Rigorous Characterization of Allosteric Modulation of the Human Metabotropic Glutamate Receptor 1 reveals probe and assay dependent pharmacology

Allosteric modulation of mGlu1 represents a viable therapeutic target for treating numerous CNS disorders. While multiple chemically distinct mGlu1 positive (PAMs) and negative (NAMs) allosteric modulators have been identified, drug discovery paradigms have not included rigorous pharmacological analysis. In the present study, we hypothesised existing mGlu1 allosteric modulators possess unappreciated probe dependent or biased pharmacology. Using HEK293A cells stably expressing human mGlu1, we screened mGlu1 PAMs and NAMs from divergent chemical scaffolds for modulation of different mGlu1 orthosteric agonists in intracellular calcium (iCa2+) mobilisation and inositol monophosphate (IP1) accumulation assays. Operational models of agonism and allosterism were used to derive estimates for important pharmacological parameters such as affinity, efficacy and cooperativity. Modulation of glutamate and quisqualate-mediated iCa2+ mobilisation revealed probe dependence at the level of affinity and cooperativity for both mGlu1 PAMs and NAMs. We also identified the previously described mGlu5 selective NAM PF-06462894 as an mGlu1 NAM with a different pharmacological profile to other NAMs. Differential profiles were also observed when comparing ligand pharmacology between iCa2+ mobilisation and IP1 accumulation. The PAMs Ro674853 and CPPHA displayed apparent negative cooperativity for modulation of quisqualate affinity, and the NAMs CPCCOEt and PF-06462894 had a marked reduction in cooperativity with quisqualate in IP1 accumulation and upon extended incubation in iCa2+ mobilisation assays. These data highlight the importance of rigorous assessment of mGlu1 modulator pharmacology to inform future drug discovery programs for mGlu1 allosteric modulators.