title: Molecular mechanism of positive allosteric modulation of the metabotropic glutamate receptor 2 by JNJ-46281222.
abstract: OBJECTIVE
  Allosteric modulation of the mGlu2 receptor is a potential strategy for treatment of various neurological and psychiatric disorders. Here, we describe the in vitro characterization of the mGlu2 positive allosteric modulator (PAM) JNJ-46281222 and its radiolabelled counterpart [(3) H]-JNJ-46281222. Using this novel tool, we also describe the allosteric effect of orthosteric glutamate binding and the presence of a bound G protein on PAM binding and use computational approaches to further investigate the binding mode.
  
  
  METHODS
  We have used radioligand binding studies, functional assays, site-directed mutagenesis, homology modelling and molecular dynamics to study the binding of JNJ-46281222.
  
  
  RESULTS
  JNJ-46281222 is an mGlu2 -selective, highly potent PAM with nanomolar affinity (KD = 1.7 nM). Binding of [(3) H]-JNJ-46281222 was increased by the presence of glutamate and greatly reduced by the presence of GTP, indicating the preference for a G protein bound state of the receptor for PAM binding. Its allosteric binding site was visualized and analysed by a computational docking and molecular dynamics study. The simulations revealed amino acid movements in regions expected to be important for activation. The binding mode was supported by [(3) H]-JNJ-46281222 binding experiments on mutant receptors.
  
  
  CONCLUSIONS
  Our results obtained with JNJ-46281222 in unlabelled and tritiated form further contribute to our understanding of mGlu2 allosteric modulation. The computational simulations and mutagenesis provide a plausible binding mode with indications of how the ligand permits allosteric activation. This study is therefore of interest for mGlu2 and class C receptor drug discovery.
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research (LACDR), Leiden University, Leiden, The Netherlands.
authors.forename: Maarten L J
authors.initials: ML
authors.lastname: Doornbos
authors.affiliation: Janssen Research and Development, Toledo, Spain.|Laboratori de Medicina Computacional Unitat de Bioestadistica, Facultat de Medicina, Universitat Autonoma de Barcelona, Bellaterra, Spain.
authors.forename: Laura
authors.initials: L
authors.lastname: Pérez-Benito
authors.affiliation: Janssen Research and Development, Toledo, Spain.
authors.forename: Gary
authors.initials: G
authors.lastname: Tresadern
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research (LACDR), Leiden University, Leiden, The Netherlands.
authors.forename: Thea
authors.initials: T
authors.lastname: Mulder-Krieger
authors.affiliation: Janssen Research and Development, Beerse, Belgium.
authors.forename: Ilse
authors.initials: I
authors.lastname: Biesmans
authors.affiliation: Janssen Research and Development, Toledo, Spain.
authors.forename: Andrés A
authors.initials: AA
authors.lastname: Trabanco
authors.affiliation: Janssen Research and Development, Toledo, Spain.
authors.forename: Jose María
authors.initials: JM
authors.lastname: Cid
authors.affiliation: Janssen Research and Development, Beerse, Belgium.
authors.forename: Hilde
authors.initials: H
authors.lastname: Lavreysen
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research (LACDR), Leiden University, Leiden, The Netherlands.
authors.forename: Adriaan P
authors.initials: AP
authors.lastname: IJzerman
authors.affiliation: Division of Medicinal Chemistry, Leiden Academic Centre for Drug Research (LACDR), Leiden University, Leiden, The Netherlands.
authors.forename: Laura H
authors.initials: LH
authors.lastname: Heitman
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D000818:Animals; D016466:CHO Cells; D003412:Cricetulus; D006801:Humans; D008958:Models, Molecular; D010880:Piperidines / Q000494:pharmacology*; D011725:Pyridines / Q000494:pharmacology*; D018094:Receptors, Metabotropic Glutamate / Q000235:genetics / Q000378:metabolism*; D014230:Triazoles / Q000494:pharmacology*
chemical_list: C000611822:3-(cyclopropylmethyl)-7-((4-phenyl-1-piperidinyl)methyl)-8-(trifluoromethyl)-1,2,4-triazolo(4,3-a)pyridine; D010880:Piperidines; D011725:Pyridines; D018094:Receptors, Metabotropic Glutamate; D014230:Triazoles; C108822:metabotropic glutamate receptor 2