Allosteric modulators of the metabotropic glutamate receptor subtype 5 (mGlu 5 ) have been proposed as potential therapies for various CNS disorders. These ligands bind to sites distinct from the orthosteric (or endogenous) ligand, often with improved subtype selectivity and spatiotemporal control over receptor responses. We recently revealed that mGlu 5 allosteric agonists and positive allosteric modulators exhibit biased agonism and/or modulation. To establish whether negative allosteric modulators (NAMs) engender similar bias, we rigorously characterized the pharmacology of eight diverse mGlu 5 NAMs. Radioligand inhibition binding studies revealed novel modes of interaction with mGlu 5 for select NAMs, with biphasic or incomplete inhibition of the radiolabeled NAM, [ 3 H]methoxy-PEPy. We assessed mGlu 5 -mediated intracellular Ca 2+ (iCa 2+ ) mobilization and inositol phosphate (IP 1 ) accumulation in HEK293A cells stably expressing low levels of mGlu 5 (HEK293A-rat mGlu 5 -low) and mouse embryonic cortical neurons. The apparent affinity of acetylenic NAMs, MPEP, MTEP and dipraglurant, was dependent on the signaling pathway measured, agonist used, and cell type (HEK293A-rat mGlu 5low versus mouse cortical neurons). In contrast, the acetylenic partial NAM, M-5MPEP, and structurally distinct NAMs (VU0366248, VU0366058, fenobam), had similar affinity estimates irrespective of the assay or cellular background. Biased modulation was evident for VU0366248 in mouse cortical neurons where it was a NAM for DHPG-mediated iCa 2+ mobilization, but neutral with DHPG in IP 1 accumulation assays. Overall, this study highlights the inherent complexity in mGlu 5 NAM pharmacology that we hypothesize may influence interpretation when translating

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

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Pinnatoxins are members of the cyclic imine group of marine phycotoxins that are highly toxic in in vivo rodent bioassays, causing rapid death due to respiratory depression. Recent studies have shown that pinnatoxins E, F and G, found in New Zealand and Australian shellfish, act as antagonists at muscletype nicotinic acetylcholine receptors (nAChRs) at the neuromuscular junction. In the present study, binding affinities and modes of these pinnatoxin isomers at neuronal and muscle nAChRs were assessed using radioligand binding, electrophysiological and molecular modelling techniques. Radioligand-binding studies revealed that all three pinnatoxins bound with high affinity to muscle-type nAChRs, as well as to the a7 and a4b2 neuronal receptors, with an order of affinity of muscle type > a7 > a4b2.