Positive Allosteric Modulators of Metabotropic Glutamate Receptor 5 as Tool Compounds to Study Signaling Bias

Positive allosteric modulation of metabotropic glutamate subtype 5 (mGlu₅) receptor has emerged as a potential new therapeutic strategy for the treatment of schizophrenia and cognitive impairments. However, positive allosteric modulator (PAM) agonist activity has been associated with adverse side effects, and neurotoxicity has also been observed for pure PAMs. The structural and pharmacological basis of therapeutic versus adverse mGlu₅ PAM in vivo effects remains unknown. Thus, gaining insights into the signaling fingerprints, as well as the binding kinetics of structurally diverse mGlu₅ PAMs, may help in the rational design of compounds with desired properties. We assessed the binding and signaling profiles of N-methyl-5-(phenylethynyl)pyrimidin-2-amine (MPPA), 3-cyano-N-(2,5-diphenylpyrazol-3-yl)benzamide (CDPPB), and 1-[4-(4-chloro-2-fluoro-phenyl)piperazin-1-yl]-2-(4-pyridylmethoxy)ethenone [compound 2c, a close analog of 1-(4-(2-chloro-4-fluorophenyl)piperazin-1-yl)-2-(pyridin-4-ylmethoxy)ethanone] in human embryonic kidney 293A cells stably expressing mGlu₅ using Ca²⁺ mobilization, inositol monophosphate (IP₁) accumulation, extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, and receptor internalization assays. Of the three allosteric ligands, only CDPPB had intrinsic agonist efficacy, and it also had the longest receptor residence time and highest affinity. MPPA was a biased PAM, showing higher positive cooperativity with orthosteric agonists in ERK1/2 phosphorylation and Ca²⁺ mobilization over IP₁ accumulation and receptor internalization. In primary cortical neurons, all three PAMs showed stronger positive cooperativity with (S)-3,5-dihydroxyphenylglycine (DHPG) in Ca²⁺ mobilization over IP₁ accumulation. Our characterization of three structurally diverse mGlu₅ PAMs provides further molecular pharmacological insights and presents the first assessment of PAM-mediated mGlu₅ internalization. SIGNIFICANCE STATEMENT: Enhancing metabotropic glutamate receptor subtype 5 (mGlu₅) activity is a promising strategy to treat cognitive and positive symptoms in schizophrenia. It is increasingly evident that positive allosteric modulators (PAMs) of mGlu₅ are not all equal in preclinical models; there remains a need to better understand the molecular pharmacological properties of mGlu₅ PAMs. This study reports detailed characterization of the binding and functional pharmacological properties of mGlu₅ PAMs and is the first study of the effects of mGlu₅ PAMs on receptor internalization.