title: Structural determinants of allosteric antagonism at metabotropic glutamate receptor 2: mechanistic studies with new potent negative allosteric modulators.
abstract: OBJECTIVE
  Altered glutamatergic neurotransmission is linked to several neurological and psychiatric disorders. Metabotropic glutamate receptor 2 (mGlu₂) plays an important role on the presynaptic control of glutamate release and negative allosteric modulators (NAMs) acting on mGlu₂/₃ receptors are under assessment for their potential as antidepressants, neurogenics and cognitive enhancers. Two new potent mGlu₂/₃ NAMs, RO4988546 and RO5488608, are described in this study and the allosteric binding site in the transmembrane (TM) domain of mGlu₂ is characterized.
  
  
  METHODS
  Site directed mutagenesis, functional measurements and β₂-adrenoceptor-based modelling of mGlu₂ were employed to identify important molecular determinants of two new potent mGlu₂/₃ NAMs.
  
  
  RESULTS
  RO4988546 and RO5488608 affected both [³H]-LY354740 agonist binding at the orthosteric site and the binding of a tritiated positive allosteric modulator (³H-PAM), indicating that NAMs and PAMs could have overlapping binding sites in the mGlu₂ TM domain. We identified eight residues in the allosteric binding pocket that are crucial for non-competitive antagonism of agonist-dependent activation of mGlu₂ and directly interact with the NAMs: Arg³·²⁸, Arg³·²⁹, Phe³·³⁶, His(E2.52) , Leu⁵·⁴³, Trp⁶·⁴⁸, Phe⁶·⁵⁵ and Val⁷·⁴³. The mGlu₂ specific residue His(E2.52) is likely to be involved in selectivity and residues located in the outer part of the binding pocket are more important for [³H]-LY354740 agonist binding inhibition, which is independent of the highly conserved Trp⁶·⁴⁸ residue.
  
  
  CONCLUSIONS
  This is the first complete molecular investigation of the allosteric binding pocket of mGlu₂ and Group II mGluRs and provides new information on what determines mGlu₂ NAMs selective interactions and effects.
authors.affiliation: Neuroscience Research, F. Hoffmann-La Roche Ltd, Basel, Switzerland.
authors.forename: L
authors.initials: L
authors.lastname: Lundström
authors.forename: C
authors.initials: C
authors.lastname: Bissantz
authors.forename: J
authors.initials: J
authors.lastname: Beck
authors.forename: J G
authors.initials: JG
authors.lastname: Wettstein
authors.forename: T J
authors.initials: TJ
authors.lastname: Woltering
authors.forename: J
authors.initials: J
authors.lastname: Wichmann
authors.forename: S
authors.initials: S
authors.lastname: Gatti
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001643:Bridged Bicyclo Compounds / Q000737:chemistry / Q000494:pharmacology; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D006224:Cricetinae; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D051381:Rats; D018094:Receptors, Metabotropic Glutamate / Q000037:antagonists & inhibitors* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment / Q000379:methods; D013329:Structure-Activity Relationship
chemical_list: D001643:Bridged Bicyclo Compounds; D018094:Receptors, Metabotropic Glutamate; C108822:metabotropic glutamate receptor 2; C104753:eglumetad; D002118:Calcium