Reprint of Pharmacological and molecular characterization of the positive allosteric modulators of metabotropic glutamate receptor 2.

The metabotropic glutamate receptor 2 (mGlu2) plays an important role in the presynaptic control of glutamate release and several mGlu2 positive allosteric modulators (PAMs) have been under assessment for their potential as antipsychotics. The binding mode of mGlu2 PAMs is better characterized in functional terms while few data are available on the relationship between allosteric and orthosteric binding sites. Pharmacological studies characterizing binding and effects of two different chemical series of mGlu2 PAMs are therefore carried out here using the radiolabeled mGlu2 agonist 3[H]-LY354740 and mGlu2 PAM 3[H]-2,2,2-TEMPS. A multidimensional approach to the PAM mechanism of action shows that mGlu2 PAMs increase the affinity of 3[H]-LY354740 for the orthosteric site of mGlu2 as well as the number of 3[H]-LY354740 binding sites. 3[H]-2,2,2-TEMPS binding is also enhanced by the presence of LY354740. New residues in the allosteric rat mGlu2 binding pocket are identified to be crucial for the PAMs ligand binding, among these Tyr3.40 and Asn5.46. Also of remark, in the described experimental conditions S731A (Ser5.42) residue is important only for the mGlu2 PAM LY487379 and not for the compound PAM-1: an example of the structural differences among these mGlu2 PAMs. This study provides a summary of the information generated in the past decade on mGlu2 PAMs adding a detailed molecular investigation of PAM binding mode. Differences among mGlu2 PAM compounds are discussed as well as the mGlu2 regions interacting with mGlu2 PAM and NAM agents and residues driving mGlu2 PAM selectivity. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'.

Pharmacological and molecular characterization of the positive allosteric modulators of metabotropic glutamate receptor 2.

The metabotropic glutamate receptor 2 (mGlu2) plays an important role in the presynaptic control of glutamate release and several mGlu2 positive allosteric modulators (PAMs) have been under assessment for their potential as antipsychotics. The binding mode of mGlu2 PAMs is better characterized in functional terms while few data are available on the relationship between allosteric and orthosteric binding sites. Pharmacological studies characterizing binding and effects of two different chemical series of mGlu2 PAMs are therefore carried out here using the radiolabeled mGlu2 agonist 3[H]-LY354740 and mGlu2 PAM 3[H]-2,2,2-TEMPS. A multidimensional approach to the PAM mechanism of action shows that mGlu2 PAMs increase the affinity of 3[H]-LY354740 for the orthosteric site of mGlu2 as well as the number of 3[H]-LY354740 binding sites. 3[H]-2,2,2-TEMPS binding is also enhanced by the presence of LY354740. New residues in the allosteric rat mGlu2 binding pocket are identified to be crucial for the PAMs ligand binding, among these Tyr3.40 and Asn5.46. Also of remark, in the described experimental conditions S731A (Ser5.42) residue is important only for the mGlu2 PAM LY487379 and not for the compound PAM-1: an example of the structural differences among these mGlu2 PAMs. This study provides a summary of the information generated in the past decade on mGlu2 PAMs adding a detailed molecular investigation of PAM binding mode. Differences among mGlu2 PAM compounds are discussed as well as the mGlu2 regions interacting with mGlu2 PAM and NAM agents and residues driving mGlu2 PAM selectivity.

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 (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. EXPERIMENTAL APPROACH: Site directed mutagenesis, functional measurements and ß2-adrenoceptor-based modelling of mGlu2 were employed to identify important molecular determinants of two new potent mGlu2/3 NAMs. KEY 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 AND IMPLICATIONS: 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.

Protein-based virtual screening of chemical databases. 1. Evaluation of different docking/scoring combinations.

Three different database docking programs (Dock, FlexX, Gold) have been used in combination with seven scoring functions (Chemscore, Dock, FlexX, Fresno, Gold, Pmf, Score) to assess the accuracy of virtual screening methods against two protein targets (thymidine kinase, estrogen receptor) of known three-dimensional structure. For both targets, it was generally possible to discriminate about 7 out of 10 true hits from a random database of 990 ligands. The use of consensus lists common to two or three scoring functions clearly enhances hit rates among the top 5% scorers from 10% (single scoring) to 25-40% (double scoring) and up to 65-70% (triple scoring). However, in all tested cases, no clear relationships could be found between docking and ranking accuracies. Moreover, predicting the absolute binding free energy of true hits was not possible whatever docking accuracy was achieved and scoring function used. As the best docking/consensus scoring combination varies with the selected target and the physicochemistry of target-ligand interactions, we propose a two-step protocol for screening large databases: (i) screening of a reduced dataset containing a few known ligands for deriving the optimal docking/consensus scoring scheme, (ii) applying the latter parameters to the screening of the entire database.