Ca(2+) flux and inositol phosphate generation in vitro, respectively. When administered to male rats in vivo, compound 1 robustly increased serum PTH levels. The stimulation of PTH secretion was rapid and transient when administered either iv or orally. The pharmacokinetic profile of compound 1 after oral administration revealed that maximal plasma levels of compound were reached within 1 h and the half-life of the compound to be approximately 2 h in rats. These data describe a representative compound of a novel chemical class than previously described allosteric modulators that offer a new avenue for the development of improved treatments of osteoporosis.