Rigorous Characterization of Allosteric Modulation of the Human Metabotropic Glutamate Receptor 1 Reveals Probe- and Assay-Dependent Pharmacology.

Abstract

Allosteric modulation of metabotropic glutamate receptor subtype 1 (mGlu₁) represents a viable therapeutic target for treating numerous central nervous system disorders. Although 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 hypothesized that existing mGlu₁ allosteric modulators possess unappreciated probe-dependent or biased pharmacology. Using human embryonic kidney 293 (HEK293A) cells stably expressing human mGlu_1, we screened mGlu₁ PAMs and NAMs from divergent chemical scaffolds for modulation of different mGlu₁ orthosteric agonists in intracellular calcium (iCa²⁺) mobilization 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⁺ mobilization 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 from other NAMs. Differential profiles were also observed when comparing ligand pharmacology between iCa²⁺ mobilization and IP₁ accumulation. The PAMs Ro67-4853 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⁺ mobilization assays. These data highlight the importance of rigorous assessment of mGlu₁ modulator pharmacology to inform future drug discovery programs for mGlu₁ allosteric modulators. SIGNIFICANCE STATEMENT: Metabotropic glutamate receptor subtype 1 (mGlu₁) positive and negative allosteric modulators have therapeutic potential in multiple central nervous system disorders. We show that chemically distinct modulators display differential pharmacology with different orthosteric ligands and across divergent signaling pathways at human mGlu₁. Such complexities in allosteric ligand pharmacology should be considered in future mGlu₁ allosteric drug discovery programs.