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

Abstract

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.

Detailed In Vitro Pharmacological Characterization of Clinically Tested Negative Allosteric Modulators of the Metabotropic Glutamate Receptor 5

“…NAMs Inhibit Glutamate-Induced mGlu 5 Internalization. Beyond acute activation of intracellular signal transduction pathways, allosteric ligands may also differentially influence receptor regulatory processes (Hellyer et al, 2019). Therefore, we next sought to assess the effect of mGlu 5 NAMs on L-glutamate-induced receptor internalization.…”

The comparative effects of mGlu5 receptor positive allosteric modulators VU0409551 and VU0360172 on cognitive deficits and signalling in the sub-chronic PCP rat model for schizophrenia

“…In HEK293 cells stably transfected to express mGlu5 receptors, VU0360172 has been reported to activate both Gαq-and Gβγ-mediated mGlu5 receptor signalling, whereas VU0409551 has been reported to be "biased" as it preferentially stimulates Gαq-mGlu5 receptor signalling (Sengmany et al, 2017). Furthermore, in cortical neurons and cell lines, VU0409551 shows significant bias away from phospho-ERK1/2 (p-ERK1/2) (relative to IP 1 ) and a lack of agonist efficacy for intracellular calcium mobilization compared to VU0360172 (Sengmany et al, 2017) and there are differences in effects on DHPG ((S)-3,5-Dihydroxyphenylglycine) receptor activation (Hellyer et al, 2019). Although these differences have been observed in vitro it is unclear what effects these compounds will have in vivo.…”

International Union of Basic and Clinical Pharmacology. CXI. Pharmacology, Signaling, and Physiology of Metabotropic Glutamate Receptors

“…Furthermore, differential cooperativity has also been noted for mGlu 5 receptor NAMs, where N-(3-chloro-2-fluorophenyl)-3-cyano-5-fluoro-benzamide inhibits iCa 2+ mobilization but is an NAL with respect to IP 1 accumulation (Sengmany et al, 2019). Recent studies indicate biased agonism and modulation of mGlu 5 receptor NAMs and PAMs extends to receptor regulatory processes such as internalization and desensitization (Hellyer et al, 2019;Arsova et al, 2020). Another contributing factor to biased modulator pharmacology at mGlu 5 receptors may be ligand binding kinetics as suggested by two recent studies on structurally diverse NAMs (Sengmany et al, 2019;Arsova et al, 2020).…”

“…Where intrinsic efficacy differs between pathways relative to a reference agonist, this is referred to as biased agonism and can be quantified as discussed previously for biased orthosteric agonists. For mGlu 5 receptors, PAM agonists from diverse scaffolds are biased agonists relative to DHPG in both recombinant and native cells; however, the bias profile differs between scaffolds, with 5- [2-(2-(3-fluorophenyl)ethynyl]-N-[(1R)-2-hydroxy-1,2-dimethylpropyl]-2-pyridinecarboxamide, DPFE,and [6,oxazolo [5,4-c]pyridin-5(4H)yl](fluorophenyl)methanone (VU0409551) each exhibiting different bias profiles for mGlu 5 receptor signaling and receptor desensitization (Sengmany et al, 2017;Hellyer et al, 2019). However, biased modulation is also possible, where the direction or magnitude of modulation of the same agonist differs between pathways and may manifest either as differential apparent affinity or cooperativity (Sengmany et al, 2019).…”

“…The first report for context-dependent pharmacology was for the mGlu 7 receptor NAM, MMPIP, which shows different magnitudes of inhibition of mGlu 7 receptor activity for the same agonist between different recombinant cell lines . For mGlu 5 receptor modulators, both PAMs and NAMs have context-dependent pharmacology, manifested as distinct biased agonism profiles (Sengmany et al, 2017;Hellyer et al, 2019) or differential apparent affinities (Sengmany et al, 2019) or potencies (Jong et al, 2019) of NAMs between recombinant and native cells from different brain regions. Quantitative pharmacological differences between cell types may be a consequence of different stimulus-response coupling efficiencies, the presence or absence of receptor interacting proteins (other GPCRs, transducers, or scaffolding partners), or differences in receptor subcellular compartmentalization and relative accessibility by different ligands.…”