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Mutational Analysis and Molecular Modeling of the Binding Pocket of the Metabotropic Glutamate 5 Receptor Negative Modulator 2-Methyl-6-(phenylethynyl)-pyridine

Pharma Division, Discovery Research CNS (P.M., M.-T.Z., J.P., C.D., C.K., C.F., R.H.P.P.) and Chemistry (N.K.), F. Hoffmann-La Roche Ltd., Basel, Switzerland

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. Using site-directed mutagenesis, [³H]MPEP binding, a functional Ca²⁺ mobilization assay, and rhodopsin-based homology modeling, we identified eight residues (Pro-654^3.36, Tyr-658^3.40, Leu-743^5.47, Thr-780^6.44, Trp-784^6.48, Phe-787^6.51, Tyr-791^6.55, and Ala-809^7.47) that are crucial for MPEP-binding to rat mGlu5 receptors. Four mutations, Y658^3.40V, W784^6.48A, F787^6.51A, and A809^7.47V, caused complete loss of [³H]MPEP binding and also blocked the MPEP-mediated inhibition of quisqualate-induced intracellular Ca²⁺ mobilization. To visualize these experimental findings, we have constructed a homology model based on the X-ray crystal of bovine rhodopsin and have suggested a possible binding mode of MPEP. We propose that MPEP via its interactions with a network of the aromatic residues including Phe-658^3.40 in transmembrane (TM) 3 helix and Trp-798^6.48, Phe-787^6.51, and Tyr-791^6.55 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.

Address correspondence to: Dr. Pari Malherbe, F. Hoffmann-La Roche Ltd., PRBN-D, Bldg. 69/327, CH-4070 Basel, Switzerland. E-mail: parichehr.malherbe{at}roche.com

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