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

ABSTRACT: Altered glutamatergic neurotransmission is linked to several neurological and psychiatric disorders. Metabotropic glutamate receptor 2 (mGlu?) plays an important role on the presynaptic control of glutamate release and negative allosteric modulators (NAMs) acting on mGlu?/? receptors are under assessment for their potential as antidepressants, neurogenics and cognitive enhancers. Two new potent mGlu?/? NAMs, RO4988546 and RO5488608, are described in this study and the allosteric binding site in the transmembrane (TM) domain of mGlu? is characterized.Site directed mutagenesis, functional measurements and ??-adrenoceptor-based modelling of mGlu? were employed to identify important molecular determinants of two new potent mGlu?/? NAMs.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 mGlu? TM domain. We identified eight residues in the allosteric binding pocket that are crucial for non-competitive antagonism of agonist-dependent activation of mGlu? and directly interact with the NAMs: Arg³·²?, Arg³·²?, Phe³·³?, His(E2.52) , Leu?·?³, Trp?·??, Phe?·?? and Val?·?³. The mGlu? 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 Trp?·?? residue.This is the first complete molecular investigation of the allosteric binding pocket of mGlu? and Group II mGluRs and provides new information on what determines mGlu? NAMs selective interactions and effects.

SUBMITTER: Lundstrom L

PROVIDER: S-EPMC3188907 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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

Lundström L L Bissantz C C Beck J J Wettstein J G JG Woltering T J TJ Wichmann J J Gatti S S

British journal of pharmacology 20110901 2b

<h4>Background and purpose</h4>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 ...[more]

PMID: 21470207

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Project description:Negative allosteric modulation of the metabotropic glutamate 5 (mGlu5) receptor has emerged as a potential strategy for the treatment of neurologic disorders. Despite the success in preclinical studies, many mGlu5 negative allosteric modulators (NAMs) that have reached clinical trials failed due to lack of efficacy. In this study, we provide a detailed in vitro pharmacological characterization of nine clinically and preclinically tested NAMs. We evaluated inhibition of l-glutamate-induced signaling with Ca2+ mobilization, inositol monophosphate (IP1) accumulation, extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, and real-time receptor internalization assays on rat mGlu5 expressed in HEK293A cells. Moreover, we determined association rates (kon) and dissociation rates (koff), as well as NAM affinities with [3H]methoxy-PEPy binding experiments. kon and koff values varied greatly between the nine NAMs (34- and 139-fold, respectively) resulting in long receptor residence times (>400 min) for basimglurant and mavoglurant, medium residence times (10-30 min) for AZD2066, remeglurant, and (RS)-remeglurant, and low residence times (<10 mins) for dipraglurant, F169521, F1699611, and STX107. We found that all NAMs inhibited l-glutamate-induced mGlu5 receptor internalization, generally with a similar potency to IP1 accumulation and ERK1/2 phosphorylation, whereas Ca2+ mobilization was less potently inhibited. Operational model of allosterism analyses revealed that dipraglurant and (RS)-remeglurant were biased toward (affinity) receptor internalization and away (cooperativity) from the ERK1/2 phosphorylation pathway, respectively. Our study is the first to measure mGlu5 NAM binding kinetics and negative allosteric modulation of mGlu5 receptor internalization and adds significant new knowledge about the molecular pharmacology of a diverse range of clinically relevant NAMs. SIGNIFICANCE STATEMENT: The metabotropic glutamate 5 (mGlu5) receptor is important in many brain functions and implicated in several neurological pathologies. Negative allosteric modulators (NAMs) have shown promising results in preclinical models but have so far failed in human clinical trials. Here we provide the most comprehensive and comparative molecular pharmacological study to date of nine preclinically/clinically tested NAMs at the mGlu5 receptor, which is also the first study to measure ligand binding kinetics and negative allosteric modulation of mGlu5 receptor internalization.

Dataset Information

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

Publications

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

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