Allosteric modulators, that exhibit no intrinsic agonist activity, offer the advantage of spatial and temporal fine-tuning of endogenous agonist activity, allowing the potential for increased selectivity, reduced adverse effects and improved clinical outcomes. Some allosteric ligands can differentially activate and/or modulate distinct signaling pathways arising from the same receptor, phenomena referred to as ’biased agonism’ and ’biased modulation’. Emerging evidence for CNS disorders with glutamatergic dysfunction suggests the metabotropic glutamate receptor subtype 5 (mGlu5) is a promising target. Current mGlu5 allosteric modulators have largely been classified based on modulation of intracellular calcium (iCa2+) responses to orthosteric agonists alone. We assessed eight mGlu5 allosteric modulators previously classified as mGlu5 PAMs or PAM-agonists representing four distinct chemotypes across multiple measures of receptor activity, to explore their potential for engendering biased agonism and/or modulation. Relative to the reference orthosteric agonist, DHPG, the eight allosteric ligands exhibited distinct biased agonism fingerprints for iCa2+ mobilization, IP1 accumulation and ERK1/2 phosphorylation in HEK293A cells stably expressing mGlu5 and in cortical neuron cultures. VU0424465, DPFE and VU0409551 displayed the most disparate biased signaling fingerprints in both HEK293A cells and cortical neurons that may account for the marked differences observed previously for these ligands in vivo. Select mGlu5 allosteric ligands also showed ‘probe dependence’ with respect to their cooperativity with different orthosteric agonists, as well as biased modulation for the magnitude of positive cooperativity observed. Unappreciated biased agonism and modulation may contribute to unanticipated effects (both therapeutic and adverse) when translating from recombinant systems to preclinical models.

The metabotropic glutamate receptor subtype 5 (mGlu5 ) is a family C GPCR that has been implicated in various neuronal processes and, consequently, in several CNS disorders. Over the past few decades, GPCR-based drug discovery, including that for mGlu5 receptors, has turned considerable attention to targeting allosteric binding sites. Modulation of endogenous agonists by allosteric ligands offers the advantages of spatial and temporal fine-tuning of receptor activity, increased selectivity and reduced adverse effects with the potential to elicit improved clinical outcomes. Further, with greater appreciation of the multifaceted nature of the transduction of mGlu5 receptor signalling, it is increasingly apparent that drug discovery must take into consideration unique receptor conformations and the potential for stimulus-bias. This novel paradigm proposes that different ligands may differentially modulate distinct signalling pathways arising from the same receptor. We review our current understanding of the complexities of mGlu5 receptor signalling and regulation, and how these relate to allosteric ligands. Ultimately, a deeper appreciation of these relationships will provide the foundation for targeted drug design of compounds with increased selectivity, not only for the desired receptor but also for the desired signalling outcome from the receptor. Linked Articles This article is part of a themed section on Molecular Pharmacology of G Protein-Coupled Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v173.20/issuetoc.

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

Allosteric modulators of metabotropic glutamate receptor 5 (mGlu5) are a promising therapeutic strategy for a number of neurological disorders. Multiple mGlu5‐positive allosteric modulator (PAM) chemotypes have been discovered that act as either pure PAMs or as PAM‐agonists in recombinant and native cells. While these compounds have been tested in paradigms of receptor activation, their effects on receptor regulatory processes are largely unknown. In this study, acute desensitization of mGlu5 mediated intracellular calcium mobilization by structurally diverse mGlu5 orthosteric and allosteric ligands was assessed in human embryonic kidney 293 cells and primary murine neuronal cultures from both striatum and cortex. We aimed to determine the intrinsic efficacy and modulatory capacity of diverse mGlu5 PAMs [(R)‐5‐((3‐fluorophenyl)ethynyl)‐N‐(3‐hydroxy‐3‐methylbutan‐2‐yl)picolinamide (VU0424465), N‐cyclobutyl‐6‐((3‐fluorophenyl)ethynyl)picolinamide (VU0360172), 1‐(4‐(2,4‐difluorophenyl)piperazin‐1‐yl)‐2‐((4‐fluorobenzyl)oxy)ethanone (DPFE), ((4‐fluorophenyl) (2‐(phenoxymethyl)‐6,7‐dihydrooxazolo[5,4‐c]pyridin‐5(4H)‐yl)methanone) (VU0409551), 3‐Cyano‐N‐(1,3‐diphenyl‐1H‐pyrazol‐5‐yl)benzamide (CDPPB)] on receptor desensitization and whether cellular context influences receptor regulatory processes. Only VU0424465 and VU0409551 induced desensitization alone in human embryonic kidney 293‐mGlu5 cells, while all PAMs enhanced (S)‐3,5‐dihydroxyphenylglycine (DHPG)‐induced desensitization. All mGlu5 PAMs induced receptor desensitization alone and enhanced DHPG‐induced desensitization in striatal neurons. VU0424465 and VU0360172 were the only PAMs that induced desensitization alone in cortical neurons. With the exception of (CDPPB), PAMs enhanced DHPG‐induced desensitization in cortical neurons. Moreover, differential apparent affinities, efficacies, and cooperativities with DHPG were observed for VU0360172, VU0409551, and VU0424465 when comparing receptor activation and desensitization in a cell type‐dependent manner. These data indicate that biased mGlu5 allosteric modulator pharmacology extends to receptor regulatory processes in a tissue dependent manner, adding yet another layer of complexity to rational mGlu5 drug discovery.

No abstract