Identification of Phe313 of the gonadotropin-releasing hormone (GnRH) receptor as a site critical for the binding of nonpeptide GnRH antagonists

The dog GnRH receptor was cloned to facilitate the identification and characterization of selective nonpeptide GnRH antagonists. The dog receptor is 92% identical to the human GnRH receptor. Despite such high conservation, the quinolone-based nonpeptide GnRH antagonists were clearly differentiated by each receptor species. By contrast, peptide antagonist binding and functional activity were not differentiated by the two receptors. The basis of the differences was investigated by preparing chimeric receptors followed by site-directed mutagenesis. Remarkably, a single substitution of Phe313 to Leu313 in the dog receptor explained the major differences in binding affinities and functional activities. The single amino acid replacement of Phe313 of the human receptor with Leu313 resulted in a 160-fold decrease of binding affinity of the nonpeptide antagonist compound 1. Conversely, the replacement of Leu313 of the dog receptor with Phe313 resulted in a 360-fold increase of affinity for this compound. These results show that Phe313 of the GnRH receptor is critical for the binding of this structural class of GnRH antagonists and that the dog receptor can be "humanized" by substituting Leu for Phe. This study provides the first identification of a critical residue in the binding pocket occupied by nonpeptide GnRH antagonists and reinforces cautious extrapolation of ligand activity across highly conserved receptors.     

Differential regulation of gonadotropin-releasing hormone by corticotropin-releasing factor family peptides in hypothalamic N39 cells

Corticotropin-releasing factor (CRF) is involved in a variety of physiological functions including regulation of hypothalamo-pituitary-adrenal axis activity during stressful periods. Urocortins (Ucns) are known to be members of the CRF family peptides. CRF has a high affinity for CRF receptor type 1 (CRF(1) receptor). Both Ucn2 and Ucn3 have very high affinity for CRF receptor type 2 (CRF(2) receptor) with little or no binding affinity for the CRF(1) receptor. Gonadotropin-releasing hormone (GnRH) is known to be involved in the regulation of the stress response. Gonadotropin-inhibitory hormone (GnIH) neurons interact directly with GnRH neurons, and the action of GnIH is mediated by a novel G-protein coupled receptor, Gpr147. This study aimed to explore the possible function of CRF family peptides and the regulation of GnRH mRNA in hypothalamic GnRH cells. Both mRNA and protein expression of the CRF(1) receptor and CRF(2) receptor were found in hypothalamic GnRH N39 cells. CRF suppressed GnRH mRNA levels via the CRF(1) receptor, while Ucn2 increased the levels via the CRF(2) receptor. Both CRF and Ucn2 increased Gpr147 mRNA levels. The results indicate that CRF and Ucn2 can modulate GnRH mRNA levels via each specific CRF receptor subtype. Finally, CRF suppressed GnRH protein levels, while Ucn2 increased the levels. Differential regulation of GnRH by CRF family peptides may contribute to the stress response and homeostasis in GnRH cells.     

Synthesis and biological evaluation of piperazinyl heterocyclic antagonists of the gonadotropin releasing hormone (GnRH) receptor

Antagonism of the gonadotropin releasing hormone (GnRH) receptor has resulted in positive clinical results in reproductive tissue disorders such as endometriosis and prostate cancer. Following the recent discovery of orally active GnRH antagonists based on a 4-piperazinylbenzimidazole template, we sought to investigate the properties of heterocyclic isosteres of the benzimidazole template. We report here the synthesis and biological activity of eight novel scaffolds, including imidazopyridines, benzothiazoles and benzoxazoles. The 2-(4-tert-butylphenyl)-8-(piperazin-1-yl)imidazo[1,2-a]pyridine ring system was shown to have nanomolar binding potency at the human and rat GnRH receptors as well as functional antagonism in vitro. Additional structure–activity relationships within this series are reported along with a pharmacokinetic comparison to the benzimidazole-based lead molecule.     

Binding properties of solubilized gonadotropin-releasing hormone receptor: role of carboxylic groups

The interaction of 125I-buserelin, a superactive agonist of gonadotropin-releasing hormone (GnRH), with solubilized GnRH receptor was studied. The highest specific binding of 125I-buserelin to solubilized GnRH receptor is evident at 4 degrees C, and equilibrium is reached after 2 h of incubation. The soluble receptor retained 100% of the original binding activity when kept at 4 or 22 degrees C for 60 min. Mono- and divalent cations inhibited, in a concentration-dependent manner, the binding of 125I-buserelin to solubilized GnRH receptor. Monovalent cations require higher concentrations than divalent cations to inhibit the binding. Since the order of potency within the divalent cations was identical with that of their association constants to dicarboxylic compounds, it is suggested that there are at least two carboxylic groups of the receptor that participate in the binding of the hormone. The carboxyl groups of sialic acid residues are not absolutely required for GnRH binding since the binding of 125I-buserelin to solubilized GnRH receptor was only slightly affected by pretreatment with neuraminidase and wheat germ agglutinin. The finding that polylysines stimulate luteinizing hormone (LH) release from pituitary cell cultures with the same efficacy as GnRH suggests that simple charge interactions can induce LH release. According to these results, we propose that the driving force for the formation of the hormone-receptor complex is an ionic interaction between the positively charged amino acid arginine in position 8 and the carboxyl groups in the binding site.     

Novel cyclic azo-bridged analogs of gonadotropin-releasing hormone.

Five linear analogs of GnRH containing a p-aminophenylalanine (Pap) residue in their sequence and their six corresponding azo-bridged cyclic derivatives were synthesized. The precyclic peptides were prepared on solid-support, while azo-cyclization was performed in solution by diazotization of the p-aminophenylalanine residue followed by intramolecular coupling of the formed diazo salt with either tyrosine or histidine side chains present in the sequence. All peptides were examined for their binding ability to the GnRH receptor expressed on rat pituitary membranes and for their LH-release activity from dispersed rat pituitary cells. Linear analogs 1 i.e [Pap(5)] GnRH and 3, i.e. [Tyr(3), Pap(5)] GnRH, were found to bind to the GnRH receptors only slightly less avidly than native GnRH. Their cyclization, however, led to a marked reduction in the binding capacity, i.e. from IC(50) of 10(-9) M to the 10(-7) M range, and in biopotency, i.e. LH-release. All other linear and cyclic peptides were found to bind selectively to the GnRH receptor only in the low microM range. Only peptide 1 was found comparable to native GnRH in respect to LH-release activity and thus may potentially be a good agonist of the parent peptide. Peptides 1-4, the most potent GnRH receptor binders, were examined for their conformational properties using CD. Cyclic-azo peptides 2 and 4 were further evaluated by NMR spectroscopy in solution combined with molecular modeling. The structural information obtained explains in part the GnRH-like biological activity observed.     

Identification and initial structure-activity relationships of a novel non-peptide quinolone GnRH receptor antagonist.

Screening of the Merck sample collection for non-peptide compounds with binding affinity for the rat GnRH receptor led to the identification of the substituted quinolone (1) as a lead compound in the search for a non-peptide GnRH receptor antagonist. Substantial improvements in potency (approximately 300 fold) were achieved by addition of an alkyl amine at the 4-position, a 3,5-dimethylphenyl group at the 3-position and 6-nitro-7-chloro-substitution of the 1 H-quinolone core.     

Hormonal regulation of gonadotropin-releasing hormone receptors and messenger RNA activity in ovine pituitary culture

Previous studies demonstrate that gonadotroph responsiveness to GnRH, GnRH binding, and the apparent number of GnRH receptors are all increased by 17 beta-estradiol (E) or inhibin (IN) in ovine pituitary cultures. Progesterone (P) attenuates these effects. To explore differences between the effects of IN and E on GnRH binding, a detailed time-course study was performed. The results indicate that after 48 h, IN had a greater effect on binding of a GnRH agonist (5-fold increase) than E (3-fold increase), but was slower to act initially. A combined treatment of IN and E gave a partially additive effect at 48 h (6.5-fold increase). The mechanism of receptor regulation in this system is not known, but could involve synthesis, recycling, or modification of GnRH receptors. To investigate the contribution of altered receptor biosynthesis to the regulation of receptor levels, a functional Xenopus oocyte-based assay for GnRH receptor mRNA activity was employed. After 48 h of treatment, IN or E each led to a 7- to 8-fold increase in GnRH receptor mRNA activity. Treatment with both hormones led to a 19-fold increase. The increase in mRNA activity induced by either hormone was greatly attenuated by P. Modulation of GnRH receptor mRNA levels suggests that IN, E, and P regulate responsiveness to GnRH in the ovine pituitary at least in part by altering de novo synthesis of GnRH receptors. The differing time courses of action, as assayed by GnRH binding, and the additivity of effects at the mRNA level suggest that IN and E alter mRNA levels via different mechanisms.     

Functional relationship between receptor binding and biological activity for analogs of mammalian and salmon gonadotropin-releasing hormones in the pituitary of goldfish (Carassius auratus)

The relationship between gonadotropin-releasing hormone (GnRH) receptor binding and biological activity in the goldfish pituitary for mammalian and salmon GnRH (sGnRH) analogs with structural modification at the C terminus involving replacement of glycine amide with an alkyl amine and replacement of the Gly6 residue with D amino acids was examined. The GnRH receptor binding data were analyzed with a computerized curve-fitting program (LIGAND) for a single as well as two classes of binding sites; analysis based on one site fit estimated binding affinity and capacity for one class of binding site, and analysis based on two-site fit estimated binding affinity and capacity for two classes of binding sites (high-affinity/low-capacity and low-affinity/high-capacity binding sites). The estimated receptor affinity values were then used to determine the correlation between binding affinity and gonadotropin (GTH)-release potency in vitro. The highest correlation between biological activity and receptor binding affinity was obtained for the high-affinity/low-capacity binding sites and GnRH analogs containing Trp7 and Leu8 residues (i.e., the salmon GnRH structural format) (R = 0.940 +/- 0.150). For the same group of GnRH analogs, there was no significant correlation between the relative GTH-release potency and binding affinity of the low-affinity/high-capacity sites (R = 0.159 +/- 0.434), or that obtained from a one-site fit (R = 0.198 +/- 0.431). Similarly, for mammalian GnRH analogs, significant correlation between binding affinity and biological activity (R = 0.406 +/- 0.049) was only obtained for the high-affinity sites, although the degree of correlation was significantly lower than that obtained for salmon GnRH analogs. The present findings provide strong support for the hypothesis that high-affinity GnRH receptors are involved in the control of GTH release in the goldfish pituitary. In addition, the results demonstrate clearly that the presence of Trp7, Leu8 residues in salmon GnRH molecule, a native peptide in goldfish, is important for recognition of the ligand by the GnRH receptors in the goldfish pituitary, and that structural modifications at positions 6 and 10 in this peptide can increase receptor binding affinity and biological activity at the pituitary level. The most active sGnRH analog identified to date is [D-Arg6, Pro9-NEt]-sGnRH.     

Two mutations in extracellular loop 2 of the human GnRH receptor convert an antagonist to an agonist

GnRH regulates the reproductive system through cognate G protein-coupled receptors in vertebrates. Certain GnRH analogs that are antagonists at mammalian receptors behave as agonists at Xenopus laevis and chicken receptors. This phenomenon provides the opportunity to elucidate interactions and the mechanism underlying receptor activation. A D-Lys(iPr) in position 6 of the mammalian GnRH receptor antagonist is required for this agonist activity (inositol phosphate production) in the chicken and X. laevis GnRH receptors. Chimeric receptors, in which extracellular loop domains of the human GnRH receptor were substituted with the equivalent domains of the X. laevis GnRH receptor, identified extracellular loop 2 as the determinant for agonist activity of one of the mammalian antagonists: antagonist 135-18. Site-directed mutagenesis of nine nonconserved residues in the C-terminal domain of extracellular loop 2 of the human GnRH receptor showed that a minimum of two mutations (Val(5.24(197))Ala and Trp(5.32(205))His) is needed in this region for agonist activity of antagonist 135-18. Agonist activity of antagonist 135-18 was markedly decreased by low pH (<7.0) compared with GnRH agonists. These findings indicate that D-Lys(iPr)(6) forms a charge-supported hydrogen bond with His(5.32(205)) to stabilize the receptor in the active conformation. This discovery highlights the importance of EL-2 in ligand binding and receptor activation in G protein-coupled receptors.     

Gonadotropin releasing hormone activation is mediated by dimerization of occupied receptors

A dinitrophenyl (DNP)-derivative of a gonadotropin releasing hormone (GnRH) antagonist was prepared by chemical modification of the epsilon amino group in position 6 of [D-pGlu1,D-Phe2,D-Trp3,D-Lys6]GnRH with 1-fluoro-2, 4-dinitrobenzene. The DNP-antagonist D-pGlu-D-Phe-D-Trp-Ser-Tyr-D-Lys(N epsilon-DNP)-Leu-Arg-Pro-Gly-NH2, retained high affinity binding to the GnRH receptor of pituitary membrane preparations and exhibited antagonistic activity when assayed in cultured pituitary cells. Both antibodies against DNP and their Fab fragments were able to bind the DNP-antagonist. However, only the addition of bivalent antibodies (and not the Fab fragments) converted the DNP-antagonist to an agonist. These results suggest that divalency is a critical factor in GnRH action.     

Binding of agonist but not antagonist leads to fluorescence resonance energy transfer between intrinsically fluorescent gonadotropin-releasing hormone receptors

We have used spot fluorescence photobleaching recovery methods to measure the lateral diffusion of GnRH receptor (GnRHR) fused at its C terminus to green fluorescent protein (GFP) after binding of either GnRH agonists or antagonist. Before ligand binding, GnRHR-GFP exhibited fast rates of lateral diffusion (D = 18 +/- 2.8 x 10(-10)cm2 x sec(-1)) and high values for fractional fluorescence recovery (%R) after photobleaching (73 +/- 1%). Increasing concentrations of agonists, GnRH or D-Ala6-GnRH, caused a dose-dependent slowing of receptor lateral diffusion as well as a decreased fraction of mobile receptors. Increasing concentrations of the GnRH antagonist Antide slowed the rate of receptor diffusion but had no effect on the fraction of mobile receptors, which remained high. To determine whether the decrease in %R caused by GnRH agonists was due, in part, to increased receptor self-association, we measured the fluorescence resonance energy transfer efficiency between GnRHR-GFP and yellow fluorescent protein-GNRHR: There was no energy transfer between GnRHR on untreated cells. Treatment of cells with GnRH agonists led to a concentration-dependent increase in the energy transfer between GnRH receptors to a maximum value of 16 +/- 1%. There was no significant energy transfer between GnRH receptors on cells treated with Antide, even at a concentration of 100 nM. These data provide direct evidence that, before binding of ligand, GnRHR exists as an isolated receptor and that binding of GnRH agonists, but not antagonist, leads to formation of large complexes that exhibit slow diffusion and contain receptors that are self-associated.     

Evolution of constrained gonadotropin-releasing hormone ligand conformation and receptor selectivity

Gonadotropin-releasing hormone (GnRH) is the central regulator of reproduction in vertebrates. GnRHs have recently been identified in protochordates and retain the conserved N- and C-terminal domains involved in receptor binding and activation. GnRHs of the jawed vertebrates have a central achiral amino acid (glycine) that favors a type II' beta-turn such that the N- and C-terminal domains are closely apposed in binding the GnRH receptor. However, protochordate GnRHs have a chiral amino acid in this position, suggesting that they bind their receptors in a more extended form. We demonstrate here that a protochordate GnRH receptor does not distinguish GnRHs with achiral or chiral amino acids, whereas GnRH receptors of jawed vertebrates are highly selective for GnRHs with the central achiral glycine. The poor activity of the protochordate GnRH was increased >10-fold at vertebrate receptors by replacement of the chiral amino acid with glycine or a d-amino acid, which favor the type II' beta-turn. Structural analysis of the GnRHs using ion mobility-mass spectrometry and molecular modeling showed a greater propensity for a type II' beta-turn in GnRHs with glycine or a d-amino acid, which correlates with binding affinity at vertebrate receptors. These findings indicate that the substitution of glycine for a chiral amino acid in GnRH during evolution allows a more constrained conformation for receptor binding and that this subtle single amino acid substitution in a site remote from the ligand functional domains has marked effects on its structure and activity.     

Gonadotropin releasing-hormone receptors: photoaffinity labeling with an antagonist

A photoaffinity antagonist of gonadotropin releasing hormone (GnRH), D pGlu-D-Phe-D-Trp-Ser-D-Lys6(N epsilon-azidobenzoyl)-Leu-Arg-Pro-Gly-NH2 (photoaffinity antagonist) was prepared by reacting [D-pGlu1, D-Phe2, D-Trp3, D-Lys6]GnRH with the N-hydroxysuccinimide ester of 4-azidobenzoic acid. The analog appeared homogeneous when analyzed by thin-layer chromatography and its photoreactivity was demonstrated by spectral changes when exposed to light. The photoaffinity antagonist retained high affinity binding to the GnRH receptor of pituitary membrane preparations and exhibited antagonistic activity when assayed in vitro in whole pituitaries. Pituitary membrane preparations were incubated with the radioactive photoaffinity GnRH antagonist and irradiated with light. Sodium dodecyl sulfate gel electrophoresis after solubilization and reduction showed the specific labeling of a single specific protein with an apparent molecular weight of 60,000 daltons. These results indicate that GnRH agonists and antagonists bind to the same receptor.     

Conserved amino acid residues that are important for ligand binding in the type I gonadotropin-releasing hormone (GnRH) receptor are required for high potency of GnRH II at the type II GnRH receptor

GnRH I regulates reproduction. A second form, designated GnRH II, selectively binds type II GnRH receptors. Amino acids of the type I GnRH receptor required for binding of GnRH I (Asp2.61(98), Asn2.65(102), and Lys3.32(121)) are conserved in the type II GnRH receptor, but their roles in receptor function are unknown. We have delineated their functions using mutagenesis, signaling and binding assays, immunoblotting, and computational modeling. Mutating Asp2.61(97) to Glu or Ala, Asn2.65(101) to Ala, or Lys3.32(120) to Gln decreased potency of GnRH II-stimulated inositol phosphate production. Consistent with proposed roles in ligand recognition, mutations eliminated measurable binding of GnRH II, whereas expression of mutant receptors was not decreased. In detailed analysis of how these residues affect ligand-dependent signaling, [Trp2]-GnRH I showed lesser decreases in potency than GnRH I at the Asp2.61(97)Glu mutant. In contrast, [Trp2]-GnRH II showed the same loss of potency as GnRH II at this mutant. This suggests that Asp2.61(97) contributes to recognition of His2 of GnRH I, but not of GnRH II. GnRH II showed a large decrease in potency at the Asn2.65(101)Ala mutant compared with analogs lacking the CO group of Gly10NH2. This suggests that Asn2.65(101) recognizes Gly10NH2 of GnRH II. GnRH agonists showed large decreases in potency at the Lys3.32(120)Gln mutant, but antagonist activity was unaffected. This suggests that Lys3.32(120) recognizes agonists, but not antagonists, as in the type I receptor. These data indicate that roles of conserved residues are similar, but not identical, in the type I and II GnRH receptors.     

Synthesis and structure-activity relationships of thieno[2,3-d]pyrimidine-2,4-dione derivatives as potent GnRH receptor antagonists

The synthesis and SAR studies of thieno[2,3-d]pyrimidine-2,4-diones as human GnRH receptor antagonists to treat reproductive diseases are discussed. It was found that the 2-(2-pyridyl)ethyl group on the 5-aminomethyl functionality of the core structure was a key feature for good receptor binding activity. SAR study of the 6-(4-aminophenyl) group suggests that hydrophobic substituents were preferred. The best compound from this series had binding affinity (K(i)) of 0.4 nM to the human GnRH receptor.     

Enhanced biological activity of dimeric gonadotropin releasing hormone

The biological activities of a series of dimeric analogs of des-Gly10-[D-Lys6]GnRH-NHEt cross-linked at Lys6 by malonic acid and elongated by Gly, i.e., HO-Glyn-CO-CH2-CO-Glyn-OH (n = 0, 1, 2), were analyzed in vitro and in vivo. All three dimeric analogs displayed increased activity in receptor binding and in LH release assays than the original monomer, and dimer Ib (n = 1) showed the highest potency in vitro. This compound also showed the highest activity in the in vivo postcoital assay, in which GnRH agonist potency is measured by inhibition of pregnancy. These results indicate that GnRH receptor activation is substantially enhanced by dimerization of the agonist ligand.     

2-(3,5-Dimethylphenyl)tryptamine derivatives that bind to the GnRH receptor

A series of 2-(3,5-dimethylphenyl)tryptamine derivatives was prepared and evaluated on a rat gonadotropin releasing hormone receptor assay. Some para-substituents on the 4-phenylbutyl side chain attached to the tryptamine nitrogen led to compounds with potent GnRH receptor binding. The study has helped define structural requirements for GnRH receptor binding for the 2-aryltryptamine GnRH antagonists.