Detailed In Vitro Pharmacological Characterization of Clinically Tested Negative Allosteric Modulators of the...

Negative allosteric modulation of the metabotropic glutamate 5 (mGlu₅) receptor has emerged as a potential strategy for the treatment of neurologic disorders. Despite the success in preclinical studies, many mGlu₅ negative allosteric modulators (NAMs) that have reached clinical trials failed due to lack of efficacy. In this study, we provide a detailed in vitro pharmacological characterization of nine clinically and preclinically tested NAMs. We evaluated inhibition of l-glutamate-induced signaling with Ca²⁺ mobilization, inositol monophosphate (IP₁) accumulation, extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, and real-time receptor internalization assays on rat mGlu₅ expressed in HEK293A cells. Moreover, we determined association rates (k_on) and dissociation rates (k_off), as well as NAM affinities with [³H]methoxy-PEPy binding experiments. k_on and k_off values varied greatly between the nine NAMs (34- and 139-fold, respectively) resulting in long receptor residence times (>400 min) for basimglurant and mavoglurant, medium residence times (10-30 min) for AZD2066, remeglurant, and (RS)-remeglurant, and low residence times (<10 mins) for dipraglurant, F169521, F1699611, and STX107. We found that all NAMs inhibited l-glutamate-induced mGlu₅ receptor internalization, generally with a similar potency to IP₁ accumulation and ERK1/2 phosphorylation, whereas Ca²⁺ mobilization was less potently inhibited. Operational model of allosterism analyses revealed that dipraglurant and (RS)-remeglurant were biased toward (affinity) receptor internalization and away (cooperativity) from the ERK1/2 phosphorylation pathway, respectively. Our study is the first to measure mGlu₅ NAM binding kinetics and negative allosteric modulation of mGlu₅ receptor internalization and adds significant new knowledge about the molecular pharmacology of a diverse range of clinically relevant NAMs. SIGNIFICANCE STATEMENT: The metabotropic glutamate 5 (mGlu₅) receptor is important in many brain functions and implicated in several neurological pathologies. Negative allosteric modulators (NAMs) have shown promising results in preclinical models but have so far failed in human clinical trials. Here we provide the most comprehensive and comparative molecular pharmacological study to date of nine preclinically/clinically tested NAMs at the mGlu₅ receptor, which is also the first study to measure ligand binding kinetics and negative allosteric modulation of mGlu₅ receptor internalization.