title: Mutational analysis and molecular modeling of the allosteric binding site of a novel, selective, noncompetitive antagonist of the metabotropic glutamate 1 receptor.
abstract: A model of the rmGlu1 seven-transmembrane domain complexed with a negative allosteric modulator, 1-ethyl-2-methyl-6-oxo-4-(1,2,4,5-tetrahydro-benzo[d]azepin-3-yl)- 1,6-dihydro-pyrimidine-5-carbonitrile (EM-TBPC) was constructed. Although the mGlu receptors belong to the family 3 G-protein-coupled receptors with a low primary sequence similarity to rhodopsin-like receptors, the high resolution crystal structure of rhodopsin was successfully applied as a template in this model and used to select residues for site-directed mutagenesis. Three mutations, F801(6.51)A, Y805(6.55)A, and T815(7.39)M caused complete loss of the [(3)H]EM-TBPC binding and blocked the EM-TBPC-mediated inhibition of glutamate-evoked G-protein-coupled inwardly rectifying K(+) channel current and [Ca(2+)](i) response. The mutation W798(6.48)F increased the binding affinity of antagonist by 10-fold and also resulted in a marked decrease in the IC(50) value (4 versus 128 nm) compared with wild type. The V757(5.47)L mutation led to a dramatic reduction in binding affinity by 13-fold and a large increase in the IC(50) value (1160 versus 128 nm). Two mutations, N7474(5.51)A and N7504(5.54)A, increased the efficacy of the EM-TBPC block of the glutamate-evoked [Ca(2+)](i) response. We observed a striking conservation in the position of critical residues. The residues Val-757(5.47), Trp-798(6.48), Phe-801(6.51), Tyr-805(6.55), and Thr-815(7.39) are critical determinants of the EM-TBPC-binding pocket of the mGlu1 receptor, validating the rhodopsin crystal structure as a template for the family 3 G-protein-coupled receptors. In our model, the aromatic ring of EM-TBPC might interact with the cluster of aromatic residues formed from Trp-798(6.48), Phe-801(6.51), and Tyr-805(6.55), thereby blocking the movement of the TM6 helix, which is crucial for receptor activation.
authors.affiliation: Pharma Division, Discovery Research CNS and Chemistry, F. Hoffmann-La Roche Ltd., CH-4070 Basel, Switzerland. parichehr.malherbe@roche.com
authors.forename: Pari
authors.initials: P
authors.lastname: Malherbe
authors.forename: Nicole
authors.initials: N
authors.lastname: Kratochwil
authors.forename: Frédéric
authors.initials: F
authors.lastname: Knoflach
authors.forename: Marie-Thérèse
authors.initials: MT
authors.lastname: Zenner
authors.forename: James N C
authors.initials: JN
authors.lastname: Kew
authors.forename: Claudia
authors.initials: C
authors.lastname: Kratzeisen
authors.forename: Hans P
authors.initials: HP
authors.lastname: Maerki
authors.forename: Geo
authors.initials: G
authors.lastname: Adam
authors.forename: Vincent
authors.initials: V
authors.lastname: Mutel
mesh_terms: D000495:Allosteric Site; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018094:Receptors, Metabotropic Glutamate / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D018094:Receptors, Metabotropic Glutamate