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Positive allosteric modulation of the GABAB receptor is a promising alternative to direct activation of the receptor as a therapeutic approach for treatment of addiction, chronic pain, anxiety, epilepsy, autism, Fragile X syndrome, and psychosis. Here we describe in vitro and in vivo characterization of a novel, potent and selective GABAB positive allosteric modulator (PAM) N-(5-(4-(4-chloro-3-fluorobenzyl)-6-methoxy-3,5-dioxo-4,5-dihydro-1,2,4-triazin-2(3H)-yl)-2-fluorophenyl)acetamide (ADX71441). In vitro, Schild plot and reversibility tests at the target confirmed PAM properties of the compound. In mice and rats ADX71441 is bioavailable after oral administration and is brain penetrant. A single dose of ADX71441 had an anxiolytic-like profile in the mouse marble burying test (minimum effective dose; MED 3 mg/kg) as well as in the elevated plus maze test in mice and rats (both MED 3 mg/kg). Also, in mice, acute administration of ADX71441 reduced visceral pain-associated behaviors in the acetic acid-induced writhing test. ADX71441 dose-dependently reduced time on rotarod in rats (MED 10 mg/kg) indicative of muscle-relaxant qualities. ADX71441 reduced locomotor activity in mice (10 mg/kg) and rats (3 mg/kg) after single dose; however, following sub-chronic administration in mice, 30 mg/kg ADX71441 was associated with normal locomotor activity. While acute administration of ADX71441 reduced body temperature in rats and mice (both MED 10 mg/kg), the effect in the former was transient, rapidly returning to normal levels despite high concentrations of the compound remaining in plasma. Thus, the GABAB PAM ADX71441 represents a valid therapeutic approach for development of novel treatment of anxiety, pain and spasticity.

Background and Purpose The GABAB receptor agonist, baclofen, has shown promising effects in patients suffering from pain, post-traumatic stress disorder, alcoholism, overactive bladder and gastroesophageal reflux disease. However, baclofen's short duration of action and side effects limit its wider use. Here we characterized a novel, GABAB receptor positive allosteric modulator (PAM) ADX71943. Experimental Approach In vitro, ADX71943 was assessed for pharmacological activity and selectivity using recombinant and native GABAB receptors. In vivo ADX71943 was assessed in the acetic acid-induced writhing (AAW) test in mice and formalin tests (FTs) in mice and rats. Marble burying (MB) and elevated plus maze (EPM) tests, rotarod, spontaneous locomotor activity (sLMA) and body temperature (BT) tests in mice and rats were used to investigate centrally-mediated effects. Key Results In vitro, in the presence of GABA, ADX71943 increased the potency and efficacy of agonists and showed selectivity at the GABAB receptor. ADX71943 reduced pain-associated behaviours in AAW; an effect blocked by GABAB receptor antagonist CGP63360. ADX71943 reduced pain in the FT in mice and rats, but was inactive in the MB and EPM despite reaching high concentrations in plasma. ADX71943 had no effect on BT, rotarod and sLMA. Conclusions and Implications ADX71943 showed consistent and target-related efficacy in tests of disorders that have a significant peripheral component (acute and chronic pain), while having no effect in those associated with centrally-mediated anxiety-like reactivity and side effects. Thus, ADX71943 is a useful pharmacological tool for delineation of peripherally- versus centrally-mediated effects of GABAB receptor activation.

BACKGROUND: Blocking metabotropic glutamate receptor type 5 (mGluR5) has been proposed as a target for levodopa-induced dyskinesias (LID) in Parkinson's disease (PD). We assessed the effect on LID of dipraglurant, a potent selective mGluR5 receptor negative allosteric modulator in the gold-standard LID macaque model. METHODS: Dipraglurant (3, 10, and 30 mg/kg, by mouth) was tested in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) macaque model of LID in a four-way crossover, single-dose, controlled study (n = 8). RESULTS: Dipraglurant inhibited dyskinesias in the LID macaque model, with best effect reached at 30 mg/kg dose with no alteration of levodopa efficacy. CONCLUSION: Acute challenges of dipraglurant were efficacious on choreic and dystonic LID in the MPTP-macaque model. Dipraglurant pharmacokinetic variables were similar to those of levodopa, suggesting that both drugs can be co-administered simultaneously in further studies.

Activation of G-protein-coupled receptors (GPCRs) results in a variety of cellular responses, such as binding to the same receptor of different ligands that activate distinct downstream cascades. Additional signaling complexity is achieved when two or more receptors are integrated into one signaling unit. Lateral receptor interactions can allosterically modulate the receptor response to a ligand, which creates a mechanism for tissue-specific fine tuning, depending on the cellular receptor coexpression pattern. GPCR homomers or heteromers have been explored widely for GPCR classes A and C but to lesser extent for class B. In the present study, we used bioluminescence resonance energy transfer (BRET) techniques, calcium flux measurements, and microscopy to study receptor interactions within the glucagon receptor family. We found basal BRET interactions for some of the receptor combinations tested that decreased upon ligand binding. A BRET increase was observed exclusively for the gastric inhibitory peptide (GIP) receptor and the glucagon-like peptide 1 (GLP-1) receptor upon binding of GLP-1 that could be reversed with GIP addition. The interactions of GLP-1 receptor and GIP receptor were characterized with BRET donor saturation studies, shift experiments, and tests of glucagon-like ligands. The heteromer displayed specific pharmacological characteristics with respect to GLP-1-induced β-arrestin recruitment and calcium flux, which suggests a form of allosteric regulation between the receptors. This study provides the first example of ligand-induced heteromer formation in GPCR class B. In the body, the receptors are functionally related and coexpressed in the same cells. The physiological evidence for this heteromerization remains to be determined.

Allosteric modulators of metabotropic glutamate receptors (mGluR) subtypes 1-8 have been shown to offer a valid way to develop small molecule non aminoacid-like therapeutics that can be administered orally and that readily cross the blood-brain barrier. Allosteric modulators of glutamatergic receptors and in particular mGluR5 have emerged as a novel and highly desirable class of compounds for the treatment of central nervous system (CNS) disorders and peripheral disorders. This article provides medicinal chemistry highlights around the chemical classes of potent and highly selective mGluR5 negative allosteric modulators (NAMs) and their therapeutic potential. In addition, it describes the medicinal chemistry approach from the discovery to the clinical candidate selection of a new series of heteroaryl-butynylpyridines targeting mGluR5. The multiparametric optimization of the initial starting point which ended in the selection of potential clinical candidates combining the best pharmacophoric features is presented. The pharmacological properties are reported and support the interest of these agents for new therapeutic approaches. Furthermore, a summary of the diverse mGluR5 Positron Emission Tomography (PET) radioligands is reported.

Altered glutamatergic synaptic transmission is among the key events defining the course of Alzheimer's disease (AD). mGlu2 receptors, a subtype of group II metabotropic glutamate receptors, regulate (as autoreceptors) fast synaptic transmission in the CNS via the controlled release of the excitatory amino acid glutamate. Since their pharmacological manipulation in rodents has been reported to affect cognition, they are potential drug targets for AD therapy. We examined the fate of these receptors in cases of AD as well as in aging PS2APP mice—a proposed model of the disease. In vitro binding of [3H]LY354740, a selective group II agonist (with selective affinity for mGlu2 receptors, under the assay conditions used) and quantitative radioautography revealed a partial, but highly significant, loss of receptors in amyloid-affected discrete brain regions of AD cases and PS2APP mice. Among the mouse brain regions affected were, above all, the subiculum but also frontolateral cortex, dentate gyrus, lacunosum moleculare and caudate putamen. In AD, significant receptor losses were registered in entorhinal cortex and lacunosum moleculare (40% and 35%, respectively). These findings have implications for the development of selective ligands for symptomatic therapy in AD and for its diagnosis.

Small molecule mGluR1 enhancers, which are 9H-xanthene-9-carboxylic acid [1,2,4]oxadiazol-3-yl- and (2H-tetrazol-5-yl)-amides, have been previously reported. Fluorinated 9H-xanthene-9-carboxylic acid oxazol-2-yl-amides with improved pharmacokinetic properties have been designed and synthesized as useful pharmacological tools for the study of the physiological roles mediated by mGlu1 receptors. The synthesis and the structure-activity relationship of this class of positive allosteric modulators of mGlu1 receptors will be discussed in detail.

We have investigated the binding properties of [(3)H]quisqualate to rat metabotropic glutamate (mGlu) 1a and 5a receptors and to rat and human brain sections. Saturation isotherms gave K:(D) values of 27 +/- 4 and 81 +/- 22 nM: for mGlu1a and mGlu5a receptors, respectively. Several compounds inhibited the binding to mGlu1a and mGlu5a receptors concentration-dependently. (S:)-4-Carboxyphenylglycine, (S:)-4-carboxy-3-hydroxyphenylglycine, and (R,S)-1-aminoindan-1,5-dicarboxylic acid, which completely inhibited [(3)H]quisqualate binding to the mGlu5a receptor, were inactive in a functional assay using this receptor. The distribution and abundance of binding sites in rat and human brain sections were studied by quantitative receptor radioautography and image analysis. Using 10 nM: [(3)H]quisqualate, a high density of binding was detected in various brain regions with the following rank order of increasing levels: medulla, thalamus, olfactory bulb, cerebral cortex, spinal cord dorsal horn, olfactory tubercle, dentate gyrus molecular layer, CA1-3 oriens layer of hippocampus, striatum, and cerebellar molecular layer. The ionotropic component of this binding could be inhibited by 30 microM: kainate, revealing the distribution of mGlu1+5 receptors. The latter were almost completely inhibited by the group I agonist (S:)-3,5-dihydroxyphenylglycine. The binding profile correlated well with the cellular sites of synthesis and regional expression of the respective group I receptor proteins revealed by in situ hybridization histochemistry and immunohistochemistry, respectively.

The asymmetric synthesis and receptor pharmacology of (1S,2R,3R,5R,6S)-2-amino-3-Hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid (+)-9 (HYDIA) and a few of its O-alkylated derivatives are described. The key step of the synthesis utilizes Sharpless' asymmetric dihydroxylation (AD-beta) for the kinetic resolution of a bicyclic racemic precursor olefin. In contrast to the bicyclic glutamate analogue LY354740, which is a potent and selective agonist for the group II metabotropic glutamate receptors (mGluRs), these new conformationally restricted and also hydroxylated or alkoxylated glutamate analogues are potent and selective antagonists for the group II mGluRs.

Fenobam [N-(3-chlorophenyl)-N'-(4,5-dihydro-1-methyl-4-oxo-1H-imidazole-2-yl)urea] is an atypical anxiolytic agent with unknown molecular target that has previously been demonstrated both in rodents and human to exert anxiolytic activity. Here, we report that fenobam is a selective and potent metabotropic glutamate (mGlu)5 receptor antagonist acting at an allosteric modulatory site shared with 2-methyl-6-phenylethynyl-pyridine (MPEP), the protypical selective mGlu5 receptor antagonist. Fenobam inhibited quisqualate-evoked intracellular calcium response mediated by human mGlu5 receptor with IC(50) = 58 +/- 2 nM. It acted in a noncompetitive manner, similar to MPEP and demonstrated inverse agonist properties, blocking 66% of the mGlu5 receptor basal activity (in an over expressed cell line) with an IC(50) = 84 +/- 13 nM. [(3)H]Fenobam bound to rat and human recombinant receptors with K(d) values of 54 +/- 6 and 31 +/- 4 nM, respectively. MPEP inhibited [(3)H]fenobam binding to human mGlu5 receptors with a K(i) value of 6.7 +/- 0.7 nM, indicating a common binding site shared by both allosteric antagonists. Fenobam exhibits anxiolytic activity in the stress-induced hyperthermia model, Vogel conflict test, Geller-Seifter conflict test, and conditioned emotional response with a minimum effective dose of 10 to 30 mg/kg p.o. Furthermore, fenobam is devoid of GABAergic activity, confirming previous reports that fenobam acts by a mechanism distinct from benzodiazepines. The non-GABAergic activity of fenobam, coupled with its robust anxiolytic activity and reported efficacy in human in a double blind placebo-controlled trial, supports the potential of developing mGlu5 receptor antagonists with an improved therapeutic window over benzodiazepines as novel anxiolytic agents.

A major determinant of neuronal homeostasis is the proper integration of cell signaling pathways recruited by a variety of neuronal and non-neuronal factors. By taking advantage of a neuroectodermal cell line (1C11) endowed with the capacity to differentiate into serotonergic (1C115-HT) or noradrenergic (1C11NE) neurons, we identified serotonin (5-hydroxytryptamine, 5-HT)- and norepinephrine (NE)-dependent signaling cascades possibly involved in neuronal functions. First, we establish that 5-HT2B receptors and 1D adrenoceptors are functionally coupled to reactive oxygen species (ROS) synthesis through NADPH oxidase activation in 1C115-HT and 1C11NE cells. This observation constitutes the prime evidence that bioaminergic autoreceptors take part in the control of the cellular redox equilibrium in a neuronal context. Second, our data identify TACE (TNF- Converting Enzyme), a member of a disintegrin and metalloproteinase (ADAM) family, as a downstream target of the 5-HT2B and 1D receptor-NADPH oxidase signaling pathways. Upon 5-HT2B or 1D receptor stimulation, ROS fully govern TNF- shedding in the surrounding milieu of 1C115-HT or 1C11NE cells. Third, 5-HT2B and 1Dreceptor couplings to the NADPH oxidase-TACE cascade are strictly restricted to 1C11-derived progenies that have implemented a complete serotonergic or noradrenergic phenotype. Overall, these observations suggest that 5-HT2B and 1D autoreceptors may play a role in the maintenance of neuron- and neurotransmitter-associated functions. Eventually, our study may have implications regarding the origin of oxidative stress as well as up-regulated expression of proinflammatory cytokines in neurodegenerative disorders, which may relate to the deviation of normal signaling pathways.

The inducible serotonergic 1C115-HT cell line expresses a defined set of serotonergic receptors of the 5-HT2B, 5-HT1B/D, and 5-HT2A subtypes, which sustain a regulation of serotonergic associated functions through G-protein-dependent signaling. 1C115-HT cells have been instrumental to assign a signaling function to the cellular prion protein PrPC. Here, we establish that antibody-mediated ligation of PrPC concomitant to agonist stimulation of 5-HT receptors modulates the couplings of all three serotonergic receptors present on 1C115-HT cells. Specific impacts of PrP antibodies were monitored depending on the receptor and pathway considered. PrPC ligation selectively cancels the 5-HT2A-PLC response, decreases the 5-HT1B/D negative coupling to adenylate cyclase, and potentiates the 5-HT2B-PLA2 coupling. As a result, PrPC ligation disturbs the functional interactions occurring between the signaling pathways of the three receptor subtypes. In 1C115-HT cells, antagonizing cross-talks arising from 5-HT2B and 5-HT2A receptors control the 5-HT1B/D function. PrPC ligation reinforces the negative regulation exerted by 5-HT2B on 5-HT1B/D receptors. On the other hand it abrogates the blocking action of 5-HT2A on the regulatory loop linking 5-HT1B/D receptors. We propose that the ligation of PrPC affects the potency or dynamics of G-protein activation by agonist-bound serotonergic receptors. Finally, the PrPC-dependent modulation of 5-HT receptor couplings is restricted to 1C115-HT cells expressing a complete serotonergic phenotype. It critically involves a PrPC-caveolin platform implemented on the neurites of 1C115-HT cells during differentiation. Our findings define PrPC as a modulator of 5-HT receptor coupling to G-proteins and thereby as a protagonist contributing to the homeostasis of serotonergic neurons. They provide a foundation for uncovering the impact of prion infection on serotonergic functions.

Atrophy of the medial temporal lobes, including the glutamatergic cortical-hippocampal circuitry, is an early event in Alzheimer's disease (AD) and probably contributes to the characteristic short-term mnemonic decline. Pharmacological strategies directly targeted to ameliorating this functional decline may represent a novel approach for the symptomatic treatment of AD. Presynaptic group II metabotropic glutamate receptors (i.e. mGlu2 and mGlu3) exert a powerful modulatory influence on the function of these pathways, in particular the perforant pathway. Using a combination of mGlu2 receptor knockout mice and the group II agonist LY354740, we show that activation of mGlu2 receptors produces a cognitive impairment, i.e. a delay-dependent deficit in delayed matching and non-matching to position, and impaired spatial learning in a Morris water maze. Conversely, a group II antagonist, LY341495, improved acquisition of spatial learning. LY354740 potently reduced field excitatory postsynaptic potentials in hippocampal slices from wild type but not mGlu2 receptor knockout mice. Taken together, these results suggest that activation of mGlu2 receptors evokes a powerful inhibitory effect on hippocampal synaptic transmission and mGlu2 agonists produce a cognitive deficit consistent with this change. Conversely, mGlu2 receptor antagonists may improve certain aspects of cognition and thus represent a novel approach for the symptomatic treatment of AD.

Following the molecular cloning in the early 1990s of the metabotropic glutamate receptors (mGlu1-8), research that focused on the physiology, pharmacology and function of these receptors revealed their potential role in CNS disorders. Numerous psychiatric and neurological dis-orders are indeed linked to changes in excitatory processes, in which glutamate plays a key role. In contrast to ligand-gated ion channels [N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) and kainate], which are responsible for fast excitatory transmission, mGlu receptors have a more modulatory role, by contributing to fine-tuning of synaptic efficacy, and control of the accuracy and sharpness of the transmission. Given the fact that the mGlu receptors are G-protein coupled, they obviously constitute new 'drugable' targets for the treatment of various CNS disorders. Due to the recent emergence of subtype-specific ligands for Group I and II mGlu receptors, this review will concentrate on the molecular characteristics, brain localization, pharmacology and physiological role of these receptors, in order to provide further insights into their therapeutic potential.

Recently, we disclosed 4-aminoquinolines as structurally novel NR1/2B subtype selective NMDA receptor antagonists. We would now like to report our findings on structurally related pyridine analogues. The SAR developed in this series resulted in the discovery of high affinity antagonists which are selective (vs alpha1 and M1 receptors) and active in vivo.

Metabotropic glutamate (mGlu) receptors have progressively evolved from the status of a ‘solution looking for problems’ to being the focus of major drug discovery programmes in an increasing number of large pharmaceutical companies. The steady increase in interest for these targets was primarily due to their relatively recent discovery and consequently, to the lack of selective systemically-active ligands to allow precise assessment of their physiological role and therapeutical potential. Fortunately, strong faith in the potential of modulatory receptors of the major excitatory transmitters in the mammalian brain, glutamate, led tenacious pioneers to design molecules which have been extremely useful to validate valuable targets for medicinal chemistry development. In addition, the increasing use of recently available high-throughput functional assays allowed the identification of ligands for these targets with novel mechanisms of action. This review will concentrate on recent progress in the field, focuss...

The in vitro binding of a new subtype-selective NMDA receptor antagonist, [ 3 H]Ro 25-6981, to rat brain membranes and sections was characterized. The compound bound to a single site on the membranes with a K D of 3 nM and a B max of 1.6 pmol/mg of protein. Specific binding, defined with a new NR2B-specific antagonist, Ro 04-5595 [1-(4-chlorophenyl)-2-methyl-6-methoxy-7-hydroxy-1,2,3,4-tetrahydroisoquinoline], at 10 μM, was fully inhibited by several compounds with the following rank order of affinities-Ro 25-6981 > CP-101,606 > Ro 04-5595 = ifenprodil » eliprodil > haloperidol > spermine > spermidine > MgCl 2 > CaCl 2 -and partially inhibited by competitive glutamate recognition site antagonists. A high density of binding sites was detected, radioautographically, in several layers of the cerebral cortex, in the hippocampus, dentate gyrus, tuberculum olfactorium, caudate putamen, medium densities in the globus pallidus, thalamus, spinal cord dorsal horn, and motoneurons, whereas the cerebellum, pons, and medulla were, with a few exceptions, e.g., locus coeruleus, poorly labeled. Overall, the distribution of [ 3 H] Ro 25-6981 binding sites correlated well with that of NR2B (but not NR2A) transcripts, revealed by in situ hybridization histochemistry. The high affinity of [ 3 H]Ro 25-6981 for NR2B-containing receptors renders this compound the ligand of choice to study the regulation of NR2B-containing receptor expression.

A new AMPA receptor antagonist, Ro 48-8587, was characterized pharmacologically in vitro. It is highly potent and selective for AMPA receptors as shown by its effects on [3H]AMPA, [3H] kainate, and [3H] MK-801 binding to rat brain membranes and on AMPA- or NMDA-induced depolarization in rat cortical wedges. [3H]Ro 48-8587 bound with a high affinity (KD = 3 nM) to a single population of binding sites with a Bmax of 1 pmol/mg of protein in rat whole brain membranes. [3H]Ro 48-8587 binding to rat whole brain membranes was inhibited by several compounds with the following rank order of potency: Ro 48-8587 > 6-nitro-7-sulphamoylbenzo[f] quinoxaline-2,3-dione (NBQX) > YM 90K > 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) > quisqualate > AMPA > glutamate > kainate > NMDA. The distribution and abundance of specific binding sites (approximately 95% of total) in sections of rat CNS, revealed by quantitative receptor radioautography and image analysis, indicated a very discrete localization. Highest binding values were observed in cortical layers (binding in layers 1 and 2 > binding in layers 3-6), hippocampal formation, striatum, dorsal septum, reticular thalamic nucleus, cerebellar molecular layer, and spinal cord dorsal horn. At 1 nM, the values for specific binding were highest in the cortical layers 1 and 2 and lowest in the brainstem (approximately 2.6 and 0.4 pmol/mg of protein, respectively). Ro 48-8587 is a potent and selective AMPA receptor antagonist with improved binding characteristics (higher affinity, selectivity, and specific binding) compared with those previously reported.

The cause of schizophrenia is unknown, but it has a significant genetic component. Pharmacologic studies, studies of gene expression in man, and studies of mouse mutants suggest involvement of glutamate and dopamine neurotransmitter systems. However, so far, strong association has not been found between schizophrenia and variants of the genes encoding components of these systems. Here, we report the results of a genomewide scan of schizophrenia families in Iceland; these results support previous work, done in five populations, showing that schizophrenia maps to chromosome 8p. Extensive fine-mapping of the 8p locus and haplotype-association analysis, supplemented by a transmission/disequilibrium test, identifies neuregulin 1 (NRG1) as a candidate gene for schizophrenia. NRG1 is expressed at central nervous system synapses and has a clear role in the expression and activation of neurotransmitter receptors, including glutamate receptors. Mutant mice heterozygous for either NRG1 or its receptor, ErbB4, show a behavioral phenotype that overlaps with mouse models for schizophrenia. Furthermore, NRG1 hypomorphs have fewer functional NMDA receptors than wild-type mice. We also demonstrate that the behavioral phenotypes of the NRG1 hypomorphs are partially reversible with clozapine, an atypical antipsychotic drug used to treat schizophrenia.

Group II metabotropic glutamate (mGlu) receptors can act as presynaptic autoinhibitory receptors at perforant path inputs to the hippocampus under conditions of high frequency synaptic activation. We have used mGlu2 -/- mice to examine the relative roles of mGlu2 and mGlu3 in the regulation of perforant path synaptic transmission mediated by both the selective group II receptor agonist, DCG-IV, and by synaptically released glutamate. Field excitatory postsynaptic potentials evoked by stimulation of either the perforant path inputs to the dentate gyrus mid-moleculare or the CA1 stratum lacunosum moleculare were inhibited by DCG-IV with IC(50) values and maximum percentage inhibition of: 169 nM (60%) and 41 nM (72%) in wild-type mice and 273 nM (19%) and 116 nM (49%) in mGlu2 -/- mice, respectively. Activation of presynaptic group II mGlu autoreceptors by synaptically released glutamate, as revealed by a LY341495-mediated increase in the relative amplitude of a test fEPSP evoked after a conditioning burst, was observed in both the dentate gyrus and the stratum lacunosum of wild-type, but not mGlu2 -/- mice. These observations demonstrate that activation of mGlu3 receptors can regulate synaptic transmission at perforant path synapses but suggest that mGlu2 is the major presynaptic group II autoreceptor activated by synaptically released glutamate.

Positive allosteric modulation of the GABA

We have identified two chemical series of compounds acting as selective positive allosteric modulators (enhancers) of native and recombinant metabotropic glutamate 1 (mGlu1) receptors. These compounds did not directly activate mGlu1 receptors but markedly potentiated agonist-stimulated responses, increasing potency and maximum efficacy. Binding of these compounds increased the affinity of a radiolabeled glutamate-site agonist at its extracellular N-terminal binding site. Chimeric and mutated receptors were used to localize amino acids in the receptor transmembrane region critical for these enhancing properties. Finally, the compounds potentiated synaptically evoked mGlu1 receptor responses in rat brain slices. The discovery of selective positive allosteric modulators of mGlu1 receptors opens up the possibility to develop a similar class of compounds for other family 3 G protein-coupled receptors.

We used ligand binding to ascertain whether the pharmacological actions of RO 25-6981 [(R:(*), S:(*))-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidinepropanol] match those of other NR2B (epsilon2) subunit specific agents. RO 25-6981 inhibited binding of 125I-MK801 [iodo-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohept-5,10-imine maleate] to receptors made from NR1a/epsilon2 but not NR1a/epsilon1. Increasing the concentration of spermidine did not change the efficacy of RO 25-6981 and minimally changed the IC(50) value. Chimeric epsilon1/epsilon2 receptors demonstrated that the structural determinants for high affinity actions of RO 25-6981 were contained completely within the first 464 amino acids, but no receptor retained wildtype features when the size of the epsilon2 component was decreased further. Epsilon1Q336R receptors were more inhibited by ifenprodil and RO 25-9681 than wildtype epsilon1 receptors in ligand binding assays but not in functional assays. Selected mutations of epsilon2E200 and epsilon2E201 also decreased the sensitivity of receptors to ifenprodil and RO 25-6981. These results suggest that RO 25-6981 shares structural determinants with ifenprodil and other modulators in the NR2B subunit.

Pharmacological activation of metabotropic glutamate receptors (mGluRs) can inhibit synaptic transmission; however, relatively little evidence exists regarding the physiological conditions under which such autoreceptors are activated by synaptically released glutamate. Bath application of selective group II mGluR agonists profoundly inhibited field excitatory postsynaptic potentials (fEPSPs) evoked by stimulation of the perforant path inputs to both the mid-molecular layer of the dentate gyrus and the stratum lacunosum moleculare of the CA1. Application of the group II selective mGluR antagonist LY341495 resulted in an increase in the relative amplitude of a test fEPSP evoked 200 ms after a conditioning burst, but not after a single conditioning stimulus, in both pathways. Antagonist application also resulted in a marked increase in the relative amplitude of test population spikes evoked in the dentate gyrus following a conditioning burst. These observations are consistent with a presynaptic autoinhibitory action of group II metabotropic receptors that is revealed following burst stimulation of the pathway, consistent with their localisation in the preterminal zone. Activation of group II mGluRs during theta-gamma pattern discharge of projection neurones in the entorhinal cortex is likely to play an important role in the regulation of synaptic transmission and plasticity in the perforant pathway.

We have examined the influence of reducing conditions on the activity of group-I or -II metabotropic glutamate receptors. In cultured cerebellar granule cells or in hippocampal slices, the reducing agent dithiothreitol (DTT) inhibited the stimulation of polyphosphoinositide (PPI) hydrolysis elicited by group-I mGlu receptor agonists without affecting responses to norepinephrine or carbamylcholine. Similarly, DTT reduced the increase in intracellular free Ca(2+) induced by glutamate in HEK-293 cells expressing mGlu5 receptors. In adult hippocampal slices, the selective group-II mGlu receptor agonist, (2S,1'R,2'R,3'R)-2-(2, 3-dicarboxycyclopropyl)glycine (DCG-IV) had no effect per se on PPI hydrolysis, but potentiated the response to quisqualate. Although DTT substantially attenuated the action of quisqualate, it did not affect the potentiation by DCG-IV, suggesting that group-II mGlu receptors are resistant to extracellular reduction. Accordingly, DTT did not affect the inhibition of forskolin-stimulated cAMP formation induced by maximally effective concentrations of group-II mGlu receptor agonists in hippocampal slices or in CHO cells expressing mGlu2 receptors. At structural level, DTT differentially affected the aggregation state of mGlu1a, -2/3 or -5 receptors. In immunoblots performed under non-reducing conditions, mGlu1a, -2/3 or -5 antibodies labeled exclusively a high-molecular weight band, corresponding to receptor dimers. Under reducing conditions, mGlu1a or -5 receptors were detected as monomers, whereas a large proportion of mGlu2/3 receptors was still present in a dimeric form. We conclude that reducing conditions differentially influence the aggregation state of group-I and -II mGlu receptors and suggest that dimerization affects the functional activity of native mGlu receptors.

We have used site-directed mutagenesis in conjunction with homologous recombination to generate two mouse lines carrying point mutations in the glycine binding site of the NMDAR1 subunit (Grin1). Glycine concentration–response curves from acutely dissociated hippocampal neurons revealed a 5- and 86-fold reduction in receptor glycine affinity in mice carryingGrin1D481NandGrin1K483Qmutations, respectively, whereas receptor glutamate affinity remained unaffected. Homozygous mutantGrin1D481Nanimals are viable and fertile and appear to develop normally. However, homozygous mutantGrin1K483Qanimals are significantly lighter at birth, do not feed, and die within a few days. No gross abnormalities in CNS anatomy were detected in eitherGrin1D481NorGrin1K483Qmice. Interestingly,in situhybridization and Western blot analysis revealed changes in the expression levels of NMDA receptor subunits inGrin1D481Nmice relative to wild type that may represent a compensatory response to the reduction in receptor glycine affinity.Grin1D481Nmice exhibited deficits in hippocampal theta burst-induced long-term potentiation (LTP) and spatial learning and also a reduction in sensitivity to NMDA-induced seizures relative to wild-type controls, consistent with a reduced activation of NMDA receptors. Mutant mice exhibited normal prepulse inhibition but showed increased startle reactivity. Preliminary analysis indicated that the mice exhibit a decreased natural aversion to an exposed environment. The lethal phenotype ofGrin1K483Qanimals confirms the critical role of NMDA receptor activation in neonatal survival. A milder reduction in receptor glycine affinity results in an impairment of LTP and spatial learning and alterations in anxiety-related behavior, providing further evidence for the role of NMDA receptor activation in these processes.

The murine F9-derived 1C11 clone exhibits a stable epithelial morphology, expresses nestin, an early neuroectodermal marker, and expresses genes involved in neuroectodermal cell fate. Upon appropriate induction, 100% of 1C11 precursor cells develop neurite extensions and acquire neuronal markers (N-CAM, synaptophysin, gammagamma-enolase, and neurofilament) as well as the general functions of either serotonergic (1C11*(/5HT)) (5HT, 5-hydroxytryptamine) or noradrenergic (1C11**(/NE)) (NE, norepinephrine) neurons. The two programs are shown to be mutually exclusive. 1C11 thus behaves as a neuroepithelial cell line with a dual bioaminergic fate. 1C11*(/5HT) cells implement a functional 5-HT transporter and thereby a complete serotonergic phenotype within 4 days, whereas 5-HT(1B/D), 5-HT(2B), and 5-HT(2A) receptors are sequentially induced. The accurate time schedule of catecholaminergic differentiation was defined. Catecholamine synthesis, storage, and catabolism are acquired within 4 days; the noradrenergic phenotype is complete at day 12 and includes a functional norepinephrine transporter and an alpha(1D)-adrenoreceptor (day 8). The time-dependent onset of neurotransmitter-associated functions proper to either program is similar to in vivo observations. Along each pathway, the selective induction of serotonergic or adrenergic receptors is shown to be an essential part of the differentiation program, since they promote an autoregulation of the corresponding phenotype.

The effect of several metabotropic ligands and di- or tripeptides were tested on the binding of [3H]-L(+)-2-amino-4-phosphonobutyric acid ([3H]-L-AP4) on rat mGlu4 receptor. For selected compounds, the functional activity was determined on this receptor using the guanosine-5′[γ-35S]-thiotriphosphate [γ-35S]-GTP binding assay. Using the scintillation proximity assay, [3H]-L-AP4 saturation analysis gave binding parameters KD and Bmax values of 150 nM and 9.3 pmoles mg−1 protein, respectively. The specific binding was inhibited concentration-dependently by several mGlu receptor ligands, and their rank order of affinity was established. Several peptides inhibited the [3H]-L-AP4 binding with the following rank order of potency: glutamate-glutamate>glutamate-glutamate-leucine=aspartate - glutamate>>glutamate - glutamate-aspartate>lactoyl-glutamate>>aspartate-aspartate. Aspartate-phenylalanine-methyl ester (aspartame) was inactive up to 1 mM and guanosine-5′-monophosphate and inosine-5′-monophosphate were inactive up to 100 μM. The [γ-35S]-GTP binding functional assay was used to determine the agonist activities of the different compounds. For the rat mGlu4 agonists, L-AP4 and L-glutamate, the correlation between their occupancy and activation of the receptor was close to one. The peptides, Glu-Glu, Asp-Glu and Glu-Glu-Asp failed to stimulate the [γ-35S]-GTP binding at receptor occupancy greater than 80% and Glu-Glu-Leu appeared to be a weak partial agonist. These peptides did not elicit a clear dose-dependent umami perception. However, Glu-lac showed a good correlation between its potency to stimulate the [γ-35S]-GTP binding and its affinity for displacement of [3H]-L-AP4 binding. These data are in agreement with the peptide taste assessment in human subjects, which showed that the acid derivatives of glutamate had characteristics similar to umami. British Journal of Pharmacology (1999) 128, 1027–1034; doi:10.1038/sj.bjp.0702885

Heterocyclic enol ethers of type 1 were studied with respect to the inhibition of 1S,3R-ACPD (10μM)-stimulated GTP γ35S binding on rat mGluR2 transfected cell membranes. The structure activity relationship with regard to the substitution pattern of the phenyl ring, the oxygen substituent and the nature of the heterocycle is discussed.

A series of 5H-thiazolo[3,2-a]pyrimidine derivatives 1 was studied with respect to the inhibition of 1S,3R-ACPD (10μM)-stimulated GTP γ 35 S binding on rat mGlu2 receptor transfected cell membranes. The influence of substituents at position 6 and 7 as well as the substitution pattern of the two phenyl-rings in position 2 and 5 on the activity is discussed.

Two new spliced variants of the human metabotropic glutamate receptor 8 (HmGluR8), designated HmGluR8b and HmGluR8c, were identified in a human fetal brain cDNA library. The HmGluR8b and c differ from previously reported HmGluR8a by the out-of-frame insertions of 55-bp and 74-bp, respectively. The 55-bp insertion which contains a stop codon resulted in substitution of the last 16 amino acids in the C-terminus of HmGluR8a with 16 different amino acids in HmGluR8b. The 74-bp insertion introduces a frame shift in the predicted translation resulting in termination of the polypeptide before the putative seven transmembrane domains. Thus, the predicted HmGluR8c protein is 501 amino acids long and could represent a secreted isoform of the receptor. The pattern of mRNA expression of mGluR8 variants in human brain were analyzed by RT-PCR, Northern blot and in situ hybridization. Both HmGluR8a and b are expressed with similar abundance in fetal and adult brains. The in situ hybridization results indicate a predominantly glial cell expression of HmGluR8c in human brain. The three isoforms were transiently expressed in CHO cells from Semliki Forest Virus vectors. [3H]l-AP4 binding was performed on the cell membranes and the saturation curves showed the presence of a binding site with KD values of 249 and 182 nM and Bmax values of 13.6 and 10.5 pmoles/mg protein for HmGluR8a and b, respectively. For the six mGluR ligands studied, a similar rank order of potency was observed on both HmGluRa and b: l-AP4>l-SOP=l-CCG I>l-glutamate>DCG IV>LY 354740.

The study of signaling cascades and of functional interactions between 5-hydroxytryptamine (5-HT) receptor pathways with heterogenous brain cell populations remains an arduous task. We took advantage of a serotonergic cell line to elucidate cross-talks between 5-HT receptors and to demonstrate the involvement of two 5-HT2 receptor subtypes in the regulation of 5-HT1B/1D function. The inducible 1C11 cell line has the unique property of acquiring within 4 days a complete serotonergic phenotype (1C11* cells), including three 5-HT receptors. 5-HT1B/1D and 5-HT2B receptors are expressed since day 2 of the serotonergic differentiation while 5-HT2A receptors are induced at day 4. We first established that 5-HT2B receptors are coupled with the phospholipase A2 (PLA2)-mediated release of arachidonic acid (AA) and that the activation of 5-HT2B receptors in 1C11*d2 cells inhibits the 5-HT1B/1D receptor function via a cyclooxygenase-dependent AA metabolite. At day 4, this 5-HT2B-mediated inhibition of the 5-HT1B/1D function can be blocked upon concomitant 5-HT2A activation although a 5-HT2A/PLA2 positive coupling was evidenced. This suggests the existence in 1C11*d4 cells of pathway(s) for 5-HT2A receptors, distinct from PLC and PLA2. Finally, this study reveals the antagonistic roles of 5-HT2A and 5-HT2B receptors in regulating the function of 5-HT1B/1D, a receptor involved in neuropsychiatric disorders and migraine pathogenesis.

Co-activation of group I metabotropic glutamate (mGlu) receptors and adenosine receptors resulted in an augmented cyclic AMP response in primary cultures of rat striatal neurones. L-glutamate and the selective group I agonist, (S)-dihydroxyphenylglycine (S-DHPG) evoked concentration-dependent potentiations of cyclic AMP accumulation stimulated by the adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA), with EC50 values of 3.41+/-0. 39 and 5.69+/-1.64 microM, respectively, and maximal augmentations of approximately 350% at concentrations of 100 microM. The S-DHPG potentiation was inhibited by group I mGlu receptor antagonists and a protein kinase C inhibitor, Ro 31-8220, implicating products of PI hydrolysis in this effect. Furthermore, L-glutamate and S-DHPG stimulated PI hydrolysis in striatal neuronal cultures with similar EC50 values to those observed for the augmentation of NECA cyclic AMP responses (5.19+/-1.18 and 3.78+/-1.42 microM, respectively). In situ hybridization and immunofluorescence techniques indicate that group I mGlu receptor-evoked potentiations are likely to be mediated via mGlu5 receptors, which are expressed at high levels in these cultures. In contrast to cross-chopped slices of neonatal rat striatum, of equivalent age, the group II mGlu receptor agonist, (2S, 2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) was without effect on NECA- or forskolin-stimulated cyclic AMP responses in primary striatal neuronal cultures. This lack of effect might be due to a low level of expression of group II mGlu receptors in cultured striatal neurones.

The in vitro binding of [3H]LY354740, the first high affinity group II-selective metabotropic glutamate (mGlu) receptor radioligand, was characterized in rat cortical, hippocampal, and thalamic membranes as well as in rat brain sections. [3H]LY354740 binding was saturable in all regions investigated. Nonspecific binding (in the presence of 10 microM DCG-IV) was approximately 8% of the total. Ionotropic glutamate receptor agonists, N-methyl-D-aspartate, (R,S)-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid/kainate, a Na+-dependent glutamate uptake blocker as well as a group I-selective mGlu receptor agonist (all up to 1 mM) did not inhibit [3H]LY354740 binding to cortical membranes. However, several known metabotropic receptor ligands inhibited the binding with the following rank order of potency: LY354740 = LY341495 > (2S,2'R, 3'R)-2-(2',3'-dicarboxycyclopropyl)glycine = (2S,1'S, 2'S)-2-(2-carboxycyclopropyl)glycine > glutamate = (1S, 3R)-1-aminocyclopentane-1,3-dicarboxylic acid > (2S,1'S, 2'S)-2-methyl-2-(2-carboxycyclopropyl)-glycine > quisqualate > ibotenate > L-2-amino-3-phosphonopropionic acid = (S)-alpha-methyl-4-carboxyphenylglycine > L-(+)-2-amino-4-phosphonobutyric acid. N-Acetyl-aspartyl-glutamate, (2S)-alpha-ethylglutamic acid, and (R, S)-alpha-methyl-4-phosphonophenylglycine inhibited [3H]LY354740 binding in a biphasic manner. Guanosine-5'-O-(3-thiotriphosphate concentration-dependently and almost completely inhibited the binding. Finally, in parasagittal sections of rat brain, a high density of specific binding was observed in the accessory olfactory bulb, cortical regions (layers 1-3 > 4-6), caudate putamen, molecular layers of the hippocampus and dentate gyrus, presubiculum, retrosplenial cortex, anteroventral thalamic nuclei, and cerebellar granular layer, reflecting its preferential (perhaps not exclusive) affinity for presynaptic and postsynaptic mGlu2 receptors. Thus, the pharmacology, tissue distribution, and sensitivity to guanosine-5'-O-(3-thiotriphosphate show that [3H]LY354740 binding probably occurs to group II mGlu receptors in rat brain.

1. The binding of the new selective group II metabotropic glutamate receptor radioligand, [3H]-(2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine ([3H]-DCG IV), was characterized in rat mGlu2 receptor-transfected CHO cell membranes. 2. [3H]-DCG IV binding was pH-dependent, but was not sensitive to temperature. Saturation analysis showed the presence of a single binding site, with a Kd value of 160 nM and a Bmax value of 10 pmol mg(-1) protein. Binding was not sensitive to Na+-dependent glutamate uptake blockers or Cl-dependent glutamate binding inhibitors. Furthermore, up to concentrations of 1 mM, the glutamate ionotropic receptor agonists, N-methyl-D-aspartic acid (NMDA), (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate, did not affect [3H]-DCG IV binding. 3. Of the compounds observed to inhibit [3H]-DCG IV binding, the most potent were the recently described selective group II agonist, (+)-2-aminobicyclo-[3.1.0]hexane-2,6-dicarboxylate (LY 354740; Ki value 16 nM) and antagonist, 2-amino-2-(2-carboxycyclopropan-1-yl)-3-(dibenzopyran-4-yl) propanoic acid (LY 341495; Ki value 19 nM). As expected, for a G-protein-coupled receptor, guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) inhibited [3H]-DCG IV binding in a concentration-dependent manner, with an IC50 value of 12 nNM. 4. A highly significant correlation was observed between the potencies of compounds able to inhibit [3H]-DCG IV binding and potencies obtained for agonist activity in a GTPgamma35S binding functional assay. In addition, these studies identified a number of compounds with previously unknown activity at mGlu2 receptors, including L(+)-2-amino-3-phosphonopropionic acid (L-AP3), L(+)-2-amino-5-phosphonopentanoic acid (L-AP5), 3-((RS)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (R-CPP), N-acetyl-L-aspartyl-L-glutamic acid (NAAG) and (RS)-alpha-methylserine-O-phosphate (MSOP).

We previously described a negative allosteric modulator (NAM) of FSHR (ADX61623) that blocked FSH-induced cAMP and progesterone production but did not block estradiol production. That FSHR NAM did not affect FSH-induced preovulatory follicle development as evidenced by the lack of an effect on the number of FSH-dependent oocytes found in the ampullae following ovulation with hCG. A goal is the development of a nonsteroidal contraceptive. Toward this end, a high-throughput screen using human FSHR identified an additional nonsteroidal small molecule (ADX68692). Although ADX68692 behaved like ADX61623 in inhibiting production of cAMP and progesterone, it also inhibited FSH-induced estradiol in an in vitro rat granulosa primary cell culture bioassay. When immature, noncycling female rats were injected subcutaneously or by oral dosing prior to exogenous FSH administration, it was found that ADX68692 decreased the number of oocytes recovered from the ampullae. The estrous cycles of mature female rats were disrupted by administration by oral gavage of 25 mg/kg and 10 mg/kg ADX68692. In the highest dose tested (25 mg/kg), 55% of animals cohabited with mature males had implantation sites compared to 33% in the 10 mg/kg group and 77% in the control group. A surprising finding was that a structural analog ADX68693, while effectively blocking progesterone production with similar efficacy as ADX68692, did not block estrogen production and despite better oral availability did not decrease the number of oocytes found in the ampullae even when used at 100 mg/kg. These data demonstrate that because of biased antagonism of the FSHR, nonsteroidal contraception requires that both arms of the FSHR steroidogenic pathway must be effectively blocked, particularly estrogen biosynthesis. Thus, a corollary to these findings is that it seems reasonable to propose that the estrogen-dependent diseases such as endometriosis may benefit from inhibition of FSH action at the ovary using the FSHR NAM approach.

3,5-Dihydroxyphenylglycine (DHPG), (S)-3-hydroxyphenylglycine and (S)-4-carboxy-3-hydroxyphenylglycine (S-4C3HPG) stimulated phosphoinositide hydrolysis in neonatal rat cortical slices, but with lower maximal effect, in comparison with 2S,1'S,2'S-2-(2'-carboxycyclopropyl)glycine (L-CCG I) or (1S,3R)-1-aminocyclo-pentane-1,3-dicarboxylic acid (1S,3R-ACPD). DHPG, 1S,3R-ACPD, and S-4C3HPG also evoked a rapidly desensitizing increase in [Ca2+]i in cortical layers of neonatal brain slices. (R,S)-alpha-methyl-4-tetrazolyl-phenylglycine (MTPG), and (R,S)-alpha-methyl-4-phosphono-phenylglycine (MPPG) inhibited the increase of phosphoinositide hydrolysis elicited by 1S,3R-ACPD but not that by R,S-DHPG. In contrast, the selective group II receptor agonist (1S,2S,5R,6S)-2-amino-bicyclo-[3.1.0]-hexane-2,6-dicarboxylate (LY 354740) potentiated the response of R,S-DHPG. Finally, 8-(4-chlorophenylthio)-cAMP, a membrane permeant analogue of cAMP, reversed the stimulatory effect of 1S,3R-ACPD and S-4C3HPG on phosphoinositide hydrolysis and [Ca2+]i mobilization, without affecting the response induced by R,S-DHPG. These data suggest that, in neonatal rat cortex, the activation of group II metabotropic glutamate receptors potentiates the phosphoinositide hydrolysis and [Ca2+]i responses mediated by group I metabotropic glutamate receptors.

1. The effects of selective agonists at group I, II and III metabotropic glutamate receptors (mGluRs) on adenosine A2 receptor-mediated cyclic AMP formation were compared in cross-chopped slices of adult and neonatal (8 days old) rat striatum, in the presence of 1 u ml(-1) adenosine deaminase. 2. The group II selective agonist, (2S,1R,2R,3R)-2-(2,3-dicarboxycyclopropyl)glycine (DCG-IV), elicited a potentiation of 5'-N-ethylcarboxamidoadenosine (NECA)-stimulated cyclic AMP production with similar potencies in adult (EC50 value 122 +/- 35 nM) and neonatal (EC50 value 285 +/-6 nM) brain. In contrast, the group I selective agonist (S)-dihydroxyphenylglycine ((S)-DHPG) augmented the NECA cyclic AMP response in neonatal striatum (EC50 value 9 +/- 1 microM), but at a concentration of 100 microM, (S)-DHPG failed to affect the NECA response in adult striatal slices. 3. The potentiation evoked by (S)-DHPG was specific for group I mGluRs as (2S,3S,4S,)-2-methyl-2-(carboxycyclopropyl)glycine (MCCG), a group II antagonist, was ineffective on the (S)-DHPG (100 microM) response at a concentration (500 microM) which reversed a similar augmentation elicited by DCG-IV (300 nM). Furthermore, a protein kinase C inhibitor (Ro 31-8220, 10 microM) markedly reversed the effect of (S)-DHPG without affecting the response to DCG-IV. 4. The mGluR agonist (2S,3S,4S,)-alpha-(carboxycyclopropyl)glycine (L-CCG-I), elicited a greater potentiation of NECA-stimulated cyclic AMP production in neonatal striatum in comparison with that observed in adult rat brain. Moreover, EC50 values obtained from adult and neonatal striatum were 2 +/-1 microM and 9 +/-1 microM, respectively. These differences in potency might reflect co-activation of both group I and group II mGluRs by L-CCG-I in neonatal striatum. 5. Distinct patterns of mGluR expression in various brain areas might account for previous conflicting data on the nature of the mGluR able to evoke such potentiated responses.

Summary A series of new 10-methoxypyrazino[1,2- a ]indoles has been prepared and shown to be 5HT 2C receptor ligands. The studied compounds 10a–j were found to act as partial agonists at the 5HT 2C receptor, binding with high affinity and moderate selectivity versus 5HT 1A and 5HT 2A receptors, but inducing only a submaximal increase in phosphoinositol formation. Compound 10j was demonstrated to be active in animal models of obsessive-compulsive disorder, depression and panic anxiety.

We previously described a negative allosteric modulator (NAM) of FSHR (ADX61623) that blocked FSH-induced cAMP and progesterone production but did not block estradiol production. That FSHR NAM did not affect FSH-induced preovulatory follicle development as evidenced by the lack of an effect on the number of FSH-dependent oocytes found in the ampullae following ovulation with hCG. A goal is the development of a nonsteroidal contraceptive. Toward this end, a high-throughput screen using human FSHR identified an additional nonsteroidal small molecule (ADX68692). Although ADX68692 behaved like ADX61623 in inhibiting production of cAMP and progesterone, it also inhibited FSH-induced estradiol in an in vitro rat granulosa primary cell culture bioassay. When immature, noncycling female rats were injected subcutaneously or by oral dosing prior to exogenous FSH administration, it was found that ADX68692 decreased the number of oocytes recovered from the ampullae. The estrous cycles of mature female rats were disrupted by administration by oral gavage of 25 mg/kg and 10 mg/kg ADX68692. In the highest dose tested (25 mg/kg), 55% of animals cohabited with mature males had implantation sites compared to 33% in the 10 mg/kg group and 77% in the control group. A surprising finding was that a structural analog ADX68693, while effectively blocking progesterone production with similar efficacy as ADX68692, did not block estrogen production and despite better oral availability did not decrease the number of oocytes found in the ampullae even when used at 100 mg/kg. These data demonstrate that because of biased antagonism of the FSHR, nonsteroidal contraception requires that both arms of the FSHR steroidogenic pathway must be effectively blocked, particularly estrogen biosynthesis. Thus, a corollary to these findings is that it seems reasonable to propose that the estrogen-dependent diseases such as endometriosis may benefit from inhibition of FSH action at the ovary using the FSHR NAM approach.

Previous work has shown that 5-hydroxytryptamine (5-HT)2A receptors can be radiolabelled with various radioligands, including partial agonists, such as [125I]-DOI and [3H]-DOB, and antagonists, such as [3H]-ketanserin and [3H]-spiperone. Because 5-HT has high affinity for the 5-HT2A receptor when displacing [3H]-DOB, the purpose of the present study was to determine whether or not the receptor could be labelled with [3H]-5-HT and what would be the effect of labelling the receptor with various radioligands having differing efficacies at the receptor. Consequently, the human 5-HT2A receptor stably expressed in NIH 3T3 cells was radiolabelled with the endogenous agonist [3H]-5-HT, the partial agonist [3H]-DOB, and the antagonist [3H]-ketanserin. The receptor could be radiolabelled with [3H]-5-HT with a Kd value of 1.3 +/- 0.1 nM and a Bmax value of 3461 +/- 186 fmoles/mg protein and the radiolabelling was sensitive to the stable guanosine 5'-triphosphate (GTP) analogue guanylyl-imidodiphosphate (GMP-PNP). Ketanserin labeled significantly more receptors (Kd = 1.1 +/- 0.1 nM: Bmax = 27,684 +/- 1500 fmoles/mg protein) than [3H]-DOB (Kd = 0.8 +/- 0.08 nM: Bmax = 8332 +/- 16 fmoles/mg protein) which, in turn, labelled significantly more receptors than [3H]-5-HT. The apparent affinity of antagonists did not change when the receptor was radiolabelled with either [3H]-agonists or [3H]-antagonists; however, agonists had a higher apparent affinity for [3H]-agonist-labeled receptors than for [3H]-antagonist-labeled receptors. Therefore, the apparent affinity of agonists for the 5-HT2A receptor estimated from displacement experiments depends on the intrinsic efficacy of the radioligand used.

Metabotropic glutamate receptor 7 (mGlu(7)) has been suggested to be a promising novel target for treatment of a range of disorders, including anxiety, post-traumatic stress disorder, depression, drug abuse, and schizophrenia. Here we characterized a potent and selective mGlu(7) negative allosteric modulator (NAM) (+)-6-(2,4-dimethylphenyl)-2-ethyl-6,7-dihydrobenzo[d]oxazol-4(5H)-one (ADX71743). In vitro, Schild plot analysis and reversibility tests at the target confirmed the NAM properties of the compound and attenuation of L-(+)-2-amino-4-phosphonobutyric acid-induced synaptic depression confirmed activity at the native receptor. The pharmacokinetic analysis of ADX71743 in mice and rats revealed that it is bioavailable after s.c. administration and is brain penetrant (cerebrospinal fluid concentration/total plasma concentration ratio at C(max) = 5.3%). In vivo, ADX71743 (50, 100, 150 mg/kg, s.c.) caused no impairment of locomotor activity in rats and mice or activity on rotarod in mice. ADX71743 had an anxiolytic-like profile in the marble burying and elevated plus maze tests, dose-dependently reducing the number of buried marbles and increasing open arm exploration, respectively. Whereas ADX71743 caused a small reduction in amphetamine-induced hyperactivity in mice, it was inactive in the mouse 2,5-dimethoxy-4-iodoamphetamine-induced head twitch and the rat conditioned avoidance response tests. In addition, the compound was inactive in the mouse forced swim test. These data suggest that ADX71743 is a suitable compound to help unravel the physiologic role of mGlu(7) and to better understand its implication in central nervous system diseases. Our in vivo tests using ADX71743, reported here, suggest that pharmacological inhibition of mGlu(7) is a valid approach for developing novel pharmacotherapies to treat anxiety disorders, but may not be suitable for treatment of depression or psychosis.

High quality gamete production in males and females requires the pituitary gonadotropin follicle stimulating hormone (FSH). In this report a novel chemical class of small molecule inhibitors of FSH receptor (FSHR) is described. ADX61623, a negative allosteric modulator (NAM), increased the affinity of interaction between (125)I-hFSH and human FSHR (hFSHR) five fold. This form of FSHR occupied simultaneously by FSH and ADX61623 was inactive for cAMP and progesterone production in primary cultures of rat granulosa cells. In contrast, ADX61623 did not block estrogen production. This demonstrates for the first time, biased antagonism at the FSHR. To determine if ADX61623 blocked FSH induction of follicle development in vivo, a bioassay to measure follicular development and oocyte production in immature female rats was validated. ADX61623 was not completely effective in blocking FSH induced follicular development in vivo at doses up to 100mg/kg as oocyte production and ovarian weight gain were only moderately reduced. These data illustrate that FSHR couples to multiple signaling pathways in vivo. Suppression of one pool of FSHR uncouples Gαs and cAMP production, and decreases progesterone production. Occupancy of another pool of FSHR sensitizes granulosa cells to FSH induced estradiol production. Therefore, ADX61623 is a useful tool to investigate further the mechanism of the FSHR signaling dichotomy. This may lead to a greater understanding of the signaling infrastructure which enables estrogen biosynthesis and may prove useful in treating estrogen dependent disease.

Heterocyclic enol ethers of type 1 were studied with respect to the inhibition of 1S,3R-ACPD (10μM)-stimulated GTP γ35S binding on rat mGluR2 transfected cell membranes. The structure activity relationship with regard to the substitution pattern of the phenyl ring, the oxygen substituent and the nature of the heterocycle is discussed.