Molecular determinants of positive allosteric modulation of the human metabotropic glutamate receptor 2

Abstract

BACKGROUND AND PURPOSEThe activation of the metabotropic glutamate receptor 2 (mGlu2) reduces glutamatergic transmission in brain regions where excess excitatory signalling is implicated in disorders such as anxiety and schizophrenia. Positive allosteric modulators (PAMs) can provide a fine-tuned potentiation of these receptors' function and are being investigated as a novel therapeutic approach. An extensive set of mutant human mGlu2 receptors were used to investigate the molecular determinants that are important for positive allosteric modulation at this receptor. EXPERIMENTAL APPROACHSite-directed mutagenesis, binding and functional assays were employed to identify amino acids important for the activity of nine PAMs. The data from the radioligand binding and mutagenesis studies were used with computational docking to predict a binding mode at an mGlu2 receptor model based on the recent structure of the mGlu1 receptor. KEY RESULTSNew amino acids in TM3 (R635, L639, F643), TM5 (L732) and TM6 (W773, F776) were identified for the first time as playing an important role in the activity of mGlu2 PAMs. CONCLUSIONS AND IMPLICATIONSThis extensive study furthers our understanding of positive allosteric modulation of the mGlu2 receptor and can contribute to improved future design of mGlu2 PAMs. Abbreviations

1,3,5‐Trisubstituted Pyrazoles as Potent Negative Allosteric Modulators of the mGlu2/3 Receptors

“…As a general trend in this pyrazole series the in vitro activity for the mGlu 2 receptor increases with lipophilicity (as exemplified by introduction of the chlorine substituent in compounds 18 – 19 ). This is not unusual, as the allosteric binding site is believed to be in the 7‐transmembrane (7TM) region . Nevertheless, this higher lipophilicity results in lower unbound brain fraction.…”

“…To prove mGlu 2 target engagement, an ex vivo occupancy assay in rat native tissue was set up . Site‐directed mutagenesis studies have shown that mGlu 2 NAMs and PAMs share multiple amino acid residues in their allosteric binding pocket which influence their activity, therefore suggesting a common binding site. Based on this, the tritiated selective mGlu 2 positive allosteric modulator (PAM) ligand [ 3 H]JNJ‐46281222 previously developed by our team, was used to conduct the ex vivo occupancy studies .…”

“…Site‐directed mutagenesis studies have shown that mGlu 2 NAMs and PAMs share multiple amino acid residues in their allosteric binding pocket which influence their activity, therefore suggesting a common binding site. Based on this, the tritiated selective mGlu 2 positive allosteric modulator (PAM) ligand [ 3 H]JNJ‐46281222 previously developed by our team, was used to conduct the ex vivo occupancy studies . The mGlu 2 receptor occupancy assay was performed via ex vivo autoradiography, 1 h after administration of the test compound.…”

Leveraging allostery to improve G protein-coupled receptor (GPCR)-directed therapeutics: cannabinoid receptor 1 as discovery target

“…In silico and modeling approaches such as docking and molecular dynamics (MD) simulation have been utilized for probing ligand fit and calculating time-dependent GPCR conformational changes in the millisecond to femtosecond range following allosteric ligand engagement, in some cases adjunctive to crystallographic analyses [14,28,99,105–108]. Experimental techniques such as nuclear magnetic resonance spectroscopy (NMR) [109,110], site-directed mutagenesis coupled with quantitative pharmacological analyses [111–114] or long-timescale MD simulations [115] have also been applied to gain structural insight into GPCR allostery. Such approaches have also helped define structure–activity relationships among GPCR-targeted allosteric scaffolds [116,117].…”

Positive allosteric modulators of metabotropic glutamate 2 receptors in schizophrenia treatment

“…Early mutagenesis studies identified three amino acid residues in transmembrane (TM) segments IV and V of mGlu 2 R to be critical for the activity of LY487379 and several other mGlu 2 R PAMs [111, 112]. A more recent extensive study identified additional residues in TM III, V, and VI to play an important role in the activity of mGlu 2 R PAMs [113]. Interestingly, 3 residues in TM IV of mGlu 2 R have been found to be essential for the heteromerization of 5-HT 2A -mGlu 2 R [68] indicating that TMD region of mGlu 2 R is implicated in different types of allosteric interactions.…”