Investigating Metabotropic Glutamate Receptor 5 Allosteric Modulator Cooperativity, Affinity, and Agonism: Enriching Structure-Function Studies and Structure-Activity Relationships

Abstract

Drug discovery programs increasingly are focusing on allosteric modulators as a means to modify the activity of G protein-coupled receptor (GPCR) targets. Allosteric binding sites are topographically distinct from the endogenous ligand (orthosteric) binding site, which allows for co-occupation of a single receptor with the endogenous ligand and an allosteric modulator that can alter receptor pharmacological characteristics. Negative allosteric modulators (NAMs) inhibit and positive allosteric modulators (PAMs) enhance the affinity and/or efficacy of orthosteric agonists. Established approaches for estimation of affinity and efficacy values for orthosteric ligands are not appropriate for allosteric modulators, and this presents challenges for fully understanding the actions of novel modulators of GPCRs. Metabotropic glutamate receptor 5 (mGlu 5 ) is a family C GPCR for which a large array of allosteric modulators have been identified. We took advantage of the many tools for probing allosteric sites on mGlu 5 to validate an operational model of allosterism that allows quantitative estimation of modulator affinity and cooperativity values. Affinity estimates derived from functional assays fit well with affinities measured in radioligand binding experiments for both PAMs and NAMs with diverse chemical scaffolds and varying degrees of cooperativity. We observed modulation bias for PAMs when we compared mGlu 5 -mediated Ca 2ϩ mobilization and extracellular signalregulated kinase 1/2 phosphorylation data. Furthermore, we used this model to quantify the effects of mutations that reduce binding or potentiation by PAMs. This model can be applied to PAM and NAM potency curves in combination with maximal fold-shift data to derive reliable estimates of modulator affinities.

Overlapping binding sites drive allosteric agonism and positive cooperativity in type 4 metabotropic glutamate receptors

“…Intrinsic allosteric agonist activity independent of the orthosteric ligand has previously been reported for certain mGlu 4 PAMs (11) and other mGlu subtypes (4,10). There is an apparent discrepancy with PHCCC, which has previously been described as a pure PAM (11,44), Statistical differences from wild type are denoted for adjusted P values as follows: *P , 0.05.…”

“…Given this interest, many mGlu 4 PAMs have been discovered within the last few years (7,9). However, some of the mGlu 4 PAMs identified thus far exhibit allosteric agonist activity (10,11), and little is known about the molecular determinants of their action. Indeed, most studies on the binding mode of allosteric modulators have been performed on mGlu 5 (12), mGlu 1 (13), and mGlu 2 (14,15), describing the existence of different pockets within the 7TM domain.…”

A Novel Metabotropic Glutamate Receptor 5 Positive Allosteric Modulator Acts at a Unique Site and Confers Stimulus Bias to mGlu5 Signaling

“…Membranes were prepared from HEK293 cells expressing rat mGlu 5 and mutants as previously described (Gregory et al, 2012). For inhibition binding experiments, membranes (20-50 mg/well) were incubated with 2 nM [ 3 H]methoxyPEPy and a range of concentrations of test ligand (100 pM to 100 mM) for 1 hour at room temperature with shaking in calcium assay buffer with 1% dimethylsulfoxide final; 10 mM MPEP was used to determine nonspecific binding.…”

“…Data were transformed and fitted using GraphPad Prism 5.01 (Graph-Pad Software, Inc., San Diego, CA). As described in Gregory et al (2012), shifts of glutamate concentration-response curves by allosteric modulators were globally fitted to an operational model of allosterism (Leach et al, 2007) with use of Eq. 2 from Gregory et al (2012).…”

“…The assay was terminated by aspiration of ligand-containing media and addition of 50 ml/well of lysis buffer. Lysates were processed as described previously (Gregory et al, 2012) and AlphaScreen signal measured using an Enspire (PerkinElmer) with standard AlphaScreen settings. Data are expressed as fold increase over basal levels of phosphorylated ERK.…”