Human GnRH Receptor Research Articles

Novel non-peptide uracil-derived human gonadotropin-releasing hormone receptor antagonists

Gonadotropin-releasing hormone (GnRH) is the main regulator of the reproductive system, acting on gonadotropic cells by binding to the GnRH1 receptor (GnRH1R). Traditionally, therapies targeting this receptor have relied on peptide modulators, which required subcutaneous or intramuscular injections. Due to the limitations of the parenteral administrations, there is a growing interest in developing oral small molecule modulators of GnRH1R as more convenient therapeutic alternatives.In this study, we examined the potential of chemically modifying elagolix, the first approved non-peptide, orally active GnRH1R antagonist, to increase its atropisomeric properties by introducing new moieties. We designed and synthesized the thio-uracil (1) and cytosine (2) derivatives of elagolix, both demonstrating GnRH1R antagonistic activities, with EC50 values of 39 and 110 nM, respectively. The atropisomers of 1 and 2 were efficiently separated using silica gel chromatography, and extensive NMR investigation, supported by Density Functional Theory (DFT) calculations, allowed us to define their conformations and rotational barriers. Docking and Molecular Dynamics (MD) studies revealed that 1 and 2 bind to GnRH1R with ΔG values comparable to elagolix, but through distinct binding modes. These results highlight the potential of non-peptide modulators to effectively modulate GnRH1R activity and pave the way for developing novel modulators.

Identification of Environmental Compounds That May Trigger Early Female Puberty by Activating Human GnRHR and KISS1R.

There has been an alarming trend toward earlier puberty in girls, suggesting the influence of an environmental factor(s). As the reactivation of the reproductive axis during puberty is thought to be mediated by the hypothalamic neuropeptides kisspeptin and gonadotropin-releasing hormone (GnRH), we asked whether an environmental compound might activate the kisspeptin (KISS1R) or GnRH receptor (GnRHR). We used GnRHR or KISS1R-expressing HEK293 cells to screen the Tox21 10K compound library, a compendium of pharmaceuticals and environmental compounds, for GnRHR and KISS1R activation. Agonists were identified using Ca2+ flux and phosphorylated extracellularly regulated kinase (p-ERK) detection assays. Follow-up studies included measurement of genes known to be upregulated upon receptor activation using relevant murine or human cell lines and molecular docking simulation. Musk ambrette was identified as a KISS1R agonist, and treatment with musk ambrette led to increased expression of Gnrh1 in murine and human hypothalamic cells and expansion of GnRH neuronal area in developing zebrafish larvae. Molecular docking demonstrated that musk ambrette interacts with the His309, Gln122, and Gln123 residues of the KISS1R. A group of cholinergic agonists with structures similar to methacholine was identified as GnRHR agonists. When applied to murine gonadotrope cells, these agonists upregulated Fos, Jun, and/or Egr1. Molecular docking revealed a potential interaction between GnRHR and 5 agonists, with Asn305 constituting the most conservative GnRHR binding site. In summary, using a Tox21 10K compound library screen combined with cellular, molecular, and structural biology techniques, we have identified novel environmental agents that may activate the human KISS1R or GnRHR.

Disruption of gonadotropin hormone biosynthesis by parabens: A potential development and reproduction-associated adverse outcome pathway

Parabens are widely used as antibacterial preservatives in foods and personal care products. The knowledge about the modes of toxic action of parabens on development and reproduction remain very limited. The present study attempted to establish a development and reproduction-associated adverse outcome pathway (AOP) by evaluating the effects of methylparaben (MP), ethylparaben (EP), propylparaben (PP) and butylparaben (BP) on the biosynthesis of gonadotropins, which are key hormones for development and reproduction. MP and BP significantly upregulated the mRNA and protein levels of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in pituitary gonadotropic cells in a concentration-dependent manner. Activation of gonadotropin-releasing hormone receptor (GnRHR) was required for gonadotropin biosynthesis induced by BP, but not MP. Molecular docking data further demonstrated the higher binding efficiency of BP to human GnRHR than that of MP, suggesting GnRHR as a potential molecular initiative event (MIE) for BP-induced gonadotropin production. L-type voltage-gated calcium channels (VGCCs) were found to be another candidate for MIE in gonadotropic cells response to both MP and BP exposure. The calcium-dependent activation of extracellular signal-regulated kinase 1 (ERK1) and ERK2 was subsequently required for MP- and BP-induced activation of GnRHR and L-type VGCCs pathways. In summary, MP and BP promoted gonadotropin biosynthesis through their interactions with cellular macromolecules GnRHR, L-type VGCCs, and subsequent key event ERK1/2. This is the first study to report the direct interference of parabens with gonadotropin biosynthesis and establish a potential AOP based on pathway-specific mechanism, which contributes to the effective screening of environmental chemicals with developmental and reproductive health risks.

A corazonin G protein-coupled receptor gene in the tick Ixodes scapularis yields two splice variants, each coding for a specific corazonin receptor

We have identified a corazonin G protein-coupled receptor (GPCR) gene in the tick Ixodes scapularis, which likely plays a central role in the physiology and behavior of this ectoparasite. This receptor gene is unusually large (1.133 Mb) and yields two corazonin (CRZ) receptor splice variants, where nearly half of the coding regions are exchanged: CRZ-Ra (containing exon 2, exon 3, and exon 4 of the gene) and CRZ-Rb (containing exon 1, exon 3, and exon 4 of the gene). CRZ-Ra codes for a GPCR with a canonical DRF sequence at the border of the third transmembrane helix and the second intracellular loop. The positively-charged R residue from the DRF sequence is important for coupling of G proteins after activation of a GPCR. CRZ-Rb, in contrast, codes for a GPCR with an unusual DQL sequence at this position, still retaining a negatively-charged D residue, but lacking a positively-charged R residue, suggesting different G protein coupling. Another difference between the two splice variants is that exon 2 from CRZ-Ra codes for an N-terminal signal sequence. Normally, GPCRs do not have N-terminal signal sequences, although a few mammalian GPCRs have. In the tick CRZ-Ra, the signal sequence probably assists with inserting the receptor correctly into the RER membrane. We stably transfected Chinese Hamster Ovary cells with each of the two splice variants and carried out bioluminescence bioassays that also included the use of the human promiscuous G protein G16. CRZ-Ra turned out to be selective for I. scapularis corazonin (EC50 = 10−8 M) and could not be activated by related neuropeptides like adipokinetic hormone (AKH) and AKH/corazonin-related peptide (ACP). Similarly, also CRZ-Rb could only be activated by corazonin, although about 4-fold higher concentrations were needed to activate it (EC50 = 4 x 10−8 M). The genomic organization of the tick corazonin GPCR gene is similar to that of the insect AKH and ACP receptor genes. This similar genomic organization can also be found in the human gonadotropin-releasing hormone (GnRH) receptor gene, confirming previous conclusions that the corazonin, AKH, and ACP receptor genes are the true arthropod orthologues of the human GnRH receptor gene.

Anti-GnRH Receptor Monoclonal Antibody, GHR106 is First-inClass GnRH Antagonist

During the last decade, a monoclonal antibody, GHR106 was generated and characterized extensively biologically and immunologically. This antibody targets specific human pan cancer marker and is being evaluated for potential therapeutic applications in cancer immunotherapy and fertility regulations. GHR106 was generated against N1-29 oligopeptide located in the extracellular domains of human GnRH receptor found either in the anterior pituitary or in most of cancer cells. In vitro culture of cancer cells revealed that this antibody can induce apoptosis of cancer cells following 24-48 hours incubations. Anti-tumor activities of GHR106 were evaluated by typical nude mouse experiments in which the effective volume reduction of implanted tumor was observed. Humanized forms of GHR106 were made available in CAR (chimeric antigen receptor) T-cell constructs. GHR106 was shown separately to induce cytotoxic killings of cancer cells in vitro by releasing cytokines following incubations of tumor cells with CAR-T cell constructs. In addition, GHR106 also acts as GnRH antagonist by a specific targeting to pituitary GnRH receptor for reversible suppressions of reproductive hormones. These were demonstrated in “Proof of Concept” rabbit experiments. A single subcutaneous injection with 1-3mg/kg of GHR106 to rabbits of either sex could result in 60 to 90% reductions of gonadotropins, estrogen and/or testosterone over a period of one to two weeks. Based on these preclinical assessments, it can be concluded that GHR106 is restricted in tissue expressions and suitable for cancer immunotherapy. It can also act as long-acting GnRH antagonist to target specifically on GnRH receptor in anterior pituitary for numerous gynecological diseases including ovulation inhibition in IVF/ART, endometriosis-premenstrual syndrome, precocious puberty, uterine fibroids and/or polycystic ovarian syndrome.

“Proof of Concept” Rabbit Experiments Demonstrating GHR106 (hIgG4) as GnRH Antagonist

“Proof of Concept” (P-o-C) experiments in rabbits were conducted to demonstrate reversible suppressions of serum reproductive hormones (LH, testosterone or E2) upon a single injection of a humanized monoclonal antibody against human GnRH receptor, GHR106 (hIgG4). In the case of a male rabbit, a single subcutaneous injection of either 1 mg/kg or 3 mg/kg of the antibody was shown to reduce concurrently the serum LH and testosterone concentrations by 60 to 80% for a period of one week or longer. The reproductive hormone levels returned to the normal range after next two weeks. Similarly, the suppressions of LH and estradiol (E2) were also observed within two days upon a single injection of GHR106 (hIgG4) in the female. The E2 levels can decrease to 30-60% of the normal over a period of one week followed by full recovery of both hormones during the next 5-10 days. P-o-C experiments were expanded to a scale of more than 30 rabbits with identical protocols to those of a single rabbit, in addition to the comparisons with those of the negative control. Reversible suppressions of reproductive hormones were observed consistently with all rabbits in the antibody injection groups, and were shown to be statistically significant. The circulation half-life of GHR106 (hIgG4) in rabbits was estimated to be 4 ± 1 day. Therefore, the results of this study revealed that GHR106(hIgG4) can be used as antibody-based long- acting GnRH antagonist with similar biological actions to those of decapeptide GnRH antagonist such as Cetrorelix or Antide. The relatively long half-life of the antibody-based GnRH antagonist, however, may be more beneficial to the treatment of gynecological and fertility-related diseases which may be caused by modulations of human GnRH receptor in humans, and also in other animal species for fertility controls.

Natural autoantibodies to the gonadotropin-releasing hormone receptor in polycystic ovarian syndrome.

ContextPolycystic ovarian syndrome (PCOS) is a complex disease with different subtypes and unclear etiology. Among the frequent comorbidities are autoimmune diseases, suggesting that autoantibodies (aAb) may be involved in PCOS pathogenesis.ObjectiveAs the gonadal axis often is dysregulated, we tested the hypothesis that aAb to the gonadotropin-releasing hormone receptor (GnRH-R) are of diagnostic value in PCOS.DesignAn in vitro assay for quantifying aAb to the GnRH-R (GnRH-R-aAb) was established by using a recombinant fusion protein of full-length human GnRH-R and firefly luciferase. A commercial rabbit antiserum to human GnRH-R was used for standardization. Serum samples of control subjects and different cohorts of European PCOS patients (n = 1051) were analyzed.ResultsThe novel GnRH-R-aAb assay was sensitive, and signals were linear on dilution when tested with the commercial GnRH-R antiserum. Natural GnRH-R-aAb were detected in one control (0.25%) and two PCOS samples (0.31%), and 12 samples were slightly above the threshold of positivity. The identification of samples with positive GnRH-R-aAb was reproducible and the signals showed no matrix interferences.ConclusionNatural GnRH-R-aAb are present in a very small fraction of adult control and PCOS subjects of European decent. Our results do not support the hypothesis that the GnRH-R constitutes a relevant autoantigen in PCOS.

Structure of the human gonadotropin-releasing hormone receptor GnRH1R reveals an unusual ligand binding mode

Gonadotrophin-releasing hormone (GnRH), also known as luteinizing hormone-releasing hormone, is the main regulator of the reproductive system, acting on gonadotropic cells by binding to the GnRH1 receptor (GnRH1R). The GnRH-GnRH1R system is a promising therapeutic target for maintaining reproductive function; to date, a number of ligands targeting GnRH1R for disease treatment are available on the market. Here, we report the crystal structure of GnRH1R bound to the small-molecule drug elagolix at 2.8 Å resolution. The structure reveals an interesting N-terminus that could co-occupy the enlarged orthosteric binding site together with elagolix. The unusual ligand binding mode was further investigated by structural analyses, functional assays and molecular docking studies. On the other hand, because of the unique characteristic of lacking a cytoplasmic C-terminal helix, GnRH1R exhibits different microswitch structural features from other class A GPCRs. In summary, this study provides insight into the ligand binding mode of GnRH1R and offers an atomic framework for rational drug design.

Discovery and Characterization of BAY 1214784, an Orally Available Spiroindoline Derivative Acting as a Potent and Selective Antagonist of the Human Gonadotropin-Releasing Hormone Receptor as Proven in a First-In-Human Study in Postmenopausal Women.

The growth of uterine fibroids is sex hormone-dependent and commonly associated with highly incapacitating symptoms. Most treatment options consist of the control of these hormonal effects, ultimately blocking proliferative estrogen signaling (i.e., oral contraceptives/antagonization of human gonadotropin-releasing hormone receptor [hGnRH-R] activity). Full hGnRH-R blockade, however, results in menopausal symptoms and affects bone mineralization, thus limiting treatment duration or demanding estrogen add-back approaches. To overcome such issues, we aimed to identify novel, small-molecule hGnRH-R antagonists. This led to the discovery of compound BAY 1214784, an orally available, potent, and selective hGnRH-R antagonist. Altering the geminal dimethylindoline core of the initial hit compound to a spiroindoline system significantly improved GnRH-R antagonist potencies across several species, mandatory for a successful compound optimization in vivo. In a first-in-human study in postmenopausal women, once daily treatment with BAY 1214784 effectively lowered plasma luteinizing hormone levels by up to 49%, at the same time being associated with low pharmacokinetic variability and good tolerability.

Treatment of Breast Cancer With Gonadotropin-Releasing Hormone Analogs.

Although significant progress has been made in the implementation of new breast cancer treatments over the last three decades, this neoplasm annually continues to show high worldwide rates of morbidity and mortality. In consequence, the search for novel therapies with greater effectiveness and specificity has not come to a stop. Among the alternative therapeutic targets, the human gonadotropin-releasing hormone type I and type II (hGnRH-I and hGnRH–II, respectively) and its receptor, the human gonadotropin-releasing hormone receptor type I (hGnRHR-I), have shown to be powerful therapeutic targets to decrease the adverse effects of this disease. In the present review, we describe how the administration of GnRH analogs is able to reduce circulating concentrations of estrogen in premenopausal women through their action on the hypothalamus–pituitary–ovarian axis, consequently reducing the growth of breast tumors and disease recurrence. Also, it has been mentioned that, regardless of the suppression of synthesis and secretion of ovarian steroids, GnRH agonists exert direct anticancer action, such as the reduction of tumor growth and cell invasion. In addition, we discuss the effects on breast cancer of the hGnRH-I and hGnRH-II agonist and antagonist, non-peptide GnRH antagonists, and cytotoxic analogs of GnRH and their implication as novel adjuvant therapies as antitumor agents for reducing the adverse effects of breast cancer. In conclusion, we suggest that the hGnRH/hGnRHR system is a promising target for pharmaceutical development in the treatment of breast cancer, especially for the treatment of advanced states of this disease.

Evaluation of 111In-Labeled GnRH-I Tracer for SPECT Tumor Imaging

Total synthesis, quality control, and preclinical evaluation of [111In]-DOTA-Triptorelin ([111In]-DOTA-TRP) for diagnostic SPECT imaging have been made. For 111In labeling, the peptide synthesis was followed by conjugation with DOTA using pSCN-Bn-DOTA. The synthesized conjugate prepared at optimized conditions was purified with a reversed-phase semipreparative column using gradient of water: acetonitrile mixture. The molecular mass was confirmed by mass spectroscopy. The conjugated Triptorelin was labeled with 500–550 MBq of 111In chloride (in 0.2 M HCl). The radiochemical purity of the product prepared under optimized conditions was about 98% (RTLC) and >95% (HPLC). The serum stability of the tracer was determined up to 24 h. The 111In-peptide chelate exhibits high stability. The binding affinity of Triptorelin peptide was determined in a binding assay for both human and rat GnRH receptors. For in vivo studies, 111In-peptide was injected intravenously via the tail vein to rats. In vitro radioligand binding assays were performed with GnRHR-expressing human cell lines using [125I]Triptorelin as the standard radioligand. The receptor affinity of the new radioligand was IC50 = 0.35 ± 0.08 nM vs. 0.13±0.01 nM for Triptorelin, and the internalization efficiency was 3.4 ± 0.7% at 1 h and 11.8 ± 1.9% at 4 h.

A New Monoclonal Antibody-Based Biosimilar GnRH Antagonist

GHR106 is a monoclonal antibody generated against an oligopeptide corresponding to that in the extra cellular domains of human GnRH receptor. The humanized forms of GHR106 exhibit almost identical biological properties to those of decapeptide GnRH antagonists such as Antide and Cetrorelix. The Fc region of humanized GHR106 has been replaced with IgG4 subtype to eliminate activities of effector function. Therefore, the newly humanized GHR106-IgG4Fc can be used clinically as biosimilar GnRH antagonists of higher molecular size, and longer half-life (hrs. vs. days) for therapeutic treatments of fertility-related health conditions without complications arising from the effector functions of immunoglobulins.

Preparation of a radiolabeled GnRH-I analogue derivative with 111 In as a new anti-proliferative agent.

The new GnRH-Ιanalogue developed in this paper was based on the D-Trp6 -GnRH-Ι-scaffold, and its potency was increased by the replacement Gly-NH2 by NH-NH2 binding to the Gly at position 10. Triptorelin-Hydrazide analogue was synthesized using solid phase. For 111 In labeling, synthesized peptide was followed by conjugation with DOTA using pSCN-Bn-DOTA. The conjugated Triptorelin-Hydrazide was labeled with 500-550MBq of 111 In-chloride (in 0.2M HCl). At optimized conditions after labeling, radio-chromatography showed radiochemical purity of approximately equal to 98% (RTLC) and greater than 95% (HPLC). The serum stability of the tracer was determined up to 24hr. Binding affinities of Triptorelin-Hydrazide analogue were determined in a binding assay for both human and rat GnRH receptors. For in vivo studies, 111 In-peptide was injected intravenously via the tail vein into rats and significant ovaries uptake consist with reported GnRH receptor mappings. In vitro radioligand binding assays performed with GnRHR-expressing human cell lines using 125 I-Triptorelin as the standard radioligand. The quantities of internalization efficiency and receptor affinity of the new radioligand were IC50 =0.20±0.04nM vs 0.13±0.08nM for Triptorelin and internalization: 3.5±0.9% at 1hr and 12.8±1.8% at 4hr of the internal reference.

Gonadotropin-releasing hormone receptor antibody activity over time in women with polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a diagnosis of exclusion with unknown etiology. We recently identified autoantibodies (AAbs) to the second extracellular loop (ECL-2) of the gonadotropin releasing hormone receptor (GnRHR) in a majority of patients with PCOS compared to ovulatory controls in baseline serum measurements. The present assay may represent the desired serological test needed to effectively screen subjects for possible PCOS. Currently, no data exists regarding autoantibody activity over time or with changes in the hormonal milieu associated with infertility treatments, specifically controlled ovarian hyperstimulation (COH) with in vitro fertilization (IVF). To evaluate the levels of AAbs to GnRHR in the serum of eleven subjects with PCOS and infertility over time and observe for changes relative to hormonal fluctuations occurring over the course of infertility treatment. Sera samples from eleven women with PCOS and infertility drawn over multiple time points (0: not on medications, 1: down-regulation phase of an IVF cycle (oral contraceptives or lupron), 2: IVF stimulation day five, 3: day of hCG trigger, and 4: day of pregnancy test) during infertility treatments were assessed for GnRHR-AAbs. The de-identified samples were screened by ELISA for GnRHR-AAbs using a synthetic 28-mer peptide (LifeTein, Somerset, NJ) from the ECL2 of human GnRHR as coating antigen. Optical density (OD) values were read at 405 nm at 60 minutes. Covariates evaluated include age, body mass index (BMI), anti-Mullerian hormone (AMH) level, antral follicle count (AFC), IVF protocol (GnRH antagonist or agonist), estradiol level, and hormonal down-regulation during IVF preparation. Statistical analyses were performed using generalized estimating equations within a generalized linear modeling framework to account for the repeated measures within each patient. AAb levels did not differ significantly by patient age (range 25-40 years; p=0.85) or BMI (range 19.3-41.8; p=0.89). After taking into account inter-patient variation and inter-time point variation, there was an association between GnRHR-AAb levels and estradiol levels (higher AAb level with lower estradiol; p<0.0001). Also, the GnRH antagonist protocol was associated with lower AAb levels (p=0.019). Most inter-patient variation could not be attributed to any of the patient characteristics. However, after taking into account inter-time point variation only, inter-patient variation in AAb levels was partially attributable to an interaction between BMI and AMH (p<0.0001) but not to age (p=0.70) or AFC (p=0.97). There was significant intra-patient variation in AAb levels at both baseline (time point 0, not on meds, p=0.042) and during the down-regulation phase (time point 1, p<0.0001) that could not be attributed to any of the measured covariates. Our initial investigations have shown that most patients in our study with PCOS have activating AAbs to GnRHR compared to ovulatory controls. Our pilot study has demonstrated that there is inter-patient variation of GnRHR-AAb levels on no medications and while in the down-regulation phase in preparation for IVF. Additionally, the fluctuation in AAb levels with estradiol levels in GnRH antagonist protocol would suggest an association between the two clinical time points and potentially a relationship between estradiol and the GnRHR-AAbs.

Synthesis and biological evaluation of 3-(2-aminoethyl) uracil derivatives as gonadotropin-releasing hormone (GnRH) receptor antagonists

We investigated a series of uracil analogues by introducing various substituents on the phenyl ring of the N-3 aminoethyl side chain and evaluated their antagonistic activity against human gonadotropin-releasing hormone (GnRH) receptors. Analogues with substituents at the ortho or meta position demonstrated potent in vitro antagonistic activity. Specifically, the introduction of a 2-OMe group enhanced nuclear factor of activated T-cells (NFAT) inhibition up to 6-fold compared to the unsubstituted analogue. We identified compound 12c as a highly potent GnRH antagonist with moderate CYP inhibition. Compound 12c showed potent and prolonged LH suppression after a single dose was orally administered in castrated monkeys compared to a known antagonist, Elagolix. We believe that our SAR study offers useful insights to design GnRH antagonists as a potential treatment option for endometriosis.

Gonadotropin-Releasing Hormone (GnRH) Receptor Structure and GnRH Binding.

Gonadotropin-releasing hormone (GnRH) regulates reproduction. The human GnRH receptor lacks a cytoplasmic carboxy-terminal tail but has amino acid sequence motifs characteristic of rhodopsin-like, class A, G protein-coupled receptors (GPCRs). This review will consider how recent descriptions of X-ray crystallographic structures of GPCRs in inactive and active conformations may contribute to understanding GnRH receptor structure, mechanism of activation and ligand binding. The structures confirmed that ligands bind to variable extracellular surfaces, whereas the seven membrane-spanning α-helices convey the activation signal to the cytoplasmic receptor surface, which binds and activates heterotrimeric G proteins. Forty non-covalent interactions that bridge topologically equivalent residues in different transmembrane (TM) helices are conserved in class A GPCR structures, regardless of activation state. Conformation-independent interhelical contacts account for a conserved receptor protein structure and their importance in the GnRH receptor structure is supported by decreased expression of receptors with mutations of residues in the network. Many of the GnRH receptor mutations associated with congenital hypogonadotropic hypogonadism, including the Glu2.53(90) Lys mutation, involve amino acids that constitute the conserved network. Half of the ~250 intramolecular interactions in GPCRs differ between inactive and active structures. Conformation-specific interhelical contacts depend on amino acids changing partners during activation. Conserved inactive conformation-specific contacts prevent receptor activation by stabilizing proximity of TM helices 3 and 6 and a closed G protein-binding site. Mutations of GnRH receptor residues involved in these interactions, such as Arg3.50(139) of the DRY/S motif or Tyr7.53(323) of the N/DPxxY motif, increase or decrease receptor expression and efficiency of receptor coupling to G protein signaling, consistent with the native residues stabilizing the inactive GnRH receptor structure. Active conformation-specific interhelical contacts stabilize an open G protein-binding site. Progress in defining the GnRH-binding site has recently slowed, with evidence that Tyr6.58(290) contacts Tyr5 of GnRH, whereas other residues affect recognition of Trp3 and Gly10NH2. The surprisingly consistent observations that GnRH receptor mutations that disrupt GnRH binding have less effect on “conformationally constrained” GnRH peptides may now be explained by crystal structures of agonist-bound peptide receptors. Analysis of GPCR structures provides insight into GnRH receptor function.

Activation of human gonadotropin-releasing hormone receptor promotes down regulation of ARHGAP18 and regulates the cell invasion of MDA-MB-231 cells

The Gonadotropin-Releasing Hormone Receptor (GnRHR) is expressed mainly in the gonadotrope membrane of the adenohypophysis and its natural ligand, the Gonadotropin-Releasing Hormone (GnRH), is produced in anterior hypothalamus. Furthermore, both molecules are also present in the membrane of cells derived from other reproductive tissues such as the breast, endometrium, ovary, and prostate, as well as in tumors derived from these tissues. The functions of GnRH receptor and its hormone in malignant cells have been related with the decrease of proliferation and the invasiveness of those tumors however, little is known about the molecules associated with the signaling pathways regulated by both molecules in malignant cells. To further analyze the potential mechanisms employed by the GnRHR/GnRH system to reduce the tumorigenesis of the highly invasive breast cancer cell line MDA-MB-231, we performed microarrays experiments to evaluated changes in genes expression and validate these modifications by functional assays. We show that activation of human GnRHR is able to diminish the expression and therefore functions of the Rho GTPase-Activating Protein 18 (ARHGAP18). Decrease of this GAP following GnRHR activation, correlates to the higher of cell adhesion and also with reduction of tumor cell invasion, supporting the notion that GnRHR triggers intracellular signaling pathways that acts through ARHGAP18. On the contrary, although a decline of cellular proliferation was observed during GnRHR activation in MDA-MB-231, this was independent of ARHGAP18 showing the complex system in which is involved the signaling pathways regulated by the GnRHR/GnRH system.

Small molecule piperazinyl-benzimidazole antagonists of the gonadotropin-releasing hormone (GnRH) receptor.

In this communication, we report the synthesis and characterization of a library of small molecule antagonists of the human gonadotropin releasing hormone receptor based upon the 2-(4-tert-butylphenyl)-4-piperazinyl-benzimidazole scaffold via Cu-catalysed azide alkyne cycloaddition. Our main purpose was to find a more soluble compound based on the WAY207024 lead with nanomolar potency to inhibit the GnRH receptor. A late stage diversification by the use of click chemistry was, furthermore developed to allow for expansion of the library in future optimisations. All compounds were tested in a functional assay to determine the individual potency of inhibiting stimulation of the receptor by the endogenous agonist GnRH. In conclusion, we found that compound 8a showed improved solubility compared to WAY207024 and nanomolar affinity to GnRH receptor.

Discovery of an Orally Bioavailable Gonadotropin-Releasing Hormone Receptor Antagonist.

We developed a compound library for orally available gonadotropin-releasing hormone (GnRH) receptor antagonists that were based on a uracil scaffold. On the basis of in vitro activity and CYP inhibition profile, we selected 18a (SKI2496) for further in vivo studies. Compound 18a exhibited more selective antagonistic activity toward the human GnRH receptors over the GnRHRs in monkeys and rats, and this compound also showed inhibitory effects on GnRH-mediated signaling pathways. Pharmacokinetic and pharmacodynamic evaluations of 18a revealed improved bioavailability and superior gonadotropic suppression activity compared with Elagolix, the most clinically advanced compound. Considering that 18a exhibited highly potent and selective antagonistic activity toward the hGnRHRs along with favorable pharmacokinetic profiles, we believe that 18a may represent a promising candidate for an orally available hormonal therapy.

Brain penetrant small molecule 18F-GnRH receptor (GnRH-R) antagonists: Synthesis and preliminary positron emission tomography imaging in rats

IntroductionThe gonadotropin releasing hormone receptor (GnRH-R) has a well-described neuroendocrine function in the anterior pituitary. However, little is known about its function in the central nervous system (CNS), where it is most abundantly expressed in hippocampus and amygdala. Since peptide ligands based upon the endogenous decapetide GnRH do not pass the blood–brain-barrier, we are seeking a high-affinity small molecule GnRH-R ligand suitable for brain imaging by positron emission tomography. We have previously reported the radiosynthesis and in vitro evaluation of two novel [18F]fluorinated GnRH-R ligands belonging to the furamide class of antagonists, with molecular weight less than 500 Da. We now extend this work using palladium coupling for the synthesis of four novel radioligands, with putatively reduced polar surface area and hydrophilicity relative to the two previously described compounds, and report the uptake of these 18F-labeled compounds in brain of living rats. MethodsWe synthesized reference standards of the small molecule GnRH-R antagonists as well as mesylate precursors for 18F-labeling. The antagonists were tested for binding affinity for both human and rat GnRH-R. Serum and blood stability in vitro and in vivo were studied. Biodistribution and PET imaging studies were performed in male rats in order to assess brain penetration in vivo. ResultsA palladium coupling methodology served for the synthesis of four novel fluorinated furamide GnRH receptor antagonists with reduced heteroatomic count. Radioligand binding assays in vitro revealed subnanomolar affinity of the new fluorinated compounds for both human and rat GnRH-R. The 18F-GnRH antagonists were synthesized from the corresponding mesylate precursors in 5–15% overall radiochemical yield. The radiolabeled compounds demonstrated good in vivo stability. PET imaging with the 18F-radiotracers in naive rats showed good permeability into brain and rapid washout, but absence of discernible specific binding in vivo. ConclusionsThe novel small molecule 18F-fluorinated GnRH-R antagonist compounds show high receptor affinity in vitro, and may prove useful for quantitative autoradiographic studies in vitro. The compounds were permeable to the blood–brain barrier, but nonetheless failed to reveal significant specific binding in brain of living rats. Nonetheless, our approach may serve as a foundation for designing PET ligands suitable to image the GnRH-R distribution in brain.