Numerous positive and negative allosteric modulators (PAMs and NAMs) of class C G protein-coupled receptors (GPCRs) have been developed as valuable preclinical pharmacologic tools and therapeutic agents. Although many class C GPCR allosteric modulators have undergone subtype selectivity screening, most assay paradigms have failed to perform rigorous pharmacologic assessment. Using mGlu as a representative class C GPCR, we tested the hypothesis that allosteric modulator selectivity was based on cooperativity rather than affinity. Specifically, we aimed to identify ligands that bound to mGlu but exhibited neutral cooperativity with mGlu agonists. We additionally evaluated the potential for these ligands to exhibit biased pharmacology. Radioligand binding, intracellular calcium (iCa) mobilization, and inositol monophosphate (IP) accumulation assays were undertaken in human embryonic kidney cells expressing low levels of rat mGlu (HEK293A-mGlu-low) for diverse allosteric chemotypes. Numerous "non-mGlu" class C GPCR allosteric modulators incompletely displaced allosteric mGlu radioligand [H]methoxy-PEPy binding, consistent with a negative allosteric interaction. Affinity estimates for CPCCOEt (mGlu ligand), PHCCC (mGlu ligand), GS39783 (GABA ligand), AZ12216052 (mGlu ligand), and CGP7930 (GABA ligand) at mGlu were within 10-fold of their target receptor. Most class C GPCR allosteric modulators had neutral cooperativity with both orthosteric and allosteric mGlu agonists in functional assays; however, NPS2143 (calcium-sensing receptor (CaSR) NAM), cinacalcet (CaSR PAM), CGP7930, and AZ12216052 were partial mGlu agonists for IP accumulation, but not iCa mobilization. By using mGlu as a model class C GPCR, we find that for many class C GPCR allosteric modulators, subtype selectivity is driven by cooperativity and misinterpreted owing to unappreciated bias.

Allosteric modulators of G protein-coupled receptors have the potential to achieve greater receptor subtype selectivity compared with ligands targeting the orthosteric site of this receptor family. However, the high attrition rate in GPCR drug discovery programs has highlighted the need to better characterize lead compounds in terms of their allosteric action, as well as the signals they elicit. Recently, the use of label-free technologies has been proposed as an approach to overcome some limitations of endpoint-based assays and detect global changes in the ligand-stimulated cell. In this study, we assessed the ability of an impedance-based label-free technology, xCELLigence, to detect allosteric modulation in a neuronal cell line natively expressing rodent M4 muscarinic acetylcholine receptors. We were able to demonstrate that positive allosteric modulation of the endogenous M4 muscarinic acetylcholine receptor can be detected using this technology. Importantly, the allosteric parameters estimated from the label-free approach are comparable to those estimated from endpoint-based assays.

The metabotropic glutamate receptor 2 (mGlu2) is a transmembrane-spanning class C G protein-coupled receptor that is an attractive therapeutic target for multiple psychiatric and neurological disorders. A key challenge has been deciphering the contribution of mGlu2 relative to other closely related mGlu receptors in mediating different physiological responses, which could be achieved through the utilization of subtype selective pharmacological tools. In this respect, allosteric modulators that recognize ligand-binding sites distinct from the endogenous neurotransmitter glutamate offer the promise of higher receptor-subtype selectivity. We hypothesized that mGlu2-selective positive allosteric modulators could be derivatized to generate bifunctional pharmacological tools. Here we developed clickable photoaffinity probes for mGlu2 based on two different positive allosteric modulator scaffolds that retained similar pharmacological activity to parent compounds. We demonstrate successful probe-dependent incorporation of a commercially available clickable fluorophore using bioorthogonal conjugation. Importantly, we also show the limitations of using these probes to assess in situ fluorescence of mGlu2 in intact cells where significant nonspecific membrane binding is evident.

Allosteric modulators bind sites distinct from orthosteric ligands, allowing for improved spatiotemporal control of receptors and greater subtype selectivity. However, we recently showed that allosteric ligands previously classified as selective for select Class C G protein‐coupled receptors (GPCRs) had unappreciated activity at other off‐target receptors, in some cases higher affinity, within the class. Here, we extended our investigation of off‐target activity of “selective” allosteric ligands for the sweet taste receptor. Using metabotropic glutamate receptor 5 (mGlu5) as a representative of Class C GPCR, we assessed the sweet protein, monellin and the small‐molecule artificial sweetener, NHDC. We found that monellin, but not NHDC, is an agonist for mGlu5. Radioligand binding and functional assays performed in cells expressing N‐terminally truncated mGlu5 demonstrated that monellin agonism was not mediated via the “common” allosteric binding site in the transmembrane domain but required the presence of the large extracellular N‐terminal domain of mGlu5. Monellin displayed neutral functional cooperativity with orthosteric ligands. However, monellin positively modulated the mGlu5 PAM‐agonist, VU0424465, activity in intracellular calcium assays, but the interaction was neutral in inositol phosphate accumulation assays. Furthermore, monellin mGlu5 agonism was positively modulated by the mGlu5 pure PAM, VU0360172. Taken together, these data indicate that monellin is an allosteric agonist for mGlu5, binding to an allosteric binding site on the N‐terminus that is functionally linked to the common Class C GPCR allosteric site in a biased manner. This is the first evidence of a naturally derived proteinaceous allosteric ligand for the mGlu receptor family.