mGlu3 Metabotropic Glutamate Receptors as a Target for the Treatment of Absence Epilepsy: Preclinical and Human Genetics Data.

BACKGROUND: Previous studies suggest that different metabotropic glutamate (mGlu) receptor subtypes are potential drug targets for treating absence epilepsy. However, no information is available on mGlu3 receptors. OBJECTIVE: To examine whether (i) changes of mGlu3 receptor expression/signaling are found in the somatosensory cortex and thalamus of WAG/Rij rats developing spontaneous absence seizures; (ii) selective activation of mGlu3 receptors with LY2794193 affects the number and duration of spikewave discharges (SWDs) in WAG/Rij rats; and (iii) a genetic variant of GRM3 (encoding the mGlu3 receptor) is associated with absence epilepsy. METHODS: Animals: immunoblot analysis of mGlu3 receptors, GAT-1, GLAST, and GLT-1; realtime PCR analysis of mGlu3 mRNA levels; assessment of mGlu3 receptor signaling; EEG analysis of SWDs; assessment of depressive-like behavior. Humans: search for GRM3 and GRM5 missense variants in 196 patients with absence epilepsy or other Idiopathic Generalized Epilepsy (IGE)/ Genetic Generalized Epilepsy (GGE) and 125,748 controls. RESULTS: mGlu3 protein levels and mGlu3-mediated inhibition of cAMP formation were reduced in the thalamus and somatosensory cortex of pre-symptomatic (25-27 days old) and symptomatic (6-7 months old) WAG/Rij rats compared to age-matched controls. Treatment with LY2794193 (1 or 10 mg/kg, i.p.) reduced absence seizures and depressive-like behavior in WAG/Rij rats. LY2794193 also enhanced GAT1, GLAST, and GLT-1 protein levels in the thalamus and somatosensory cortex. GRM3 and GRM5 gene variants did not differ between epileptic patients and controls. CONCLUSION: We suggest that mGlu3 receptors modulate the activity of the cortico-thalamo-cortical circuit underlying SWDs and that selective mGlu3 receptor agonists are promising candidate drugs for absence epilepsy treatment.

Metabotropic Glutamate Receptors Modulate Exocytotic Tau Release and Propagation.

Using synaptosomes purified from the brains of two transgenic mouse models overexpressing mutated human tau (TgP301S and Tg4510) and brains of patients with sporadic Alzheimer's disease, we showed that aggregated and hyperphosphorylated tau was both present in purified synaptosomes and released in a calcium- and synaptosome-associated protein of 25 kDa (SNAP25)-dependent manner. In all mouse and human synaptosomal preparations, tau release was inhibited by the selective metabotropic glutamate receptor 2/3 (mGluR2/3) agonist LY379268, an effect prevented by the selective mGlu2/3 antagonist LY341495. LY379268 was also able to block pathologic tau propagation between primary neurons in an in vitro microfluidic cellular model. These novel results are transformational for our understanding of the molecular mechanisms mediating tau release and propagation at synaptic terminals in Alzheimer's disease and suggest that these processes could be inhibited therapeutically by the selective activation of presynaptic G protein-coupled receptors. SIGNIFICANCE STATEMENT: Pathological tau release and propagation are key neuropathological events underlying cognitive decline in Alzheimer's disease patients. This paper describes the role of regulated exocytosis, and the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) protein SNAP25, in mediating tau release from rodent and human synaptosomes. This paper also shows that a selective mGluR2/3 agonist is highly effective in blocking tau release from synaptosomes and tau propagation between neurons, opening the way to the discovery of novel therapeutic approaches to this devastating disease.

Behavioural and biochemical responses to methamphetamine are differentially regulated by mGlu2 and mGlu3 metabotropic glutamate receptors in male mice.

Group II metabotropic glutamate receptors (mGlu2 and mGlu3 receptors) shape mechanisms of methamphetamine addiction, but the individual role played by the two subtypes is unclear. We measured methamphetamine-induced conditioned place preference (CPP) and motor responses to single or repeated injections of methamphetamine in wild-type, mGlu2-/-, and mGlu3-/-mice. Only mGlu3-/-mice showed methamphetamine preference in the CPP test. Motor response to the first methamphetamine injection was dramatically reduced in mGlu2-/-mice, unless these mice were treated with the mGlu5 receptor antagonist, MTEP. In contrast, methamphetamine-induced sensitization was increased in mGlu3-/-mice compared to wild-type mice. Only mGlu3-/-mice sensitized to methamphetamine showed increases in phospho-ERK1/2 levels in the nucleus accumbens (NAc) and free radical formation in the NAc and medial prefrontal cortex. These changes were not detected in mGlu2-/-mice. We also measured a series of biochemical parameters related to the mechanism of action of methamphetamine in naïve mice to disclose the nature of the differential behavioural responses of the three genotypes. We found a reduced expression and activity of dopamine transporter (DAT) and vesicular monoamine transporter-2 in the NAc and striatum of mGlu2-/-and mGlu3-/-mice, whereas expression of the DAT adaptor, syntaxin 1A, was selectively increased in the striatum of mGlu3-/-mice. Methamphetamine-stimulated dopamine release in striatal slices was largely reduced in mGlu2-/-, but not in mGlu3-/-, mice. These findings suggest that drugs that selectively enhance mGlu3 receptor activity or negatively modulate mGlu2 receptors might be beneficial in the treatment of methamphetamine addiction and associated brain damage.

Structural Basis for ( S)-3,4-Dicarboxyphenylglycine (DCPG) As a Potent and Subtype Selective Agonist of the mGlu8 Receptor.

( S)-3,4-Dicarboxyphenylglycine (DCPG) was first reported in 2001 as a potent orthosteric agonist with high subtype selectivity for the mGlu8 receptor, but the structural basis for its high selectivity is not well understood. We have solved a cocrystal structure of recombinant human mGlu8 amino terminal domain (ATD) protein bound to ( S)-DCPG, which possesses the largest lobe opening angle observed to date among known agonist-bound mGlu ATD crystal structures. The binding conformation of ( S)-DCPG observed in the crystal structure is significantly different from that in the homology model built from an l-glutamate-bound rat mGlu1 ATD crystal structure, which has a smaller lobe opening angle. This highlights the importance of considering various lobe opening angles when modeling mGlu ATD-ligand complex. New homology models of other mGlu receptors based on the ( S)-DCPG-bound mGlu8 ATD crystal structure were explored to rationalize ( S)-DCPG's high mGlu8 receptor subtype selectivity.

Metabotropic Glutamate2 Receptors Play a Key Role in Modulating Head Twitches Induced by a Serotonergic Hallucinogen in Mice.

There is substantial evidence that glutamate can modulate the effects of 5-hydroxytryptamine2A (5-HT2A) receptor activation through stimulation of metabotropic glutamate2/3 (mGlu2/3) receptors in the prefrontal cortex. Here we show that constitutive deletion of the mGlu2 gene profoundly attenuates an effect of 5-HT2A receptor activation using the mouse head twitch response (HTR). MGlu2 and mGlu3 receptor knockout (KO) as well as age-matched ICR (CD-1) wild type (WT) mice were administered (±)1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and observed for head twitch activity. DOI failed to produce significant head twitches in mGlu2 receptor KO mice at a dose 10-fold higher than the peak effective dose in WT or mGlu3 receptor KO mice. In addition, the mGlu2/3 receptor agonist LY379268, and the mGlu2 receptor positive allosteric modulator (PAM) CBiPES, potently blocked the HTR to DOI in WT and mGlu3 receptor KO mice. Conversely, the mGlu2/3 receptor antagonist LY341495 (10 mg/kg) increased the HTR produced by DOI (3 mg/kg) in mGlu3 receptor KO mice. Finally, the mGlu2 receptor potentiator CBiPES was able to attenuate the increase in the HTR produced by LY341495 in mGlu3 receptor KO mice. Taken together, all of these results are consistent with the hypothesis that that DOI-induced head twitches are modulated by mGlu2 receptor activation. These results also are in keeping with a critical autoreceptor function for mGlu2 receptors in the prefrontal cortex with differential effects of acute vs. chronic perturbation (e.g., constitutive mGlu2 receptor KO mice). The robust attenuation of DOI-induced head twitches in the mGlu2 receptor KO mice appears to reflect the critical role of glutamate in ongoing regulation of 5-HT2A receptors in the prefrontal cortex. Future experiments with inducible knockouts for the mGlu2 receptor and/or selective mGlu3 receptor agonists/PAMs/antagonists could provide an important tools in understanding glutamatergic modulation of prefrontal cortical 5-HT2A receptor function.

Determination of L-AP4-bound human mGlu8 receptor amino terminal domain structure and the molecular basis for L-AP4's group III mGlu receptor functional potency and selectivity.

L-2-Amino-4-phosphonobutyric acid (L-AP4) is a known potent and selective agonist for the Group III mGlu receptors. However, it does not show any selectivity among the individual group III mGlu subtypes. In order to understand the molecular basis for this group selectivity, we solved the first human mGlu8 amino terminal domain (ATD) crystal structures in complex with L-glu and L-AP4. In comparison with other published L-glu-bound mGlu ATD structures, we have observed L-glu binds in a significantly different manner in mGlu1. Furthermore, these new structures provided evidence that both the electronic and steric nature of the distal phosphate of L-AP4 contribute to its exquisite Group III functional agonist potency and selectivity.

Synthesis and Pharmacological Characterization of C4ß-Amide-Substituted 2-Aminobicyclo[3.1.0]hexane-2,6-dicarboxylates. Identification of (1 S,2 S,4 S,5 R,6 S)-2-Amino-4-[(3-methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid (LY2794193), a Highly Potent and Selective mGlu3 Receptor Agonist.

Multiple therapeutic opportunities have been suggested for compounds capable of selective activation of metabotropic glutamate 3 (mGlu3) receptors, but small molecule tools are lacking. As part of our ongoing efforts to identify potent, selective, and systemically bioavailable agonists for mGlu2 and mGlu3 receptor subtypes, a series of C4ß-N-linked variants of (1 S,2 S,5 R,6 S)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 1 (LY354740) were prepared and evaluated for both mGlu2 and mGlu3 receptor binding affinity and functional cellular responses. From this investigation we identified (1 S,2 S,4 S,5 R,6 S)-2-amino-4-[(3-methoxybenzoyl)amino]bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 8p (LY2794193), a molecule that demonstrates remarkable mGlu3 receptor selectivity. Crystallization of 8p with the amino terminal domain of hmGlu3 revealed critical binding interactions for this ligand with residues adjacent to the glutamate binding site, while pharmacokinetic assessment of 8p combined with its effect in an mGlu2 receptor-dependent behavioral model provides estimates for doses of this compound that would be expected to selectively engage and activate central mGlu3 receptors in vivo.

Translational Pharmacology of the Metabotropic Glutamate 2 Receptor-Preferring Agonist LY2812223 in the Animal and Human Brain.

LY2812223 [(1R,2S,4R,5R,6R)-2-amino-4-(1H-1,2,4-triazol-3-ylsulfanyl)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid] was identified via structure-activity studies arising from the potent metabotropic glutamate mGlu2/3 receptor agonist LY354740 [(+)-2-aminobicyclo[3.1.0] hexane-2,6-dicarboxylic acid] as an mGlu2-preferring agonist. This pharmacology was determined using stably transfected cells containing either the human mGlu2 or mGlu3 receptor. We extended the pharmacological evaluation of LY2812223 to native brain tissues derived from relevant species used for preclinical drug development as well as human postmortem brain tissue. This analysis was conducted to ensure pharmacological translation from animals to human subjects in subsequent clinical studies. A guanosine 5'-O-(3-[35S]thio)triphosphate (GTPγS) functional binding assay, a method for measuring Gi-coupled signaling that is inherent to the group 2 mGlu receptors, was used to evaluate LY2812223 pharmacology of native mGlu receptors in mouse, rat, nonhuman primate, and human cortical brain tissue samples. In native tissue membranes, LY2812223 unexpectedly acted as a partial agonist across all species tested. Activity of LY2812223 was lost in cortical membranes collected from mGlu2 knockout mice, but not those from mGlu3 knockout mice, providing additional support for mGlu2-preferring activity. Other signal transduction assays were used for comparison with the GTP binding assay (cAMP, calcium mobilization, and dynamic mass redistribution). In ectopic cell line-based assays, LY2812223 displayed near maximal agonist responses at the mGlu2 receptor across all assay formats, while it showed no functional agonist activity at the mGlu3 receptor except in the cAMP assay. In native brain slices or membranes that express both mGlu2 and mGlu3 receptors, LY2812223 displayed unexpected partial agonist activity, which may suggest a functional interplay between these receptor subtypes in the brain.

Broad spectrum efficacy with LY2969822, an oral prodrug of metabotropic glutamate 2/3 receptor agonist LY2934747, in rodent pain models.

BACKGROUND AND PURPOSE: A body of evidence suggests activation of metabotropic glutamate 2/3 (mGlu2/3 ) receptors would be an effective analgesic in chronic pain conditions. Thus, the analgesic properties of a novel mGlu2/3 receptor agonist prodrug were investigated. EXPERIMENTAL APPROACH: After oral absorption, the prodrug LY2969822 rapidly converts to the brain penetrant, potent and subtype-selective mGlu2/3 receptor agonist LY2934747. Behavioural assessments of allodynia, hyperalgesia and nocifensive behaviours were determined in preclinical pain models after administration of LY2969822 0.3-10 mg·kg-1 . In addition, the ability of i.v. LY2934747 to modulate dorsal horn spinal cord wide dynamic range (WDR) neurons in spinal nerve ligated (SNL) rats was assessed. KEY RESULTS: Following treatment with LY2934747, the spontaneous activity and electrically-evoked wind-up of WDR neurons in rats that had undergone spinal nerve ligation and developed mechanical allodynia were suppressed. In a model of sensitization, orally administered LY2969822 prevented the nociceptive behaviours induced by an intraplantar injection of formalin. The on-target nature of this effect was confirmed by blockade with an mGlu2/3 receptor antagonist. LY2969822 prevented capsaicin-induced tactile hypersensitivity, reversed the SNL-induced tactile hypersensitivity and reversed complete Freund's adjuvant - induced mechanical hyperalgesia. The mGlu2/3 receptor agonist prodrug demonstrated efficacy in visceral pain models, including a colorectal distension model and partially prevented the nocifensive behaviours in the mouse acetic acid writhing model. CONCLUSIONS AND IMPLICATIONS: Following oral administration of the prodrug LY2969822, the mGlu2/3 receptor agonist LY2934747 was formed and this attenuated pain behaviours across a broad range of preclinical pain models.

In vitro pharmacological and rat pharmacokinetic characterization of LY3020371, a potent and selective mGlu2/3 receptor antagonist.

Metabotropic glutamate 2/3 (mGlu2/3) receptors are of considerable interest owing to their role in modulating glutamate transmission via presynaptic, postsynaptic and glial mechanisms. As part of our ongoing efforts to identify novel ligands for these receptors, we have discovered (1S,2R,3S,4S,5R,6R)-2-amino-3-[(3,4-difluorophenyl)sulfanylmethyl]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid; (LY3020371), a potent and selective orthosteric mGlu2/3 receptor antagonist. In this account, we characterize the effects of LY3020371 in membranes and cells expressing human recombinant mGlu receptor subtypes as well as in native rodent and human brain tissue preparations, providing important translational information for this molecule. In membranes from cells expressing recombinant human mGlu2 and mGlu3 receptor subtypes, LY3020371.HCl competitively displaced binding of the mGlu2/3 agonist ligand [3H]-459477 with high affinity (hmGlu2 Ki = 5.26 nM; hmGlu3 Ki = 2.50 nM). In cells expressing hmGlu2 receptors, LY3020371.HCl potently blocked mGlu2/3 agonist (DCG-IV)-inhibited, forskolin-stimulated cAMP formation (IC50 = 16.2 nM), an effect that was similarly observed in hmGlu3-expressing cells (IC50 = 6.21 nM). Evaluation of LY3020371 in cells expressing the other human mGlu receptor subtypes revealed high mGlu2/3 receptor selectivity. In rat native tissue assays, LY3020371 demonstrated effective displacement of [3H]-459477 from frontal cortical membranes (Ki = 33 nM), and functional antagonist activity in cortical synaptosomes measuring both the reversal of agonist-suppressed second messenger production (IC50 = 29 nM) and agonist-inhibited, K+-evoked glutamate release (IC50 = 86 nM). Antagonism was fully recapitulated in both primary cultured cortical neurons where LY3020371 blocked agonist-suppressed spontaneous Ca2+ oscillations (IC50 = 34 nM) and in an intact hippocampal slice preparation (IC50 = 46 nM). Functional antagonist activity was similarly demonstrated in synaptosomes prepared from epileptic human cortical or hippocampal tissues, suggesting a translation of the mGlu2/3 antagonist pharmacology from rat to human. Intravenous dosing of LY3020371 in rats led to cerebrospinal fluid drug levels that are expected to effectively block mGlu2/3 receptors in vivo. Taken together, these results establish LY3020371 as an important new pharmacological tool for studying mGlu2/3 receptors in vitro and in vivo. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'.

Discovery of (1S,2R,3S,4S,5R,6R)-2-Amino-3-[(3,4-difluorophenyl)sulfanylmethyl]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid Hydrochloride (LY3020371·HCl): A Potent, Metabotropic Glutamate 2/3 Receptor Antagonist with Antidepressant-Like Activity.

As part of our ongoing efforts to identify novel ligands for the metabotropic glutamate 2 and 3 (mGlu2/3) receptors, we have incorporated substitution at the C3 and C4 positions of the (1S,2R,5R,6R)-2-amino-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid scaffold to generate mGlu2/3 antagonists. Exploration of this structure-activity relationship (SAR) led to the identification of (1S,2R,3S,4S,5R,6R)-2-amino-3-[(3,4-difluorophenyl)sulfanylmethyl]-4-hydroxy-bicyclo[3.1.0]hexane-2,6-dicarboxylic acid hydrochloride (LY3020371·HCl, 19f), a potent, selective, and maximally efficacious mGlu2/3 antagonist. Further characterization of compound 19f binding to the human metabotropic 2 glutamate (hmGlu2) site was established by cocrystallization of this molecule with the amino terminal domain (ATD) of the hmGlu2 receptor protein. The resulting cocrystal structure revealed the specific ligand-protein interactions, which likely explain the high affinity of 19f for this site and support its functional mGlu2 antagonist pharmacology. Further characterization of 19f in vivo demonstrated an antidepressant-like signature in the mouse forced-swim test (mFST) assay when brain levels of this compound exceeded the cellular mGlu2 IC50 value.

Synthesis and Pharmacological Characterization of C4-(Thiotriazolyl)-substituted-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylates. Identification of (1R,2S,4R,5R,6R)-2-Amino-4-(1H-1,2,4-triazol-3-ylsulfanyl)bicyclo[3.1.0]hexane-2,6-dicarboxylic Acid (LY2812223), a Highly Potent, Functionally Selective mGlu2 Receptor Agonist.

Identification of orthosteric mGlu(2/3) receptor agonists capable of discriminating between individual mGlu2 and mGlu3 subtypes has been highly challenging owing to the glutamate-site sequence homology between these proteins. Herein we detail the preparation and characterization of a series of molecules related to (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate 1 (LY354740) bearing C4-thiotriazole substituents. On the basis of second messenger responses in cells expressing other recombinant human mGlu2/3 subtypes, a number of high potency and efficacy mGlu2 receptor agonists exhibiting low potency mGlu3 partial agonist/antagonist activity were identified. From this, (1R,2S,4R,5R,6R)-2-amino-4-(1H-1,2,4-triazol-3-ylsulfanyl)bicyclo[3.1.0]hexane-2,6-dicarboxylic acid 14a (LY2812223) was further characterized. Cocrystallization of 14a with the amino terminal domains of hmGlu2 and hmGlu3 combined with site-directed mutation studies has clarified the underlying molecular basis of this unique pharmacology. Evaluation of 14a in a rat model responsive to mGlu2 receptor activation coupled with a measure of central drug disposition provides evidence that this molecule engages and activates central mGlu2 receptors in vivo.

Synthesis and pharmacological characterization of C4-disubstituted analogs of 1S,2S,5R,6S-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate: identification of a potent, selective metabotropic glutamate receptor agonist and determination of agonist-bound human mGlu2 and mGlu3 amino terminal domain structures.

As part of our ongoing research to identify novel agents acting at metabotropic glutamate 2 (mGlu2) and 3 (mGlu3) receptors, we have previously reported the identification of the C4α-methyl analog of mGlu2/3 receptor agonist 1 (LY354740). This molecule, 1S,2S,4R,5R,6S-2-amino-4-methylbicyclo[3.1.0]hexane-2,6-dicarboxylate 2 (LY541850), exhibited an unexpected mGlu2 agonist/mGlu3 antagonist pharmacological profile, whereas the C4ß-methyl diastereomer (3) possessed dual mGlu2/3 receptor agonist activity. We have now further explored this structure-activity relationship through the preparation of cyclic and acyclic C4-disubstituted analogs of 1, leading to the identification of C4-spirocyclopropane 5 (LY2934747), a novel, potent, and systemically bioavailable mGlu2/3 receptor agonist which exhibits both antipsychotic and analgesic properties in vivo. In addition, through the combined use of protein-ligand X-ray crystallography employing recombinant human mGlu2/3 receptor amino terminal domains, molecular modeling, and site-directed mutagenesis, a molecular basis for the observed pharmacological profile of compound 2 is proposed.

Levels of the Rab GDP dissociation inhibitor (GDI) are altered in the prenatal restrain stress mouse model of schizophrenia and are differentially regulated by the mGlu2/3 receptor agonists, LY379268 and LY354740.

LY379268 and LY354740, two agonists of mGlu2/3 metabotropic glutamate receptors, display different potencies in mouse models of schizophrenia. This differential effect of the two drugs remains unexplained. We performed a proteomic analysis in cultured cortical neurons challenged with either LY379268 or LY354740. Among the few proteins that were differentially influenced by the two drugs, Rab GDP dissociation inhibitor-ß (Rab GDIß) was down-regulated by LY379268 and showed a trend to an up-regulation in response to LY354740. In cultured hippocampal neurons, LY379268 selectively down-regulated the α isoform of Rab GDI. Rab GDI inhibits the activity of the synaptic vesicle-associated protein, Rab3A, and is reduced in the brain of schizophrenic patients. We examined the expression of Rab GDI in mice exposed to prenatal stress ("PRS mice"), which have been described as a putative model of schizophrenia. Rab GDIα protein levels were increased in the hippocampus of PRS mice at postnatal days (PND)1 and 21, but not at PND60. At PND21, PRS mice also showed a reduced depolarization-evoked [(3)H]d-aspartate release in hippocampal synaptosomes. The increase in Rab GDIα levels in the hippocampus of PRS mice was reversed by a 7-days treatment with LY379268 (1 or 10 mg/kg, i.p.), but not by treatment with equal doses of LY354740. These data strengthen the validity of PRS mice as a model of schizophrenia, and show for the first time a pharmacodynamic difference between LY379268 and LY354740 which might be taken into account in an attempt to explain the differential effect of the two drugs across mouse models.

Synthesis and pharmacological characterization of 4-substituted-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylates: identification of new potent and selective metabotropic glutamate 2/3 receptor agonists.

As part of our ongoing interest in identifying novel agonists acting at metabotropic glutamate (mGlu) 2/3 receptors, we have explored the effect of structural modifications of 1S,2S,5R,6S-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate (LY354740), a potent and pharmacologically balanced mGlu2/3 receptor agonist. Incorporation of relatively small substituents (e.g., F, O) at the C4 position of this molecule resulted in additional highly potent mGlu2/3 agonists that demonstrate excellent selectivity over the other mGlu receptor subtypes, while addition of larger C4-substituents (e.g., SPh) led to a loss of agonist potency and/or the appearance of weak mGlu2/3 receptor antagonist activity. Further characterization of the α-fluoro-substituted analogue (LY459477) in vivo revealed that this molecule possesses good oral bioavailability in rats and effectively suppresses phencyclidine-evoked locomotor activity at doses that do not impair neuromuscular coordination. This molecule therefore represents a valuable new addition to the arsenal of pharmacological tools competent to investigate mGlu2/3 receptor function both in vitro and in vivo.

Discovery of (1R,2R)-N-(4-(6-isopropylpyridin-2-yl)-3-(2-methyl-2H-indazol-5-yl)isothiazol-5-yl)-2-methylcyclopropanecarboxamide, a potent and orally efficacious mGlu5 receptor negative allosteric modulator.

A novel series of selective negative allosteric modulators (NAMs) for metabotropic glutamate receptor 5 (mGlu5) was discovered from an isothiazole scaffold. One compound of this series, (1R,2R)-N-(4-(6-isopropylpyridin-2-yl)-3-(2-methyl-2H-indazol-5-yl)isothiazol-5-yl)-2-methylcyclopropanecarboxamide (24), demonstrated satisfactory pharmacokinetic properties and, following oral dosing in rats, produced dose-dependent and long-lasting mGlu5 receptor occupancy. Consistent with the hypothesis that blockade of mGlu5 receptors will produce analgesic effects in mammals, compound 24 produced a dose-dependent reduction in paw licking responses in the formalin model of persistent pain.

Pharmacological profiling of native group II metabotropic glutamate receptors in primary cortical neuronal cultures using a FLIPR.

The group II metabotropic glutamate (mGlu) receptors comprised of the mGlu2 and mGlu3 receptor subtypes have gained recognition in recent years as potential targets for psychiatric disorders, including anxiety and schizophrenia. In addition to studies already indicating which subtype mediates the anxiolytic and anti-psychotic effects observed in disease models, studies to help further define the preferred properties of selective group II mGlu receptor ligands will be essential. Comparison of the in vitro properties of these ligands to their in vivo efficacy and tolerance profiles may help provide these additional insights. We have developed a relatively high-throughput native group II mGlu receptor functional assay to aid this characterisation. We have utilised dissociated primary cortical neuronal cultures, which after 7 days in vitro have formed functional synaptic connections and display periodic and spontaneous synchronised calcium (Ca(2+)) oscillations in response to intrinsic action potential bursts. We herein demonstrate that in addition to non-selective group II mGlu receptor agonists, (2R,4R)-APDC, LY379268 and DCG-IV, a selective mGlu2 agonist, LY541850, and mGlu2 positive allosteric modulators, BINA and CBiPES, inhibit the frequency of synchronised Ca(2+) oscillations in primary cultures of rat and mouse cortical neurons. Use of cultures from wild-type, mGlu2(-/-), mGlu3(-/-) and mGlu2/3(-/-) mice allowed us to further probe the contribution of mGlu2 and mGlu3, and revealed LY541850 to be a partial mGlu2 agonist and a full mGlu3 antagonist. Overnight pre-treatment of cultures with these ligands revealed a preferred desensitisation profile after treatment with a positive allosteric modulator. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

CNS distribution of metabotropic glutamate 2 and 3 receptors: transgenic mice and [³H]LY459477 autoradiography.

Group II metabotropic glutamate (mGlu) receptor agonists were efficacious in randomized clinical research trials for schizophrenia and generalized anxiety disorder. The regional quantification of mGlu(2) and mGlu(3) receptors remains unknown. A selective and structurally novel mGlu(2/3) receptor agonist, 2-amino-4-fluorobicyclo[3.1.0]hexane-2,6-dicarboxylic acid (LY459477) was tritiated and the distribution of mGlu(2) and mGlu(3) receptors was studied in transgenic mice lacking either mGlu(2), mGlu(3) or both receptors. LY459477 is an agonist with 1-2 nM potency for rodent and human mGlu(2) and mGlu(3) receptors. The functional selectivity of LY459477 was demonstrated by over 640-fold selectivity and the displacement binding selectivity was greater than 320-fold for all glutamate receptors except mGlu(6) (∼230-fold). More than 1000-fold selectivity was demonstrated for all non-glutamate receptors known to be targeted by antipsychotic drugs. Like atypical antipsychotic drugs, LY459477 reversed in vitro electrophysiological effects of a serotonergic hallucinogen and behavioral effects of phencyclidine or amphetamine. There was virtually no binding of [(3)H]LY459477 to any brain region in mice with a deletion of both mGlu(2) and mGlu(3) receptors. Regions enriched in mGlu(2) receptors included the medial prefrontal cortex, select hippocampal regions, the medial mammillary nucleus, the medial habenula, and the cerebellar granular cell layer. Regions enriched in mGlu(3) receptors were the dorsolateral entorhinal cortex, the hippocampal CA1 field, the piriform cortex, the substantia nigra, the thalamic reticular nucleus, and primary sensory thalamic nuclei. These findings suggest [(3)H]LY459477 should be a useful tool to further define the role of mGlu(2) and mGlu(3) receptors throughout the brain with respect to major neuropsychiatric syndromes. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

Differentiating the roles of mGlu2 and mGlu3 receptors using LY541850, an mGlu2 agonist/mGlu3 antagonist.

Despite the potential therapeutic relevance of group II metabotropic glutamate (mGlu) receptors, there has been a lack of pharmacological tools for separating the roles of mGlu2 and mGlu3 receptor subtypes. LY541850 was claimed from human mGlu receptors expressed in non-neuronal cells to be a selective orthosteric mGlu2 agonist and mGlu3 antagonist. We have verified this pharmacological profile of LY541850 in hippocampal slices. Field excitatory post-synaptic potentials (fEPSPs) evoked by stimulation of the temporo-ammonic path (TAP) input to CA1 stratum lacunosum moleculare (SLM) were inhibited by LY541850 in mGlu3-/- mice (EC(50) 38 nM) and wild-type littermates (EC(50) 42 nM) to a similar extent but were not significantly affected in mGlu2-/- mice. The group II agonist, DCG-IV, inhibited the fEPSP in all three genotypes. Co-application of DCG-IV and LY541850 in mGlu3-/- and wild-type littermates resulted in an additive effect, whereas in mGlu2-/- mice, LY541850 reversed the inhibitory action of DCG-IV. These results confirm the selective mGlu2 agonist and mGlu3 antagonist actions of LY541850. A similar profile of activity was seen in medial perforant path synapse to the dentate gyrus. Systemic administration of LY541850 to wild-type mice, reduced the increase in locomotor activity following both phencyclidine and amphetamine administration. These data support the hypothesis that mGlu2 receptors mediate the antipsychotic effects of mixed group II agonists. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

Improved synthesis of C4α- and C4ß-methyl analogues of 2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate.

An efficient and divergent synthesis of C4α- and C4ß-methyl-substituted analogues of 2-aminobicyclo[3.1.0]hexane 2,6-dicarboxylate, which are important tools in the study of metabotropic glutamate receptor function, has been achieved. By taking advantage of an unanticipated facial selectivity of the bicyclo[3.1.0]hexane ring system, either the C4α- or C4ß-methyl substituent was introduced in a highly stereoselective and high-yielding manner.