"Selective" Class C G Protein-Coupled Receptor Modulators Are Neutral or Biased mGlu Allosteric Ligands.

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.