Metabotropic glutamate receptor 5 (mGlu5 )-positive allosteric modulators differentially induce or potentiate desensitization of mGlu5 signaling in recombinant cells and neurons.

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.

Differential contribution of metabotropic glutamate receptor 5 common allosteric binding site residues to biased allosteric agonism.

Allosteric modulators of metabotropic glutamate receptor subtype 5 (mGlu5) represent an attractive therapeutic strategy for multiple CNS disorders. Chemically distinct mGlu5 positive allosteric modulators (PAMs) that interact with a common binding site can demonstrate biased allosteric agonism relative to the orthosteric agonist, DHPG, when comparing activity in signaling assays such as IP1 accumulation, ERK1/2 phosphorylation (pERK1/2) and iCa2+ mobilization. However, the structural basis for such biased agonism is not well understood. Therefore, we evaluated biased allosteric agonism mediated by four mGlu5 PAM-agonists from diverse chemical scaffolds (i.e., DPFE, VU0409551, VU29, and VU0424465) for three measures of mGlu5 activation (i.e., iCa2+ mobilization, IP1 accumulation and ERK1/2 phosphorylation) at eight single-point mutations within the common allosteric binding pocket of mGlu5. In particular, mGlu5 allosteric site mutations had differential effects on the intrinsic efficacy of mGlu5 PAMs for multiple signaling pathways. Specifically, a loss of agonism for iCa2+ mobilization was evident for DPFE and VU0409551 for most mutants, whereas IP1 accumulation and ERK phosphorylation were retained, albeit with reduced maximal responses. Additionally, bias profiles between iCa2+ mobilization and IP1/ERK pathways remained similar to wild type for most mutants. However, W784A and A809G mutants lost bias between IP1 accumulation and ERK phosphorylation for VU0424465, whereas a loss of bias between iCa2+ mobilization and ERK1/2 phosphorylation was evident for F787A, S808A and A809G mutants. These data provide further insight into the structural requirements for allosteric agonism across multiple mGlu5-mediated signaling pathways. An understanding of mGlu5 biased agonism at a structural level may provide the foundation for rational structure-based design of biased allosteric ligands for the treatment of neurological disorders.

Probe dependence and biased potentiation of metabotropic glutamate receptor 5 is mediated by differential ligand interactions in the common allosteric binding site.

The metabotropic glutamate receptor 5 (mGlu5) is a promising therapeutic target for multiple CNS disorders. Recent mGlu5 drug discovery has focused on targeting binding sites within the mGlu5 7-transmembrane domain (7TM) that are topographically distinct from that of the endogenous ligand. mGlu5 primarily couples to Gq/11 proteins leading to mobilization of intracellular Ca2+ (iCa2+), but also activates iCa2+ independent signaling pathways, with biased agonism/modulation operative for multiple positive allosteric modulator (PAM) and PAM-agonist chemotypes. Although several residues within the common allosteric binding pocket are key determinants of PAM activity, how these residues affect biased modulation is unknown. The current study probed the molecular basis of mGlu5 PAM biased modulation. Modulation of mGlu5 activity by four chemically distinct mGlu5 PAMs (VU0424465, DPFE, VU29 and VU0409551) was assessed across two distinct receptor endpoints (iCa2+ mobilization and ERK1/2 phosphorylation) at mGlu5 receptors containing single-point mutations of allosteric binding pocket residues informed by computational modeling. Many mutations had differential effects on PAM affinity and cooperativity across signaling endpoints, resulting in gain or reversal of bias at the level of both affinity and functional cooperativity. Additionally, mutants had differential effects on functional cooperativity between the orthosteric ligands, DHPG and glutamate, and the PAMs, DPFE and VU29, but not VU0409551, indicating that probe dependence is linked to orthosteric agonists conferring activation states that differentially influence allosteric ligand-receptor interactions in a chemotype dependent fashion. Collectively, these data provide crucial insight into the residues that govern different activation states adopted by mGlu5 in order to signal via distinct intracellular pathways when co-bound by orthosteric agonists and PAMs.

Kinetic and system bias as drivers of metabotropic glutamate receptor 5 allosteric modulator pharmacology.

Allosteric modulators of the metabotropic glutamate receptor subtype 5 (mGlu5) 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 spatio-temporal control over receptor responses. We recently revealed that mGlu5 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 mGlu5 NAMs. Radioligand inhibition binding studies revealed novel modes of interaction with mGlu5 for select NAMs, with biphasic or incomplete inhibition of the radiolabeled NAM, [3H]methoxy-PEPy. We assessed mGlu5-mediated intracellular Ca2+ (iCa2+) mobilization and inositol phosphate (IP1) accumulation in HEK293A cells stably expressing low levels of mGlu5 (HEK293A-rat mGlu5-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 mGlu5-low 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 iCa2+ mobilization, but neutral with DHPG in IP1 accumulation assays. Overall, this study highlights the inherent complexity in mGlu5 NAM pharmacology that we hypothesize may influence interpretation when translating into preclinical models and beyond in the design and development of novel therapeutics for neuropsychiatric and neurological disorders.

Biased allosteric agonism and modulation of metabotropic glutamate receptor 5: Implications for optimizing preclinical neuroscience drug discovery.

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. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'.