"Selective" Class C G Protein-Coupled Receptor Modulators Are Neutral or Biased mGlu₅ 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 mGlu₅ 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 mGlu₅ but exhibited neutral cooperativity with mGlu₅ agonists. We additionally evaluated the potential for these ligands to exhibit biased pharmacology. Radioligand binding, intracellular calcium (iCa²⁺) mobilization, and inositol monophosphate (IP₁) accumulation assays were undertaken in human embryonic kidney cells expressing low levels of rat mGlu₅ (HEK293A-mGlu₅-low) for diverse allosteric chemotypes. Numerous "non-mGlu₅" class C GPCR allosteric modulators incompletely displaced allosteric mGlu₅ radioligand [³H]methoxy-PEPy binding, consistent with a negative allosteric interaction. Affinity estimates for CPCCOEt (mGlu₁ ligand), PHCCC (mGlu₄ ligand), GS39783 (GABA_B ligand), AZ12216052 (mGlu₈ ligand), and CGP7930 (GABA_B ligand) at mGlu₅ were within 10-fold of their target receptor. Most class C GPCR allosteric modulators had neutral cooperativity with both orthosteric and allosteric mGlu₅ agonists in functional assays; however, NPS2143 (calcium-sensing receptor (CaSR) NAM), cinacalcet (CaSR PAM), CGP7930, and AZ12216052 were partial mGlu₅ agonists for IP₁ accumulation, but not iCa²⁺ mobilization. By using mGlu₅ 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.