Metabotropic glutamate (mGlu) 5 is a G-protein-coupled metabotropic glutamate receptor that plays an important role as a modulator of synaptic plasticity, ion channel activity, and excitotoxicity. 2-Methyl-6-(phenylethynyl)-pyridine (MPEP) is a highly potent, noncompetitive, selective, and systemically active antagonist of mGlu5 receptors. It binds to a novel allosteric site that resides within the seven-transmembrane domain of mGlu5 receptors. 6.55 in TM6 helix prevents the movement of TM6 helix relative to TM3 helix, a step that is required for receptor activation, and consequently stabilizes the inactive conformation of mGlu5 receptor. In the TM6 region, we observed a striking similarity between the critical residues involved in MPEP-binding site with those of previously identified as 1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)-1,6-dihydropyrimidine-5-carbonitrile-binding pocket of mGlu1, pointing to a common mechanism of inhibition shared by both antagonists.

The metabotropic glutamate receptor 5 (mGlu5) is a glutamateactivated class C G protein-coupled receptor widely expressed in the central nervous system and clinically investigated as a drug target for a range of indications, including depression, Parkinson's disease, and fragile X syndrome. Here, we present the novel potent, selective, and orally bioavailable mGlu5 negative allosteric modulator with inverse agonist properties 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine (CTEP). CTEP binds mGlu5 with low nanomolar affinity and shows Ͼ1000-fold selectivity when tested against 103 targets, including all known mGlu receptors. CTEP penetrates the brain with a brain/plasma ratio of 2.6 and displaces the tracer [ 3 H]3-(6-methyl-pyridin-2-ylethynyl)-cyclohex-2-enone-O-methyl-oxime (ABP688) in vivo in mice from brain regions expressing mGlu5 with an average ED 50 equivalent to a drug concentration of 77.5 ng/g in brain tissue. This novel mGlu5 inhibitor is active in the stress-induced hyperthermia procedure in mice and the Vogel conflict drinking test in rats with minimal effective doses of 0.1 and 0.3 mg/kg, respectively, reflecting a 30-to 100-fold higher in vivo potency compared with 2-methyl-6-(phenylethynyl)pyridine (MPEP) and fenobam. CTEP is the first reported mGlu5 inhibitor with both long half-life of approximately 18 h and high oral bioavailability allowing chronic treatment with continuous receptor blockade with one dose every 48 h in adult and newborn animals. By enabling long-term treatment through a wide age range, CTEP allows the exploration of the full therapeutic potential of mGlu5 inhibitors for indications requiring chronic receptor inhibition.

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Fenobam [N-(3-chlorophenyl)-N¢-(4,5-dihydro-1-methyl-4-oxo-1H-imidazole-2-yl)urea], a clinically validated non-benzodiazepine anxiolytic, has been shown to be a potent and non-competitive metabotropic glutamate (mGlu)-5 receptor antagonist. In the present study, we have used the site-directed mutagenesis coupled with three-dimensional receptor-based pharmacophore modelling to elucidate the interacting mode of fenobam within the seven-transmembrane domain (7TMD) of mGlu5 receptor and its comparison with that of 2-methyl-6-(phenylethynyl)pyridine (MPEP), the prototype antagonist. The common residues involved in the recognition of MPEP and fenobam include Pro654 3.36 , Tyr658 3.40 , Thr780 6.44 , Trp784 6.48 , Phe787 6.51 , Tyr791 6.55 and Ala809 7.47 . The differentiating residues between both modulators' interacting modes are Arg647 3.29 , Ser657 3.39 and Leu743 5.47 . Our data suggest that these chemically unrelated mGlu5 antagonists act similarly, probing a functionally unique region of the 7TMD. Using [ 3 H]inositol phosphates accumulation assay, we have also identified the critical residues involved in the inverse agonist effect of MPEP. The mutation W784 6.48 A completely blocked the inverse agonist activity of MPEP; two mutations F787 6.51 A and Y791 6.55 A, caused a drastic decrease in the MPEP inverse agonism. Furthermore, these three mutations led to an increased efficacy of quisqualate without having any effect on its potency. The fact that the residues Trp784 6.48 and Phe787 6.51 are essential equally in antagonism and inverse agonism effects emphasizes again the key role of these residues and the involvement of a common transmembrane network in receptor inactivation by MPEP. Keywords: fenobam, inverse agonist, metabotropic glutamate receptor, MPEP, pharmacophore modelling, phosphoinositide hydrolysis. The major excitatory neurotransmitter in the brain, glutamate, acts at two distinct classes of receptors, ionotropic (NMDA, AMPA and kainate) and metabotropic (mGlu; Pin and Acher 2002). On the basis of sequence homology, second messenger coupling and pharmacology, eight mGlu receptors are divided into three groups: group I (mGlu1 and -5) receptors couple to the activation of phospholipase C leading to phosphoinositide hydrolysis and elevation of intracellular Ca 2+ levels ([Ca 2+ ] i ), group II (mGlu2 and -3) and group III (mGlu4, are negatively coupled to cAMP production (Pin and Acher 2002).

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