Structural determinants of allosteric antagonism at metabotropic glutamate receptor 2: mechanistic studies with new potent negative allosteric modulators.

BACKGROUND AND PURPOSE Altered glutamatergic neurotransmission is linked to several neurological and psychiatric disorders. Metabotropic glutamate receptor 2 (mGlu(2)) plays an important role on the presynaptic control of glutamate release and negative allosteric modulators (NAMs) acting on mGlu(2/3) receptors are under assessment for their potential as antidepressants, neurogenics and cognitive enhancers. Two new potent mGlu(2/3) NAMs, RO4988546 and RO5488608, are described in this study and the allosteric binding site in the transmembrane (TM) domain of mGlu(2) is characterized. EXPERIMENTAL APPROACH Site directed mutagenesis, functional measurements and beta(2)-adrenoceptor-based modelling of mGlu(2) were employed to identify important molecular determinants of two new potent mGlu(2/3) NAMs. KEY RESULTS RO4988546 and RO5488608 affected both [H-3]-LY354740 agonist binding at the orthosteric site and the binding of a tritiated positive allosteric modulator (H-3-PAM), indicating that NAMs and PAMs could have overlapping binding sites in the mGlu(2) TM domain. We identified eight residues in the allosteric binding pocket that are crucial for non-competitive antagonism of agonist-dependent activation of mGlu(2) and directly interact with the NAMs: Arg(3.28), Arg(3.29), Phe(3.36), His(E2.52), Leu(5.43), Trp(6.48), Phe(6.55) and Val(7.43). The mGlu(2) 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 [H-3]-LY354740 agonist binding inhibition, which is independent of the highly conserved Trp(6.48) residue. CONCLUSIONS AND IMPLICATIONS This is the first complete molecular investigation of the allosteric binding pocket of mGlu(2) and Group II mGluRs and provides new information on what determines mGlu(2) NAMs selective interactions and effects.