Design and Synthesis of Potent Tyr(OMe)5-Gonadotropin-Releasing Hormone (GnRH) Analogues with Modifications at Positions 6, 9 and 10

Summary We have recently reported the synthesis and the conformational properties of some Gonadotropin-releasing hormone (GnRH) analogues in which the tyrosine residue at position 5 is substituted with tyrosine-O-methyl (Keramida et al., Let. Pept. Sci., 3 (1996) 257/Matsoukas et al., Eur. J. Med. Chem., 32 (1997) 927). The analogue [Tyr-(OMe)⁵]-GnRH was found to exert a lower degree of desensitization than the native GnRH peptides in terms of pituitary gonadotropin (GTH) release in goldfish. Compared to GnRH, however, [Tyr-(OMe)⁵]-GnRH exerted a lower GTH-release potency in cultured goldfish pituitary fragments, and was bound with a lower binding affinity to the rat pituitary GnRH receptors. In order to increase the potency of [Tyr-(OMe)⁵]-GnRH, we have synthesized a group of GnRH peptides containing Tyr-(OMe)⁵ in combination with other substitutions at positions 6, 9 and 10 and we have estimated their binding affinity for the rat pituitary receptors and gonadotropin (GTH) release potency in the goldfish pituitary. A selected number of these analogues was also tested for their ability to induce ovulation in seabass. The important structural modifications that increased the binding and gonadotropic activity of [Tyr(OMe)⁵]-GnRH in vitro and in vivo were found to include the replacement of the proline at position 9 with azetidine, glycine amide terminus with an alkyl amide group and Gly⁶ residue with hydrophilicd-amino acids such asd-Arg⁶. Overall, the findings provide SAR information on a group of novel GnRH peptides that can be also used to induce ovulation in teleosts.     

Design and synthesis of potent tyr(OMe)5-gonadotropin-releasing hormone (GnRH) analogues with modifications at positions 6, 9 and 10

We have recently reported the synthesis and the conformational properties of some Gonadotropin-releasing hormone (GnRH) analogues in which the tyrosine residue at position 5 is substituted with tyrosine-O-methyl (Keramida et al., Let. Pept. Sci., 3 (1996) 257/Matsoukas et al., Eur. J. Med. Chem., 32 (1997) 927). The analogue [Tyr-(OMe)⁵]-GnRH was found to exert a lower degree of desensitization than the native GnRH peptides in terms of pituitary gonadotropin (GTH) release in goldfish. Compared to GnRH, however, [Tyr-(OMe)⁵]-GnRH exerted a lower GTH-release potency in cultured goldfish pituitary fragments, and was bound with a lower binding affinity to the rat pituitary GnRH receptors. In order to increase the potency of [Tyr-(OMe)⁵]-GnRH, we have synthesized a group of GnRH peptides containing Tyr-(OMe)⁵ in combination with other substitutions at positions 6, 9 and 10 and we have estimated their binding affinity for the rat pituitary receptors and gonadotropin (GTH) release potency in the goldfish pituitary. A selected number of these analogues was also tested for their ability to induce ovulation in seabass. The important structural modifications that increased the binding and gonadotropic activity of [Tyr(OMe)⁵]-GnRH in vitro and in vivo were found to include the replacement of the proline at position 9 with azetidine, glycine amide terminus with an alkyl amide group and Gly⁶ residue with hydrophilic D-amino acids such as D-Arg⁶. Overall, the findings provide SAR information on a group of novel GnRH peptides that can be also used to induce ovulation in teleosts.     

Activity of vertebrate gonadotropin-releasing hormones and analogs with variant amino acid residues in positions 5, 7 and 8 in the goldfish pituitary.

All non-mammalian vertebrates as well as marsupial mammals have two or more forms of gonadotropin-releasing hormone (GnRH) in the brain. Goldfish brain and pituitary contains two molecular forms of GnRH, salmon GnRH ([Trp7, Leu8]m-GnRH; s-GnRH) and chicken GnRH-II ([His5, Trp7, Tyr8]m-GnRH; cII-GnRH). Both sGnRH and cII-GnRH stimulate gonadotropin (GtH) as well as growth hormone (GH) release from the goldfish pituitary. The purpose of the present study was to study the activity of the five known forms of GnRHs as well as analogs of mammalian GnRH (m-GnRH) with variant amino acid residues in positions 5, 7 and 8 in terms of binding to GnRH receptors, and release of GTH and GH from the perifused fragments of goldfish pituitary in vitro. All five vertebrate GnRH peptides stimulated both GtH and GH release in a dose-dependent manner, although their potencies were very different. cII-GnRH was somewhat more active than s-GnRH in releasing GtH, whereas s-GnRH tended to have a greater potency than cII-GnRH in terms of GH release. Both chicken GnRH-I (cI-GnRH) and lamprey GnRH (l-GnRH) were significantly less potent than mGnRH, s-GnRH and cII-GnRH in releasing GtH and GH. cII-GnRH binds with higher affinity for the high affinity binding sites compared to all other native peptides. The activity of [Trp7]-GnRH was similar to both s-GnRH and cII-GnRH in releasing GtH and GH. Substitution of His5 resulted in a significant decrease in GtH releasing potencies compared to mGnRH, sGnRH and cII-GnRH. [His5]-GnRH also had lower GH releasing potency than mGnRH and sGnRH. Tyr8, His8 and Leu8 substitutions caused significant decreases in GtH releasing potencies compared to mGnRH, s-GnRH and cII-GnRH, but did not cause a significant change in GH releasing potency. The combination of [His5, Trp7]-GnRH had GtH and GH releasing activities similar to m-GnRH, s-GnRH and cII-GnRH.(ABSTRACT TRUNCATED AT 400 WORDS)     

Primary culture of normal pituitary cells of carp (cyprinus carpio) for the study of gonadotropin release

Summary A method for preparing enzymaticlaly dispersed pituitary cell cultures of carp (Cyprinus carpio) is described. The cultures have been used to assay a synthetic analog of gonadotropin releasing hormone (GnRH) and to determine the specificity of steroids able to affect gonadotropin (GtH) release in vitro. Time course secretion studies indicated that by 48 h incubation, in the presence of 500 pM GnRH, cumulative secretion of gonadotropin (719 ng±90/2.5×10⁵ cells) had exceeded that of controls (446 ng±106/2.5×10⁵ cells). Estradiol-17β, progesterone, testosterone, and 11-ketotestosterone showed different inhibitory effects on pituitary basal GtH release. Based on the results, it was concluded that carp pituitary cell cultures can be applied to investigations of several aspects of the hypothalamo-hypophysial-gonadal system. This investigation was supported by the Deutsche Forschungsgemeinschaft, Bonn, FRG.     

Identification of Tyr(290(6.58)) of the human gonadotropin-releasing hormone (GnRH) receptor as a contact residue for both GnRH I and GnRH II: importance for high-affinity binding and receptor activation

Molecular modeling showed interactions of Tyr (290(6.58)) in transmembrane domain 6 of the GnRH receptor with Tyr (5) of GnRH I, and His (5) of GnRH II. The wild-type receptor exhibited high affinity for [Phe (5)]GnRH I and [Tyr (5)]GnRH II, but 127- and 177-fold decreased affinity for [Ala (5)]GnRH I and [Ala (5)]GnRH II, indicating that the aromatic ring in position 5 is crucial for receptor binding. The receptor mutation Y290F decreased affinity for GnRH I, [Phe (5)]GnRH I, GnRH II and [Tyr (5)]GnRH II, while Y290A and Y290L caused larger decreases, suggesting that both the para-OH and aromatic ring of Tyr (290(6.58)) are important for binding of ligands with aromatic residues in position 5. Mutating Tyr (290(6.58)) to Gln increased affinity for Tyr (5)-containing GnRH analogues 3-12-fold compared with the Y290A and Y290L mutants, suggesting a hydrogen-bond between Gln of the Y290Q mutant and Tyr (5) of GnRH analogues. All mutations had small effects on affinity of GnRH analogues that lack an aromatic residue in position 5. These results support direct interactions of the Tyr (290(6.58)) side chain with Tyr (5) of GnRH I and His (5) of GnRH II. Tyr (290(6.58)) mutations, except for Y290F, caused larger decreases in GnRH potency than affinity, indicating that an aromatic ring is important for the agonist-induced receptor conformational switch.     

Calcium ions as a mediator in GnRH action on gonadotropin release in the common carp (Cyprinus carpio L)

Collagenase-dispersed carp pituitary cells in a perifusion system were used to study the role of calcium ions in the mechanism of GnRH action on the release of maturational gonadotropin (GtH) in fish. The specific calcium chelator EGTA and the calcium antagonist manganese (Mn2+) caused a 40% inhibition in the basal GtH release and completely blocked GnRH-stimulated GtH release. Short-term application of graded doses of calcium ionophore A23187 caused a dose-dependent increase in GtH secretion. A23187 failed to stimulate GtH secretion in the presence of EGTA. Depolarization of the membrane by K+ caused a strong stimulation of GtH release similar to the action of GnRH. Stimulatory action of K+ was inhibited by EGTA. These data suggest a role for extracellular calcium as an intracellular mediator in GnRH-stimulated, as well as in basal, GtH release in carp. The stimulation of GtH release by K+ also indicates that voltage-dependent processes could be involved in this phenomenon.     

Homologous desensitization of gonadotropin-releasing hormone (GnRH) receptors in the goldfish pituitary: effects of native GnRH peptides and a synthetic GnRH antagonist.

Gonadotropin-releasing hormone (GnRH) stimulates release of gonadotropin hormone (GTH) through interaction with high affinity receptors in the goldfish pituitary. In the present study, we investigated desensitization of two native GnRH peptides, [Trp7, Leu8]-GnRH (sGnRH) and [His5, Trp7, Tyr8]-GnRH (cGnRH-II), using superfused fragments of goldfish pituitary in vitro. Pulsatile treatment with either sGnRH or cGnRH-II (2-min pulses given every 60 min) resulted in dose-dependent secretion of GTH from the goldfish pituitary; cGnRH-II had a greater GTH release potency and displayed a greater receptor binding affinity than sGnRH. Both sGnRH and cGnRH-II-induced GTH release were partially inhibited by concomitant treatment with either [D-Phe2, Pro3, D-Phe6]-GnRH or [D-pGlu1, D-Phe2, D-Trp3.6]-GnRH. These antagonists had greater receptor binding affinities than the native peptides, with no stimulatory action on GTH release in the absence of the GnRH agonists. Continuous treatment with either sGnRH or cGnRH-II (10(-7) M), rapidly desensitized pituitary GTH release in a biphasic fashion; initially there was a rapid increase in GTH release of approximately 10-20-fold (phase 1), followed by a sharp decline in GTH release, reaching a stable concentration 2-3-fold above the basal level (phase 2). Further stimulation of the pituitaries with sGnRH or cGnRH-II (10(-7) M) (second treatment) after 60 min recovery resulted in a significantly lower sGnRH or cGnRH-II-induced GTH release compared to that observed during the initial treatment period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural elucidation of Leuprolide and its analogues in solution: insight into their bioactive conformation

Leuprolide [dLeu⁶, NHEt¹⁰]GnRH, a potent gonadotropin-releasing hormone (GnRH) agonist, is used in a wide variety of hormone-related diseases like cancer and endometriosis. In this report, the conformational behaviour of Leuprolide and its linear synthetic analogues, namely [Tyr⁵(OMe), dLeu⁶, Aze⁹, NHEt¹⁰]GnRH (1) and [Tyr⁵(OMe), dLeu⁶, NHEt¹⁰]GnRH (2) have been studied in DMSO and H₂O solutions by means of 2D nuclear magnetic resonance (NMR) experiments and detailed molecular dynamics (MD) simulations. The aim was to identify the conformational requirements of GnRH analogues for agonistic activity. This approach is of value as no crystallographic data are available for the GnRH receptor (G protein-coupled receptor, GPCR). The NOE data indicate the existence of a β-turn type I in the 2–5 segments of Leuprolide and its linear analogues in the case of using DMSO-d₆ as solvent, whereas a β-turn type II in the 3–6 segments is indicated using D₂O as solvent. The final structures fulfil the conformational requirements that are known, in the literature, to play a significant role in receptor recognition and activation. Finally, the linear analogues (1) and (2) are biologically active when tested against the human breast cancer cell line, MCF-7.     

Signal transduction of the gonadotropin releasing hormone (GnRH) receptor: Cross-talk of calcium,protein kinase C (PKC), and arachidonic acid

Summary 1. The decapeptide neurohormone gonadotropin releasing hormone (GnRH) is the first key hormone of the reproductive system. Produced in the hypothalamus, GnRH is released in a pulsatile manner into the hypophysial portal system to reach the anterior pituitary and stimulates the release and synthesis of the gonadotropin hormones LH and FSH. GnRH, a Ca²⁺ mobilizing ligand, binds to its respective binding protein, which is a member of the seven transmembrane domain receptor family and activates a G-protein (Gq).2. The subunit of Gq triggers enhanced phosphoinositide turnover and the elevation of multiple second messengers required for gonadotropin release and biosynthesis.3. The messenger molecules IP₃, diacylglycerol, Ca²⁺, protein kinase C, arachidonic acid and leukotriene C₄ cross-talk in a complex networks of signaling, culminating in gonadotropin release and gene expression.     

Identification of His5,Trp7,Tyr8-GnRH (chicken GnRH II) in amphibian brain

GnRH immunoreactive and bioactive peptides in Xenopus laevis brain extract were investigated by high performance liquid chromatography (HPLC), radioimmunoassay with region-specific antisera raised against GnRH (mammalian), His5,Trp7,Tyr8-GnRH (chicken II) and Tyr3,Leu5,Glu6,Trp7,Lys8-GnRH (lamprey), and by assessment of biological activity. Two immunoreactive peptides eluted in the same positions as GnRH and His5,Trp7,Tyr8-GnRH respectively in HPLC systems which were specifically designed to separate four known natural vertebrate GnRHs (mammalian, chicken I and II, salmon). The immunological properties of these two immunoreactive peaks, determined by relative interaction with three region-specific antisera raised against mammalian GnRH and two specific His5,Trp7,Tyr8-GnRH antisera, were identical to those of GnRH and His5,Trp7,Tyr8-GnRH. The immunoreactive peak co-eluting with His5,Trp7,Tyr8-GnRH represented approximately one-third of the total brain GnRH. Both immunoreactive peaks stimulated luteinizing hormone (LH) release in a chicken dispersed pituitary cell bioassay, and the amounts of LH release stimulated by the two peaks were appropriate for these peaks being GnRH and His5,Trp7,Tyr8-GnRH. A small hydrophobic peak with GnRH immunoreactivity eluted in the same position as Trp7,Leu8-GnRH (salmon), while Gln8-GnRH (chicken I) and lamprey GnRH were not detected. Two additional rather hydrophilic peptides cross-reacted with a COOH-terminus-directed antiserum and had LH-releasing activity. LH-releasing activity was also detected in hydrophobic HPLC fractions. In summary, these data provide evidence for the presence of both GnRH and a second peptide with properties identical to His5,Trp7,Tyr8-GnRH in X. laevis brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural modifications of non-mammalian gonadotropin-releasing hormone (GnRH) isoforms: design of novel GnRH analogues

Three natural forms of vertebrate gonadotropin-releasing hormone (GnRH) provided the structural basis upon which to design new GnRH agonists: [His⁵,Trp⁷,Leu⁸]-GnRH, dogfish (df) GnRH; [His⁵,Asn⁸]-GnRH, catfish (cf) GnRH; and [His⁵,Trp⁷,Tyr⁸]-GnRH, chicken (c) GnRH-II. The synthetic peptides incorporated the position 6 dextro ( )-isomers -arginine ( -Arg) or -naphthylalanine ( -Nal) in combination with an ethylamide substitution of position 10. The in vitro potencies for LH and FSH release of these analogues were assessed using static cultures of rat anterior pituitary cells. Efficacious peptides were examined for their gonadotropin-II and growth hormone releasing abilities from perifused goldfish pituitary fragments. Rat LH and FSH release was measured using homologous radioimmunoassays, whereas goldfish growth hormone and gonadotropin-II release were determined using heterologous carp hormone radioimmunoassays. The receptor binding of the most potent analogues was determined in bovine pituitary membrane preparations. Substitution of -Nal⁶ into [His⁵,Asn⁸]-GnRH increased the potency over 2200-fold compared with the native ligand (cfGnRH) in cultured rat pituitary cells. This was equivalent to a 55-fold greater potency than that of the native mammal (m) GnRH peptide. Substitution of -Nal⁶ or -Arg⁶ into dfGnRH or cGnRH-II resulted in potencies that were related to the overall hydrophobicity of the analogues. The [ -Nal⁶,Pro⁹NEt]-cfGnRH bound to the bovine membrane preparation with an affinity statistically similar to that of [ -Nal⁶,Pro⁹NEt]-mGnRH (k_d = 0.40 ± 0.04 and 0.55 ± 0.10 nM, respectively) in cultured rat pituitary cells. All analogues tested released the same ratio of FSH to LH. In goldfish, the analogues did not possess superagonistic activity but instead desensitized the pituitary fragments at lower analogue doses than that of the sGnRH standard suggesting differences in receptor affinity or signal transduction.     

GnRH and GnRH receptors: distribution,function and evolution

Gonadotropin‐releasing hormone (GnRH) was originally identified because of its essential role in regulating reproduction in all vertebrates. Since then, three phylogenetically related GnRH decapeptides have been characterized in vertebrates and invertebrates. Almost all tetrapods investigated have at least two GnRH forms (GnRH1 and GnRH2) in the central nervous system. From distributional and functional studies in vertebrates, GnRH1 in the hypothalamus projects predominantly to the pituitary and regulates reproduction via gonadotropin release. GnRH2, which is located in the midbrain, projects to the whole brain and is thought to be involved in sexual behaviour and food intake. GnRH3, located in the forebrain, has only been found in teleost fish and appears to be involved in sexual behaviour, as well as, in some fish species, gonadotropin release. Multiple GnRH receptors (GnRH‐Rs), G‐protein‐coupled receptors regulate endocrine functions and neural transmissions in vertebrates. Phylogenetic and structural analyses of coding sequences show that all vertebrate GnRH‐Rs cluster into two main receptor types comprised of four subfamilies. This suggests that at least two rounds of GnRH receptor gene duplications may have occurred in different groups within each lineage. Functional studies suggest that two particular subfamilies of GnRH receptors have independently evolved to act as species‐specific endocrine modulators in the pituitary, and these show the greatest variety in regulating neuron networks in the brain. Given the long evolutionary history of the GnRH system, it seems likely that much more remains to be understood about its roles in behaviour and function of vertebrates.     

Lack of gonadotropin releasing-hormone action on in vivo and in vitro growth hormone release,in rainbow trout (Oncorhynchus mykiss)

In order to understand the mechanisms implicated at the hypothalamo-pituitary level in growth-reproduction interaction in salmonids, the gonadotropin-releasing hormone (GnRH) action on growth hormone (GH) release was studied, in rainbow trout (Oncorhynchus mykiss). In vivo, acute treatment with salmon GnRH (sGnRH), chicken GnRH-II (cGnRH-II) and an sGnRH analogue [(DArg6Pro9)sGnRH] was performed on catheterized fish. The different forms of GnRH have no effect on plasma GH levels of immature and mature fish, but induce a stimulation of gonadotropin (GtH) release in mature fish. In the present work we have adapted and validated a culture system for GH regulation studies. In this system, increasing doses of sGnRH, (DArg6Pro9)sGnRH and cGnRH-II are inactive on GH release (24 hr incubation) in immature or mature fish, but stimulate GtH release in a dose-dependent manner. sGnRH (10⁻⁶ M) has no action on GH release, whatever the incubation time (15 min–24 hr). In a perifusion system, sGnRH also has no action on GH release but stimulates GtH release. The present results obtained using in vivo and in vitro techniques adapted for GH regulation studies, show that GnRH does not function as a growth hormone-releasing factor in rainbow trout as it does in goldfish.     

Use of a mammalian gonadotropin‐releasing hormone (GnRH) agonist to characterize pituitary GnRH receptors in white sturgeon (Acipenser transmontanus Richardson)

Aim of the present study was to characterize the pituitary GnRH receptor in white sturgeon, Acipenser transmontanus, using a superagonist analog of mammalian GnRH [D‐Ala⁶, des‐Gly¹⁰–Pro⁹]‐ethylamide and to investigate the possible effect of estradiol‐17β treatment on the concentration and affinity of the GnRH receptor in immature white sturgeon. The binding of ¹²⁵I‐GnRH‐A to sturgeon pituitary receptors was rapid and saturable at 4°C and 20°C. However, maximal binding at 20°C was almost two‐fold greater than the highest binding noted at 4°C. Specific binding of radioligand was directly related to the amount of tissue included in the assay system over the range of 5–20 mg fresh tissue equivalents per ml. The binding capacity of ¹²⁵I‐GnRH‐A with sturgeon pituitary tissue was much greater than radiolabeled GnRH. Administration of E₂ to immature sturgeon caused an almost two‐fold increase in GnRH‐A binding capacity (E₂ treated: Bmax = 2.87 fmoles 3 mg^?1 FTE; control: Bmax = 1.70 fmoles 3 mg^?1), and did not affect GnRH‐A binding affinity (E₂ treated: Ka = 0.13 × 10¹¹ m ^?1; control: Ka = 0.15 × 10¹¹ m ^?1). Overall, the study provides evidence that the GnRH analog is effective for characterizing the GnRH receptor in white sturgeon; however, more experimentation is necessary to determine whether E₂ administration to immature white sturgeon can increase the GnRH receptor capacity.     

Involvement of voltage-dependent calcium channels (VDCC) in the action of GnRH on GtH release in common carp (Cyprinus carpio L): comparison with K+ action.

The involvement of different types of voltage-dependent calcium channels (VDCC) in the stimulatory action of GnRH (in comparison with K+) on maturational gonadotropin (GtH) release was investigated using superfused carp pituitary cells. The action of these 2 stimulants was not modified either by D600 or nifedipine (drugs blocking L-type of VDCC). Cadmium (Cd2+), which blocks all types of VDCC indifferently, provoked a dose-dependent stimulation of GtH secretion. Cd2+ action was not altered by addition of sGnRH in any of the doses. Similar results were obtained using K+ as a secretagogue, but only the highest dose of Cd2+ (200 mumol/l) was able to completely block K+ action. Low doses (0.1 and 1 mumol/l) of the L-type VDCC activator BAY-K8644 did not change basal GtH secretion and had no effect on sGnRH-stimulated GtH secretion. Surprisingly, doses (10 mumol/l and higher) of BAY-K8644 evoked dose-dependent inhibition of GtH secretion. On the other hand, a higher concentration (20 mumol/l) of nifedipine provoked a stimulation of GtH release. Our results indicate that the stimulatory action of GnRH and K+ involves activation of a certain type of cadmium-sensitive VDCC (probably T- or N-type VDCC) whereas dihydropyridine and diphenylalkylamine sensitive VDCC (L-type VDCC) does not participate in this phenomenon. The inhibitory action of BAY-K8644 and, on the other hand, the stimulatory action of nifedipine indicate that L-type VDCC probably play a role in other physiological pathways regulating GtH release in carp.     

Regulation of gonadotropin release, GnRH receptors, and gonadotrope responsiveness: a role for GnRH receptor microaggregation

Gonadotropin-releasing hormone (GnRH) stimulates luteinizing hormone (LH) and follicle-stimulating hormone (FSH) release from pituitary gonadotrope cells. Additional receptor-mediated actions of the releasing hormone include homologous regulation of both the GnRH receptor and of cell responsiveness. While it is apparent that the release mechanism is Ca²⁺ mediated, it remains unclear how this receptor-mediated action is integrated with regulation of the receptor and with cell responsiveness. It is the purpose of this review to describe the requirements for gonadotropin release as well as for receptor and response regulation in order to prepare an integrated model for these actions of the releasing hormone.     

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.     

Gonadotropin-releasing hormone analog structural determinants of selectivity for inhibition of cell growth: support for the concept of ligand-induced selective signaling

GnRH and its receptor are expressed in human reproductive tract cancers, and direct antiproliferative effects of GnRH analogs have been demonstrated in cancer cell lines. The intracellular signaling responsible for this effect differs from that mediating pituitary gonadotropin secretion. The GnRH structure-activity relationship is different for the two effects. Here we report a structure-activity relationship study of GnRH agonist antiproliferative action in model cell systems of rat and human GnRH receptors stably expressed in HEK293 cells. GnRH II was more potent than GnRH I in inhibiting cell growth in the cell lines. In contrast, GnRH I was more potent than GnRH II in stimulating inositol phosphate production, the signaling pathway in gonadotropes. The different residues in GnRH II (His(5), Trp(7), Tyr(8)) were introduced singly or in pairs into GnRH I. Tyr(5) replacement by His(5) produced the highest increase in the antiproliferative potency of GnRH I. Tyr(8) substitution of Arg(8) produced the most selective analog, with very poor inositol phosphate generation but high antiproliferative potency. In nude mice bearing tumors of the HEK293 cell line, GnRH II and an antagonist administration was ineffective in inhibiting tumor growth, but D-amino acid stabilized analogs (D-Lys(6) and D-Arg(6)) ablated tumor growth. Docking of GnRH I and GnRH II to the human GnRH receptor molecular model revealed that Arg(8) of GnRH I makes contact with Asp(302), whereas Tyr(8) of GnRH II appears to make different contacts, suggesting these residues stabilize different receptor conformations mediating differential intracellular signaling and effects on gonadotropin and cell growth. These findings provide the basis for the development of selective GnRH analog cancer therapeutics that directly target tumor cells or inhibit pituitary gonadotropins or do both.     

Homologous desensitization with gonadotropin-releasing hormone (GnRH) also diminishes gonadotrope responsiveness to maitotoxin: a role for the GnRH receptor-regulated calcium ion channel in mediation of cellular desensitization

Maitotoxin (MTX) stimulates gonadotropin release from pituitary cell cultures. The time course and efficacy of LH release in response to GnRH and to MTX are similar; both secretagogues require extracellular Ca2+ and are inhibited by the selective Ca2+ ion channel antagonist methoxyverapamil (D600). LH release in response to either GnRH or MTX is not measurably inhibited by two other chemical classes of Ca2+ ion channel inhibitors represented by nifedipine and by diltiazem. The two secretagogues are nonadditive in their action on LH release when presented at high doses and prior studies indicate that MTX has no endogenous ionophoretic activity. These observations indicate that MTX likely stimulates LH release due to activation of the GnRH receptor associated Ca2+-ion channel in the gonadotrope. We have therefore assessed the functional state of this channel during the development of homologous desensitization of the gonadotrope to GnRH by measuring the ability of MTX to stimulate LH release. Cells were desensitized with GnRH in the presence of 3 mM EGTA. Under these conditions, the cells become refractory to GnRH in the absence of gonadotropin release since the latter process, but not the former, requires extracellular Ca2+. Accordingly, this approach allows assessment of the degree of desensitization in the absence of the influence of gonadotropin depletion. Such desensitized cells are less responsive to GnRH. Desensitized pituitary cells also respond with diminished efficacy and potency to MTX three or more hours after GnRH treatment but not at an earlier time (1 h) when GnRH receptors are diminished. These data are consistent with a model in which homologous desensitization is viewed as developing in two phases.(ABSTRACT TRUNCATED AT 250 WORDS)