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

Angela Arsova, Thor C. Møller, Shane D. Hellyer, Line Vedel, Simon R. Foster, Jakob L. Hansen, Hans Bräuner-Osborne and Karen J. Gregory
Molecular Pharmacology May 2021, 99 (5) 328-341; DOI: https://doi.org/10.1124/molpharm.120.000185

Abstract

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.

Introduction

The involvement of metabotropic glutamate (mGlu) receptors in central nervous system disorders such as Parkinson disease, schizophrenia, and major depressive disorder has made these receptors interesting targets for drug discovery research (Nicoletti et al., 2015; Foster and Conn, 2017). Metabotropic glutamate receptor subtype 5 (mGlu5) is a group I mGlu receptor that is primarily coupled to Gq/11 proteins. mGlu5 is generally found postsynaptically and is important in neuronal development and synaptic plasticity—for instance, in memory formation and cognition (Valenti et al., 2002; Dhami and Ferguson, 2006; Waung and Huber, 2009). High sequence similarity in the orthosteric glutamate binding site between the eight mGlu receptor subtypes makes the discovery of selective orthosteric ligands challenging (Wellendorph and Bräuner-Osborne, 2009). Hence, mGlu5 discovery efforts have focused on targeting topographically distinct sites with allosteric modulators; many diverse scaffolds have been identified that interact with a common site within the seven-transmembrane domains (Doré et al., 2014; Christopher et al., 2018). Allosteric modulators offer higher subtype receptor selectivity and the ability to spatiotemporally regulate pre-existing receptor responses; in this way, allosteric modulators potentially avoid unwanted side effects (Melancon et al., 2012; Changeux and Christopoulos, 2017). Allosteric modulators may enhance [termed positive allosteric modulators (PAMs)] or diminish receptor activation [termed negative allosteric modulators (NAMs)] (Gentry et al., 2015). PAMs can have intrinsic agonist activity and are referred to as PAM agonists (Foster and Conn, 2017; Sengmany et al., 2017).

The first bioavailable mGlu5 PAM, 3-cyano-N-(2,5-diphenylpyrazol-3-yl)benzamide (CDPPB), had antipsychotic-like and procognitive effects in preclinical models, establishing mGlu5 PAMs as promising interventions for schizophrenia (Kinney et al., 2005; Horio et al., 2013). Subsequently, mGlu5 PAMs have also been associated with serious adverse effects such as neurotoxicity and seizure induction (Bridges et al., 2013; Rook et al., 2013; Parmentier-Batteur et al., 2014). These adverse side effects were initially attributed to PAM agonist activity, e.g., VU0424465 (5-[2-(3-fluorophenyl)ethynyl]-N-[(2R)-3-hydroxy-3-methylbutan-2-yl]pyridine-2-carboxamide) (Rook et al., 2013) and VU0403602 (N-cyclobutyl-5-((3-fluorophenyl)ethynyl)picolinamide) (Bridges et al., 2013). However, some pure PAMs may also lead to neurotoxicity, indicating that PAM agonist activity is not the only predictor of adverse effect liability (Parmentier-Batteur et al., 2014). In many drug discovery paradigms, PAM agonist activity is only tested in a single functional assay (i.e., Ca2+ mobilization). Such approaches do not detect pleiotropic mGlu5 signaling; therefore, some “pure” PAMs may in fact be agonists for different cellular responses. Investigation of biased mGlu5 signaling has thus emerged as a means to avoid unwanted side effects (Sengmany et al., 2017). Relative to a reference agonist, a “biased agonist” preferentially activates select responses relative to others activated through the same receptor (Trinh et al., 2018). Biased agonism is believed to be achieved through the stabilization of unique receptor conformations that have higher affinity for certain effector proteins over others (Kenakin and Christopoulos, 2013; Smith et al., 2018). Biased allosteric modulation is also possible, manifesting as different apparent affinities or magnitudes of cooperativity with the same orthosteric agonist depending upon the response measured (Sengmany et al., 2017; Hellyer et al., 2019; Sengmany et al., 2019).

Alongside the conformational theory for ligand bias, ligand binding kinetics are also implicated in signaling bias (Klein Herenbrink et al., 2016; Lane et al., 2017). The duration of the ligand-receptor complex is proposed to be proportional to agonist efficacy (Copeland, 2016); compounds that occupy receptors longer potentially catalyze more effector protein activation cycles (Lane et al., 2017). Therefore, increasing receptor residence time has been exploited as a strategy in rational drug design to increase ligand affinity and efficacy (Lindstrom et al., 2007; Tummino and Copeland, 2008). However, long residence times may also lead to on-target toxicity (Kapur and Seeman, 2001). To date, the contribution of ligand binding kinetics to mGlu5 biased agonism and potentiation has remained unexplored.

Here, we evaluated the signaling profiles of three structurally diverse mGlu5 PAMs using four different functional assays: Ca2+ mobilization, IP1 accumulation, ERK1/2 phosphorylation, and real-time receptor internalization. N-methyl-5-(phenylethynyl)pyrimidin-2-amine (MPPA) is a potent PAM of glutamate stimulation of intracellular Ca2+ mobilization and has efficacy in reversing amphetamine-induced hyperlocomotion in rats (Sharma et al., 2009). Discovered alongside the in vivo efficacious PAM 1-(4-(2-chloro-4-fluorophenyl)piperazin-1-yl)-2-(pyridin-4-ylmethoxy)ethenone, compound 2c has previously only been evaluated as a PAM of glutamate in mGlu5-Ca2+ mobilization assays (Xiong et al., 2010). The intrinsic efficacy and potentiation (of DHPG and l-glutamate) by these two PAMs were compared with CDPPB, a well characterized PAM agonist of glutamate activation of mGlu5 (Kinney et al., 2005; Sengmany et al., 2017). Moreover, we determined kinetics of PAM binding to mGlu5 and compared these parameters to affinity estimates obtained with functional assays and inhibition binding experiments.

Materials and Methods

The experiments presented in this paper were planned based on the availability of compounds and established assays and cell lines in the two laboratories in which the experiments were performed. The experiments were exploratory (i.e., not designed to test a prespecified statistical null hypothesis), and the reported P values should therefore be viewed as descriptive. The minimum number of independent experiments was decided beforehand based on our previous experiences with the assays and cell lines.

Materials.

MPPA, CDPPB, and compound 2c were obtained from Lundbeck (Copenhagen, Denmark). DHPG, 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester, MPEP, LY341495, and DL-TBOA were purchased from Tocris (Bristol, UK). DMEM GlutaMAX-I, FBS, dialyzed FBS, penicillin-streptomycin solution, B-27, Fungizone antimycotic, Neurobasal media, Fluo-4 AM cell permeant dye, and HBSS were purchased from Invitrogen (Carlsbad, CA). Probenecid, Pierce BCA protein assay kit, and Fluo-4 AM No Wash kit were purchased from Thermo Fisher Scientific (Waltham, MA). [3H]Methoxy-PEPy was custom-synthesized by Pharmaron (Manchester, UK). MicroScint-20 was purchased from PerkinElmer (Waltham, MA). pRK5 plasmids encoding HA- and SNAP-tagged rat mGlu5a (HA-SNAP-rmGlu5a) and excitatory amino acid transporter 3 (EAAT3) were gifts from Laurent Prézeau (Institut de Génomique Fonctionnelle, Montpellier, France) and previously described (Brabet et al., 1998; Doumazane et al., 2011); the SNAP-tag is derived from O6-alkylguanine-DNA alkyltransferase, which can be covalently labeled with a fluorophore to monitor protein localization. All of the other reagents were purchased from Sigma-Aldrich (St. Louis, MO).

Cell Culture.

A low-expressing wild-type rat mGlu5 (rmGlu5) HEK293A stable cell line (HEK293A-mGlu5-low) was maintained as described previously (Sengmany et al., 2017); when cultured in parallel with nontransfected HEK293A cells, DMEM GlutaMAX-I was used and supplemented with 10% dialyzed FBS, 1% penicillin-streptomycin, and 16 mM HEPES, and geneticin (500 μg/ml) was included to maintain stable expression of HEK293A-mGlu5-low. Cultured cells were routinely monitored for mycoplasma contamination.

Animals.

All animal experiments and procedures were approved by the Monash Institute of Pharmaceutical Sciences Animal Ethics Committee (protocol number MIPS.2014.37). The 8-week-old Asmu:Swiss outbred female wild-type mice were provided by the Monash Animal Research Platform (Clayton, Victoria, Australia). Animals were humanely sacrificed, and day 16 mixed-sex embryos were recovered for primary cell culture.

Primary Cell Culture.

Cortical neurons were isolated from embryonic day 16 Asmu:Swiss wild-type mice sacrificed by decapitation. The cortex was isolated, and neurons were mechanically dissociated in ice-cold HBSS. Cortical neurons were plated on a poly(d-lysine)– and FBS-coated transparent clear-bottom 96-well plate in Neurobasal media supplemented with 2 mM l-glutamine, 1× B-27, 50 U/ml penicillin, 50 mg/ml streptomycin, and 1.25 mg/ml Fungizone antimycotic at a density of 100,000 cells per well. Plates were stored at 37°C and 5% CO2 for 6 to 7 days before experimentation.

Radioligand Binding Assays.

Membrane preparations from HEK293A-mGlu5-low cells were prepared as described previously (Arsova et al., 2020). Inhibition of [3H]methoxy-PEPy (specific activity 85 Ci/mmol) binding assays were equilibrated for 1 hour using our previously described approach in a 96-well plate format (Arsova et al., 2020). In this assay format, ligand depletion is not a concern, as the total amount of ligand bound as a percentage of radioligand added was well under 10% for all experiments, ranging from 1.1% to 3.6%. For association binding experiments, compound and [3H]methoxy-PEPy were premixed 1:1 and added to the plate at different time points. For dissociation binding experiments, membranes were pre-equilibrated with [3H]methoxy-PEPy for 1 hour, and a saturating concentration of MPEP (1 μM) was added at different time points to determine the radioligand Koff. Membranes were harvested through GF/C filter plates using a 96-well FilterMate harvester (PerkinElmer) to separate unbound radioligand. After drying overnight at room temperature, plates were loaded with MicroScint-20 scintillation liquid and incubated at room temperature for 2 hours prior to measuring scintillation spectrometry with a MicroBeta2 microplate counter (PerkinElmer).

Ca2+ Mobilization Assay.

Ca2+ mobilization in HEK293A-mGlu5-low cells was measured as previously described (Arsova et al., 2020) and represents both release from intracellular stores as well as influx of extracellular Ca2+ (Sengmany et al., 2017). PAM potentiation of the response to 100 nM l-glutamate or DHPG was measured in assay buffer (HBSS supplemented with 20 mM HEPES, 1 mM MgCl2, and 1 mM CaCl2 with pH adjusted to 7.4) with 0.1% bovine serum albumin. Intrinsic PAM agonist activity was measured after a 3-hour incubation in assay buffer supplemented with 10 U/ml glutamic-pyruvic transaminase (GPT) and 10 mM sodium pyruvate to eliminate ambient glutamate. Cortical neurons were serum-starved for 4 hours at 37°C and 5% CO2 in starvation media (DMEM with 4500 mg/l glucose, sodium pyruvate, and sodium bicarbonate, without l-glutamine, supplemented with 16 mM HEPES) before assay initiation. Cortical neurons and HEK293A cells were incubated for 1 hour with Fluo-4 AM cell permeant dye diluted in calcium assay buffer (assay buffer as above supplemented with 2.5 mM probenecid). Compounds were diluted in calcium assay buffer to 0.3% final DMSO concentration. After dye loading, cells were washed once with calcium assay buffer. Intrinsic PAM agonist activity was assessed with and without a 15-minute preincubation with 300 µM LY341495 after dye loading. Fluorescence was measured on a FlexStation1 or Flexstation3 plate reader (Molecular Devices, San Jose, CA) at 37°C. For cortical neurons, PAMs were added simultaneously with 120 nM DHPG (or vehicle) at t = 20 seconds, and responses were measured over 120 seconds. In total, 500 nM l-glutamate was added during the final 30 seconds at t = 110 seconds to confirm neuron integrity. The peak change in fluorescence was determined after applying a five-point smoothing function, and data were expressed as a percentage of the DHPG maximal response.

IP1 Accumulation Assay.

IP1 accumulation in HEK293A-mGlu5-low cells was measured with the IP-One assay kit (Cisbio, Codolet, France) as previously described after a 3-hour incubation in IP1 assay buffer (HBSS supplemented with 20 mM HEPES, 1 mM MgCl2, 1 mM CaCl2, and 40 mM LiCl2 with pH adjusted to 7.4) supplemented with 10 U/ml GPT and 10 mM sodium pyruvate to eliminate ambient glutamate (Arsova et al., 2020). Intrinsic PAM agonist activity was measured with and without a 30-minute preincubation with 300 µM LY341495 prior to PAM addition. Potentiation of orthosteric agonist activity was measured in the presence of 500 nM l-glutamate or DHPG. Cortical neurons were starved for 4 hours in starvation media. Compounds were diluted in IP1 assay buffer to 0.3% final DMSO concentration. Compounds were incubated for 1 hour at 37°C before IP1 levels were determined.

ERK1/2 Phosphorylation Assay.

ERK1/2 phosphorylation in HEK293A-mGlu5-low cells was measured with either the Advanced phospho-ERK1/2 (Thr202/Tyr204) assay kit (Cisbio) or AlphaScreen SureFireTM kit (TGR Biosciences) as previously described after a 3-hour incubation in serum-free DMEM supplemented with 10 U/ml GPT and 10 mM sodium pyruvate to eliminate ambient glutamate (Sengmany et al., 2017; Arsova et al., 2020). Intrinsic PAM agonist activity (5-minute stimulation) was measured with and without a 30-minute preincubation with 300 µM LY341495. Potentiation of orthosteric agonist activity was measured in the presence of 500 nM l-glutamate (5-minute stimulation) or DHPG (20-minute stimulation).

Receptor Internalization Assay.

mGlu5 internalization in transiently transfected HEK293A cells was measured with a time-resolved Förster resonance energy transfer assay after labeling the receptor with SNAP-Lumi4-Tb (Cisbio) as previously described (Arsova et al., 2020). Intrinsic PAM agonist activity was measured with and without a 30-minute preincubation with 300 µM LY341495. Potentiation of orthosteric agonist activity was measured in the presence of 30 µM DL-TBOA (to measure potentiation of glutamate) or 1 µM DHPG.

Data Analysis.

Data were analyzed using GraphPad Prism software version 8 (San Diego, CA) as previously described (Arsova et al., 2020). Briefly, inhibition binding data were fitted to either a competitive binding model,Embedded Image(1)or to an allosteric binding model,Embedded ImageEmbedded Image(2)where KD is the equilibrium dissociation constant for the radioligand, KB is the equilibrium dissociation constant for the allosteric modulator, and α is the cooperativity factor. In eq. 1, the IC50 is the concentration of unlabeled inhibitor that reduces binding to 50% of the top and bottom plateaus. The IC50 was used to estimate the Ki (equilibrium dissociation constant of the unlabeled inhibitor) using the Cheng-Prusoff equation ((Cheng and Prusoff, 1973)).

Competition association binding was fitted to the kinetics of the competitive binding model:Embedded ImageEmbedded ImageEmbedded ImageEmbedded ImageEmbedded ImageEmbedded ImageEmbedded Image(3)where k1 and k2 are the radioligand kinetic association and dissociation rates, respectively; k3 and k4 are the unlabeled ligand kinetic association and dissociation rates, respectively; and Bmax is the maximum binding.

Concentration-response curves from functional assays were fitted with a four-parameter sigmoidal concentration-response curve to derive EC50 and Emax values:Embedded Image(4)Biased agonism was determined by fitting to the operational model of agonism (Black et al., 1985):Embedded Image(5)where [A] is the agonist concentration, Em is the maximal response of the system, n is the transducer slope, and τ is the coupling efficiency. System and observation bias were nullified by subtraction of the transduction coefficient log(τ/KA) of a compound from the transduction coefficient of a reference agonist to obtain ∆log(τ/KA).

Allosteric modulation of l-Glu– and DHPG-mediated responses were fitted to the operational model of allosterism:Embedded Image(6)where KA and KB are the equilibrium dissociation constants of the orthosteric ligand and allosteric modulator, respectively; α represents affinity cooperativity; β is a scaling factor representing the effect an allosteric modulator has on orthosteric agonist efficacy; and [A] and [B] are the concentrations of the orthosteric agonist and the allosteric modulator, respectively. Parameters Embedded ImageA and Embedded ImageB represent the intrinsic ability of the orthosteric and allosteric ligand, respectively, to activate the receptor, and Em and n represent the maximal system response and the transducer slope, respectively. KA for DHPG and l-glutamate were constrained to values obtained from inhibition binding studies (Mutel et al., 2000; Gregory et al., 2012). Affinity cooperativity α was constrained to 1, assuming neutral cooperativity.

Results

Affinity, Association, and Dissociation Rates for mGlu5 PAM Binding.

Although previous studies show that CDPPB and MPPA bind to the common allosteric MPEP site on mGlu5 (Chen et al., 2007; Sharma et al., 2009), there is no binding information available for compound 2c. As such, we measured displacement of the radiolabeled MPEP analog [3H]methoxy-PEPy from mGlu5 to provide insight into the binding site of compound 2c and to determine PAM affinity estimates. Membranes from HEK293A cells with low expression of mGlu5 (HEK293A-mGlu5-low) were used, which have comparable mGlu5 expression to cortical astrocytes (Noetzel et al., 2012). MPPA fully displaced the radioligand, which is consistent with a competitive interaction (Fig. 1). Only partial displacement was observed for CDPPB, which may be due to either noncompetitive interaction or solubility limits of the compound (Fig. 1). Similarly, because of limited solubility, we were unable to test sufficiently high compound 2c concentrations to determine whether it can fully displace the radioligand (Fig. 1). Radioligand displacement curves were analyzed to obtain MPPA affinity (pKI) estimates using a model of competitive binding, whereas for CDPPB, affinity (pKB) and affinity cooperativity factor (α) estimates were derived using the allosteric ternary complex model (Table 1). The compound 2c displacement curve was fitted with both models.

Fig. 1.
Fig. 1.

Inhibition of [3H]methoxy-PEPy binding using HEK293A-mGlu5-low cell membranes. Displacement by each of the three PAMs was measured after a 1-hour incubation at room temperature. Data were normalized to 0 as 0% and to 100% as the mean for the total specific binding. Data points represent means + S.D. (duplicate measurement) from four (MPPA and CDPPB) or six (compound 2c) independent experiments. For compound 2c, the displacement curve was fitted equally well with a competitive (dashed line) vs. allosteric (solid line) model.

View this table:
TABLE 1

Affinity and kinetics of binding estimates for mGlu5 PAMs obtained from competition binding experiments with [3H]methoxy-PEPy in HEK293A-mGlu5-low cells

Data represent the means and 95% CI of n independent experiments performed in duplicate.

Binding kinetics of mGlu5 PAMs have not previously been assessed but could potentially be linked to different functional profiles. Therefore, the binding kinetics of MPPA, CDPPB, and compound 2c at mGlu5 were assessed with competition association binding experiments (Table 1). Data were fitted to the association competition binding function (Motulsky and Mahan, 1984) using kinetic parameters for [3H]methoxy-PEPy determined previously (koff: 0.14 ± 0.01 minutes−1; kon: 2.34 ± 0.46 Embedded Image107 M−1 min−1; Arsova et al. (2020)) (Fig. 2). Both MPPA and compound 2c had fast binding kinetics, prohibiting accurate quantification of koff. Hence, CDPPB had the longest residence time of the three PAMs, which is also reflected in a higher affinity relative to MPPA and compound 2c. MPPA had the fastest kon, followed by CDPPB and compound 2c (Table 1).

Fig. 2.
Fig. 2.

Kinetics of binding with HEK293A-mGlu5-low cell membranes. Competition association binding with [3H]methoxy-PEPy and indicated concentrations of each PAM: A) MPPA; B) CDPPB; C) compound 2c. Data are represented as means + S.D. (duplicate measurements) from six (MPPA), four (CDPPB competition experiments), eight (CDPPB vehicle experiments), or three (compound 2c) independent experiments.

Intrinsic Agonist Activity in Signaling Assays in HEK293A-mGlu5-Low Cells.

MPPA, CDPPB, and compound 2c were assessed for intrinsic agonist activity by measuring mGlu5 activation of Ca2+ mobilization (release from intracellular stores and extracellular influx), IP1 accumulation, and ERK1/2 phosphorylation (Fig. 3). Each of the three PAMs showed mGlu5 agonist activity across all three measures. DHPG had similar potency (pEC50) in the Ca2+ mobilization, IP1 accumulation, and ERK1/2 phosphorylation assays (Supplemental Table 1). We hypothesized that intrinsic agonist activity of PAMs may be due to potentiation of ambient glutamate. Therefore, experiments were repeated in the presence of 300 µM LY341495, a nonselective mGlu orthosteric antagonist (Kingston et al., 1998). Treatment with LY341495 reduced the basal level of IP1 accumulation to 22.8% of the untreated control, indicative of inverse agonist activity or inhibition of ambient glutamate (Supplemental Fig. 1). LY341495 had no effect on basal responses for Ca2+ mobilization or ERK1/2 phosphorylation (Supplemental Fig. 1). In the presence of LY341495, only CDPPB retained agonist activity for the three measures of mGlu5 activity, indicating that apparent intrinsic agonism for MPPA and compound 2c was most likely due to modulation of ambient glutamate. CDPPB agonism was then compared with that of the orthosteric agonist DHPG. Relative to DHPG, CDPPB was a partial agonist for Ca2+ mobilization and ERK1/2 phosphorylation but achieved the same maximal response as DHPG in the absence of LY341495 in the IP1 accumulation assay (Fig. 3; Supplemental Fig. 1; Supplemental Table 2). CDPPB had significantly lower agonist potency in IP1 accumulation (12- to 30-fold) when compared with ERK1/2 phosphorylation and Ca2+ mobilization (Supplemental Fig. 1; Supplemental Table 2).

Fig. 3.
Fig. 3.

Intrinsic PAM agonist activity in HEK293A-mGlu5-low cells. (A) Peak Ca2+ mobilization measurement measured 90 seconds after PAM or DHPG addition at 37°C expressed as percent maximal DHPG response. (B) IP1 accumulation measured 1 hour after PAM or DHPG addition at 37°C expressed as percent maximal (max) DHPG response. (C) ERK1/2 phosphorylation measurement after a 5-minute incubation of PAM or DHPG addition at 37°C. In the presence of 300 µM LY341495, the response to PAMs or DHPG is diminished for intracellular Ca2+ (iCa2+) mobilization (D), IP1 accumulation (E), or ERK1/2 phosphorylation (F). Data in (D–F) are expressed as percent maximal DHPG response in the absence of LY341495, in which 0% is defined by vehicle (veh) treated in the presence of LY341495. The effect of LY341495 on basal responses in each assay is shown in Supplemental Fig 1. The dashed line in (D–F) shows the response to DHPG concentration-response relationship (from A–C) in the absence of LY341495 for reference. Data are means + S.D. (duplicate measurements) from 3–11 independent experiments (refer to Table 2 for exact numbers).

Potentiation of Orthosteric Agonists in Signaling Assays in HEK293A-mGlu5-Low Cells.

MPPA, CDPPB, and compound 2c were then tested for their ability to potentiate stimulation of mGlu5 by a low concentration of l-glutamate and DHPG in the three signaling assays (Fig. 4). All three PAMs potentiated the responses induced by both orthosteric agonists in Ca2+ mobilization, IP1 accumulation, and ERK1/2 phosphorylation signaling assays (Fig. 4). Concentration-response curves were fitted to quantify PAM potency (pPAM50) and the maximum level of potentiation (PAMmax) (Supplemental Table 3). Compound 2c potentiated the l-glutamate and DHPG responses to the same maximum response as the orthosteric agonists alone in the Ca2+ mobilization and IP1 accumulation assays. Both compound 2c and CDPPB potentiated the l-glutamate and DHPG responses above the orthosteric agonist maximal response in the ERK1/2 phosphorylation assay (Fig. 4). Compound 2c had the lowest potency in all three assays, whereas MPPA and CDPPB had similar PAM potencies (Supplemental Table 3).

Fig. 4.
Fig. 4.

Potentiation of orthosteric agonist responses in HEK293A-mGlu5-low cells. Ca2+ mobilization (intracellular: iCa2+) after stimulation with orthosteric agonist alone or simultaneous addition of PAM and 100 nM l-glutamate (Glu) (A) or DHPG (B). IP1 accumulation in response to incubation with orthosteric agonist alone or coincubation with PAM and 1 µM l-glutamate(C) or DHPG (D). Phosphorylated ERK1/2 levels after stimulation with orthosteric agonist alone or simultaneous addition of PAMs and 500 nM l-glutamate (E) or DHPG (F). Data points are means + S.D. (triplicate measurements) from three to six independent experiments (refer to Table 3 and Supplemental Table 1 for exact numbers). Data were normalized to buffer or vehicle (veh.) treated as 0% and to the maximal (max) l-glutamate (A and C), maximal DHPG (B and D), or 10% FBS (E and F) responses as 100%.

PAMs Induce mGlu5 Internalization in HEK293A Cells.

Most GPCRs are regulated by desensitization and internalization upon agonist stimulation (Ferguson, 2001). The mGlu5 receptor is internalized upon stimulation with l-glutamate (Levoye et al., 2015; Arsova et al., 2020), and several PAMs can induce and/or potentiate DHPG-stimulated mGlu5 desensitization of Ca2+ mobilization (Hellyer et al., 2019). The ability of MPPA, CDPPB, and compound 2c to induce mGlu5 internalization was characterized using a real-time internalization assay. The assay is based on time-resolved Förster resonance energy transfer between the long lifetime donor fluorophore Lumi4-Tb, covalently attached to a SNAP-tag on cell surface receptors, and the cell-impermeant acceptor fluorophore fluorescein-O′-acetic acid (Roed et al., 2014; Foster and Bräuner-Osborne, 2018). The assay requires N-terminal fusion of mGlu5 with a SNAP-tag; therefore, HEK293A cells were transiently transfected with SNAP-tagged mGlu5 (HEK293A-SNAP-mGlu5), which resulted in mGlu5 expression levels that were ∼10 times higher than in the HEK293A-mGlu5-low cell line (Arsova et al., 2020). Cells were cotransfected with the EAAT3 glutamate transporter to reduce the extracellular glutamate concentration during measurements. To measure PAM potentiation of glutamate, glutamate transport was inhibited by adding the nontransportable EAAT3 inhibitor DL-TBOA at the same time as the PAMs. However, inhibition of EAAT3 with a saturating concentration of DL-TBOA (100 µM) resulted in ∼0.9 µM extracellular l-glutamate and ∼40% of the mGlu5 internalization induced by 100 µM l-glutamate (Arsova et al., 2020). The DL-TBOA concentration was reduced to 30 µM for the l-glutamate potentiation experiments, which resulted in 24% (95% CI 23%–25%, n = 3) of the maximum l-glutamate–induced mGlu5 internalization.

DHPG induced a concentration-dependent increase in mGlu5 internalization that reached a plateau around 60 minutes after agonist addition (Supplemental Fig. 2), similar to the previously observed temporal profile for l-glutamate (Arsova et al., 2020). In the absence of added orthosteric agonist or antagonist, CDPPB and compound 2c induced mGlu5 internalization, although to a lower level than that achieved by DHPG (Fig. 5, A–C). When 300 µM LY341495 was added to block activation by ambient/released l-glutamate, only CDPPB remained a partial agonist for inducing mGlu5 internalization (Fig. 5, D–F), consistent with intrinsic efficacy in the three signaling assays (Fig. 3). LY341495 alone had no effect on the baseline level of internalization (Supplemental Fig. 1). Internalization concentration-response curves were calculated by determining the area under the curves from the 60-minute time courses (Fig. 6) and fitted to determine the Emax and pEC50 values (Supplemental Table 2). The agonist potency of CDPPB for internalization was >30-fold lower than for Ca2+ mobilization and pERK1/2, but within 3-fold of IP1 accumulation. DHPG potency for internalization was within 4-fold of the three signaling measures. Compound 2c curves were not well defined, precluding the estimation of Emax and pEC50 values (Fig. 6A).

Fig. 5.
Fig. 5.

Real-time measurement of mGlu5 internalization. HEK293A cells were transiently transfected with HA-SNAP-mGlu5 and EAAT3 (HEK293A-SNAP-mGlu5), and internalization was measured as a change in fluorescence over time. (A–C) Indicated concentrations of each PAM were added at t = 0 minutes, and surface mGlu5 levels were tracked for 66 minutes. (D–F) PAM-induced mGlu5 internalization in the presence of 300 µM LY341495. (G–I) Potentiation of l-glutamate–induced mGlu5 internalization by indicated PAMs. The l-glutamate concentration was increased by partially blocking the EAAT3 glutamate transporter with 30 µM DL-TBOA (DL-threo-β-Benzyloxyaspartic acid). (J–L) Potentiation of 1 μM DHPG-induced mGlu5 internalization by indicated PAMs. Data points are means + S.D. (triplicate measurements) from three independent experiments, and solid lines are nonlinear regression fit to an exponential model of one-phase association.

Fig. 6.
Fig. 6.

Concentration-response relationships for agonism and potentiation of mGlu5 internalization. From the kinetic measurements in Fig. 5, the area under the curve was calculated for each ligand concentration and normalized to the maximal (max) orthosteric agonist response measured in parallel. Each PAM was tested alone (A) and in the presence of 300 µM LY341495 (B). Each PAM was assessed for potentiation of l-glutamate (by partially blocking l-glutamate (Glu) transport with 30 µM DL-TBOA) (C) or 1 µM DHPG (D) induced mGlu5 internalization. Data are means + S.D. (triplicate measurement) from three or four (l-glutamate) independent experiments. For reference, the control curve for DHPG (without LY341495) is shown by the dashed line in (B). Error bars not shown lie within the dimensions of the symbol, veh. denotes vehicle.

All three PAMs potentiated l-glutamate– and DHPG-induced internalization with kinetics similar to DHPG (Fig. 5, G–L; Supplemental Fig. 3). Potentiation of both l-glutamate and DHPG by MPPA induced a lower maximum internalization than CDPPB and compound 2c (Fig. 6, C–D). Similar to the other signaling assays, compound 2c had the lowest pPAM50 value of the three PAMs in the internalization assay. CDPPB and compound 2c potentiated to similar or greater levels of internalization compared with each orthosteric ligand alone (Fig. 6, C–D).

Quantification and Comparison of PAM Affinity and Cooperativity in HEK293A Cells.

Comparisons of PAMmax and pPAM50 values between the four measures of mGlu5 function revealed assay-dependent differences for each PAM (Supplemental Fig. 4) but no evidence for probe dependence when comparing values derived from DHPG versus l-glutamate (Supplemental Fig. 5). However, PAMmax and pPAM50 values are assay-dependent composite values comprising allosteric ligand affinity, cooperativity, and efficacy. Assay-independent measures of affinity (pKA or pKB), intrinsic efficacy (τ), and cooperativity (αβ) can be derived from fitting of concentration-response curves of PAMs and a reference agonist to operational models of agonism or allosterism. CDPPB concentration-response curves in the presence of LY341495 were fitted with the operational model of agonism, in which DHPG was the reference agonist, to determine pKA and τ for different mGlu5 functional measures (Table 2). The apparent pKA was lower for the internalization pathway than for Ca2+ mobilization and ERK1/2 phosphorylation (Table 2). However, the model is limited to partial agonists, so for IP1 accumulation, in which CDPPB behaved as a full agonist, we were only able to determine the composite transduction coefficient log(τ/KA). By comparing these with the transduction coefficient of DHPG, we calculated Δlog(τ/KA) values for each pathway and found that CDPPB did not show any significant bias between functional measures (Fig. 7A; Table 2).

View this table:
TABLE 2

Affinity (pKA) and log(τ/KA) estimates for the agonist activity of DHPG or mGlu5 PAMs in HEK293A-mGlu5-low or HEK293A-SNAP-mGlu5 cells

Estimates were derived in the presence of the orthosteric antagonist LY341495 to ensure that only the intrinsic agonist activity of the PAMs was measured. Data represent the means and 95% CI of n independent experiments performed in duplicate or triplicate (internalization assay).

Fig. 7.
Fig. 7.

Assessment of biased agonism or modulation in HEK293A-mGlu5-low or HEK293A-SNAP-mGlu5 cells. (A) For CDPPB intrinsic agonism, ∆log(τ/KA) values (relative to DHPG) were derived from concentration-response curves in the presence of LY341495. (B) Cooperativity factors for each functional response are presented relative to the value calculated from Ca2+ mobilization. Cooperativity with DHPG is depicted in squares. Cooperativity with l-glutamate is depicted in triangles. For select PAMs and functional outputs, cooperativity could not be determined (not applicable: n.a.) or was indistinguishable from neutral because of intrinsic PAM agonist activity. Data are means and 95% CI from three to five independent experiments (refer to Tables 2 and 4 for exact numbers). *P < 0.05, one-way ANOVA with Tukey’s multiple comparisons test.

We used an operational model of allosterism to determine the affinity (pKB) and cooperativity (log β) from concentration-response curves of PAM potentiation of either l-glutamate or DHPG (Tables 3 and 4). The intrinsic agonist activity (τ) of CDPPB was constrained to the values determined for CDPPB in the presence of LY341495 when fitting the Ca2+ mobilization and ERK1/2 phosphorylation curves. Since τ could not be determined for IP1 accumulation of CDPPB, it was not possible to use the operational model of allosterism to analyze the corresponding potentiation curves. Furthermore, compound 2c potentiated the activity of both orthosteric agonists in IP1 accumulation and of DHPG in ERK1/2 phosphorylation above the maximum response of the orthosteric agonist. This prohibited fitting compound 2c data with the operational model of allosterism, as there was no independent means to estimate the maximal system response (Em). With the assumption that PAMs were neutral with respect to affinity cooperativity (log α), comparing the efficacy cooperativity scaling factor (log β) across the four functional pathways showed that log β was highest in the Ca2+ mobilization pathway for MPPA with l-glutamate and for the Ca2+ mobilization and ERK1/2 phosphorylation pathways for MPPA with DHPG (Fig. 7B). The pKB values were in general agreement between each functional measure and with pKI values derived from radioligand inhibition binding, although the pKB values for CDPPB and compound 2c derived from the internalization experiment were 5- to 8-fold lower than the corresponding pKI values (Supplemental Fig. 6). There was no indication of probe bias when comparing the pKB values obtained in presence of l-glutamate and DHPG, but the log β values were on average 2.09-fold (95% CI 1.70–2.48) higher in the presence of DHPG (Supplemental Fig. 5, C and D).

View this table:
TABLE 3

Affinity estimates for allosteric modulation of low concentrations of orthosteric ligand (l-glutamate or DHPG) in HEK293A-mGlu5-low or HEK293A-SNAP-mGlu5 cells

Data represent the means and 95% CI of n independent experiments performed in triplicate.

View this table:
TABLE 4

Cooperativity factors for allosteric modulation of low concentrations of orthosteric ligand (l-glutamate or DHPG) by mGlu5 PAMs in HEK293A-mGlu5-low or HEK293A cells

Data represent the means and 95% CI of n independent experiments performed in triplicate.

Agonism and Potentiation of DHPG in Cortical Neurons.

We used primary cortical neurons to study PAM Ca2+ mobilization and IP1 accumulation in cells with endogenous expression of mGlu5. In these experiments, we used DHPG as the orthosteric agonist (pEC50 values given in Supplemental Table 1), as glutamate was unsuitable because of the presence of other mGlu receptors and ionotropic glutamate receptors in cortical neurons. We preincubated cells with 30 μM 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester to inhibit activation of mGlu1, which is also a receptor for DHPG (Ito et al., 1992). In the absence of orthosteric ligand, only compound 2c induced a weak agonist response in Ca2+ mobilization (Fig. 8A). In contrast, all PAMs acted as partial agonists (50%–60% of the maximum DHPG response) in the IP1 accumulation assay (Fig. 8B; Supplemental Table 3). Although neurons endogenously express glutamate transporters, it is possible that glutamate released during the IP1 accumulation experiment could contribute to the observed stimulation, similar to what we observed in HEK293A cells, in which it was necessary to block the orthosteric binding site with a competitive antagonist to determine the agonist activity of the PAMs. However, there are no mGlu5-selective orthosteric antagonists, and since cortical neurons also express other mGlu receptors, the inclusion of a nonselective orthosteric antagonist such as LY341495 could be a further confounding factor. GPT was included to minimize the influence of ambient glutamate.

Fig. 8.
Fig. 8.

PAM agonism and potentiation of DHPG in cortical neurons. Intrinsic agonist activity of PAMs in primary cortical neurons for Ca2+ mobilization (A) and IP1 accumulation (B). (C) PAM potentiation of 120 nM DHPG was assessed in Ca2+ mobilization assays with simultaneous addition. (D) For IP1 accumulation, PAM potentiation was assessed in the presence of 1 μM DHPG, since DHPG has lower potency in this assay. Data are means + S.D. (duplicate measurements) from four to nine independent experiments (refer to Table 5 for exact numbers). Data were normalized to 0% as buffer and 100% maximal (max) DHPG response. Where iCa2+ denotes intracellular Ca2+ and veh. denotes vehicle.

All PAMs potentiated a low concentration (120 nM) of DHPG-stimulated mGlu5- Ca2+ mobilization and IP1 accumulation (Fig. 8, C and D). In Ca2+ mobilization assays, the maximum response of compound 2c potentiation of DHPG (Emax) was similar to the Emax of DHPG, whereas MPPA and CDPPB induced 40%–50% of the DHPG Emax (Supplemental Table 3). In the IP1 accumulation pathway, all three PAMs potentiated the DHPG response to 70%–80% of the DHPG Emax (Fig. 8D). CDPPB had the highest potency (pPAM50) in both assays in the presence of DHPG and the highest pEC50 in the IP1 accumulation assay without added agonist.

Again, we used operational models to derive the affinities (pKA) and transduction coefficients log(τ/KA) from PAM concentration-response curves in the absence of orthosteric ligand and the affinities (pKB) and cooperativities (log β) from DHPG potentiation curves (Table 5). Only compound 2c elicited a response in both pathways in the absence of orthosteric ligand, with similar transduction coefficients for the Ca2+ mobilization and IP1 accumulation pathways. In the presence of DHPG, the cooperativity factors (log β) of MPPA, CDPPB, and compound 2c were indistinguishable from 0 in the IP1 accumulation pathway. Therefore, log β was higher for all three PAMs in the Ca2+ mobilization pathway. Affinity estimates (pKB) were similar for the two pathways for MPPA, CDPPB, and compound 2c (Table 5).

View this table:
TABLE 5

Quantification of agonist activity of DHPG or PAMs, as well as PAM modulation of DHPG responses, in mouse cortical neurons

Data represent the means and 95% CI of n independent experiments performed in duplicate.

Discussion

Positive allosteric modulation of mGlu5 shows promise as a potential therapeutic strategy for schizophrenia. However, undesirable on-target side effects associated with mGlu5 PAMs have stalled development (Rook et al., 2013; Parmentier-Batteur et al., 2014; Foster and Conn, 2017). Development of safer and more efficacious mGlu5 PAMs is hampered by the lack of in-depth molecular pharmacological characterization to accurately link in vitro profiles to in vivo effects. We provided a detailed characterization of the mGlu5 PAMs CDPPB, MPPA, and compound 2c, representing distinct structural scaffolds; MPPA and compound 2c had previously only been assessed in mGlu5-mediated Ca2+ mobilization assays. CDPPB had a longer receptor residence time than either MPPA or compound 2c, which correlated with higher mGlu5 affinity. In addition to rigorously profiling the agonist and potentiator activity of each modulator at three measures of acute mGlu5 signaling, we assessed the influence of mGlu5 PAMs on receptor internalization for the first time. We found no evidence for biased agonism; however, MPPA was a biased modulator, with different magnitudes of positive cooperativity with l-glutamate or DHPG depending on the measure of mGlu5 function. Importantly, this biased cooperativity also translated to natively expressed mGlu5 in primary cortical neurons.

Ligand binding kinetics have been correlated with compound affinity and efficacy in vivo (Tummino and Copeland, 2008; Copeland, 2016). We report the first assessment of mGlu5 PAM binding kinetics. CDPPB has the longest receptor residence time of the three PAMs. A slower rate of dissociation may be linked to the fact that CDPPB alone showed intrinsic efficacy at all four measures of mGlu5 activity in recombinant cells. For some GPCRs, compounds with longer residence times or fast kon rates have higher efficacy and, thus, have been considered as more desirable lead compounds (Vauquelin and Charlton, 2010; Guo et al., 2014; Doornbos et al., 2017). Indeed, the efficacy (but not affinity) of select mGlu2 PAMs correlates with residence time, whereas increased kon rates correlated with increased affinities (Doornbos et al., 2017). Indeed, MPPA behaved similarly here; a fast kon rate compensated for a short residence time, giving rise to submicromolar affinity. Ligand kinetics can also determine the extent of signaling bias for the serotonin 5-HT2B and the dopamine D2 receptors (Unett et al., 2013; Klein Herenbrink et al., 2016) but not for the µ opioid receptor (Pedersen et al., 2020). In the future, it would be of interest to explore the relationship between receptor residence time and PAM agonist efficacy and biased agonism for additional structurally diverse mGlu5 ligands.

CDPPB alone retained PAM agonist activity across all four measures of mGlu5 activity in the presence of both GPT/EAAT3 and LY341495 to negate the confounding influence of ambient glutamate. These data are in keeping with previous evidence for CDPPB PAM agonist activity as well as induction of receptor desensitization and tolerance development with respect to changes in sleep architecture, although such effects are known to be context- and model-dependent (Kinney et al., 2005; Parmentier-Batteur et al., 2012; Hellyer et al., 2019). CDPPB showed robust agonist efficacy for IP1 accumulation while being a weak partial agonist for Ca2+ mobilization and ERK1/2 phosphorylation, consistent with previous work (Sengmany et al., 2017). Application of the operational model of agonism found that CDPPB was not biased relative to DHPG, in direct contrast to earlier findings in which CDPPB and a number of structurally diverse mGlu5 PAMs preferentially activated IP1 accumulation over Ca2+ mobilization relative to l-glutamate or DHPG (Sengmany et al., 2017; Hellyer et al., 2018). One possible explanation for this discrepancy may be the inclusion of the orthosteric antagonist LY341495. Notably, the same concentration of LY341495 had very different effects on the DHPG concentration-response curve, right-shifting DHPG potency in Ca2+ mobilization as expected for a competitive antagonist; however, the DHPG response was completely abolished in IP1 accumulation and pERK1/2. Moreover, LY341495 markedly reduced basal IP1 accumulation, suggestive of inverse agonist activity. The lack of apparent inverse agonism for other mGlu5 activity measures may reflect observational bias or an LY341495-specific effect. Certain mGlu5 NAMs are biased modulators (Jong et al., 2019; Sengmany et al., 2019; Arsova et al., 2020); future experiments should explore this possibility for orthosteric antagonists. Therefore, it is possible that the receptor conformations sampled when simultaneously occupied by LY341495 and CDPPB are distinct from those sampled by CDPPB alone or CDPPB with a small population occupied by the low ambient glutamate levels.

Probe dependence is operative at mGlu5, manifesting as differences in the magnitude of cooperativity depending on the orthosteric agonist used (Sengmany et al., 2017; Hellyer et al., 2020). Probe-dependent PAMs include 1‐(4‐(2,4‐difluorophenyl)piperazin‐1‐yl)‐2‐((4‐fluorobenzyl)oxy)ethanone (structurally related to compound 2c) and acetylenic PAMs (which share an overlapping pharmacophore with MPPA). Herein, CDPPB did not show probe dependence; affinity and cooperativity estimates derived from interactions with either DHPG or l-glutamate were similar, consistent with our earlier report (Sengmany et al., 2017). Further, affinity and cooperativity estimates for MPPA and compound 2c determined from potentiation curves of DHPG or l-glutamate were also similar, despite belonging to structural classes of mGlu5 PAMs that show probe dependence. These findings build on the evidence base that structurally similar mGlu5 allosteric ligands can differentially exhibit probe dependence, representing an important consideration when interpreting structure-activity relationships within a discovery program.

Related to probe dependence is the idea that allosteric modulators can engender biased modulation, as evidenced by different magnitudes of affinity or cooperativity depending on the measure of receptor activity in the presence of the same orthosteric agonist. For mGlu5, biased modulation has been observed for allosteric ligands classified as NAMs or PAMs based on Ca2+ mobilization assays (Sengmany et al., 2017, 2019; Arsova et al., 2020). Here, we show that MPPA is a biased mGlu5 PAM for which the magnitude of cooperativity with orthosteric agonists is lower/neutral when measured in IP1 accumulation or receptor internalization when compared with Ca2+ mobilization and ERK1/2 phosphorylation in HEK293A-mGlu5 cells. These data are in agreement with previous reports for mGlu5 in which cooperativity was lower when measured in IP1 accumulation assays over Ca2+ mobilization (Sengmany et al., 2017, 2019). Although mGlu5 couples predominantly to Gq/11 proteins to elevate inositol trisphosphate levels and release of Ca2+ from intracellular stores, mGlu5 also couples to Gs and modulates the activity of multiple ion channels (enabling extracellular Ca2+ influx) in a Gq/11-independent fashion [reviewed in Gregory and Goudet (2021)]. Both intracellular release and extracellular influx of Ca2+ were measured in the assays used herein. Therefore, the biased agonism and modulation observed for mGlu5 PAMs between two measures that are traditionally considered linked likely arises because of stabilizing receptor conformations that differentially favor these different effectors. A key difference between the responses for which MPPA cooperativity is greater is the temporal nature of the assays. Ca2+ mobilization and ERK1/2 phosphorylation are short-lived in comparison with IP1 accumulation and receptor internalization, which are both measured over 1 hour. Measuring a nonequilibrium response can influence how signaling bias is observed, as previously shown for the dopamine D2 receptor (Klein Herenbrink et al., 2016). In contrast, compound 2c cooperativity estimates with DHPG and l-glutamate were not significantly different across all measures. However, the intrinsic efficacy of CDPPB or high positive cooperativity of compound 2c prohibited quantification of these parameters in certain assays using the modulator titration paradigm employed here. Importantly, biased cooperativity of MPPA with DHPG between IP1 accumulation and Ca2+ mobilization translated to natively expressed mGlu5. The observation that magnitudes of cooperativity can differ depending on the measure of receptor activity may contribute to the challenges in translating in vitro profiles to efficacy in vivo, particularly in discovery pipelines in which cooperativity is determined from a single measure.

Context and cell background are important considerations when classifying mGlu5 ligand pharmacology. Pharmacological profiles in primary cortical neurons differed to recombinant cells. In primary cortical neurons, none of the three ligands showed intrinsic efficacy for Ca2+ mobilization, and all were robust partial agonists for IP1 accumulation. For select mGlu5 PAMs, agonist activity for Ca2+ mobilization has been linked to receptor expression levels in recombinant cells and is not always recapitulated in native cells (Noetzel et al., 2012). The biased agonism profiles for mGlu5 PAM agonists can be different between recombinant and native systems as well (Sengmany et al., 2017). In native systems, mGlu5 forms oligomeric complexes with 1) other GPCRs, 2) surface proteins, and 3) scaffolding proteins via the C-tail (Pin and Bettler, 2016); differing complements of effector, regulatory, and scaffolding proteins have the capacity to shape mGlu5 signaling in a cell type–dependent manner. In addition, mGlu5 is found on intracellular membranes such that the cellular response to mGlu5 activation may differ depending on where it is generated from and accessibility of ligands to different subcellular compartments (Jong et al., 2014, 2019). Recombinant versus native cells may have different ambient glutamate levels, or glutamate may be released in an activity-dependent manner. All of these factors may contribute to the mechanisms underlying biased mGlu5 modulation. Future experiments could employ selective inhibitors of coexpressed channels and transporters to decipher these underlying mechanisms.

Compound 2c consistently had the greatest degree of positive cooperativity, independent of the orthosteric ligand or response measured. Although compound 2c has not yet been tested for in vivo efficacy, related compounds have demonstrated antipsychotic efficacy and procognitive effects in preclinical models (Xiong et al., 2010; Gregory et al., 2013). The magnitude of mGlu5 PAM cooperativity with l-glutamate based on Ca2+ mobilization was recently shown to correlate with efficacy in the amphetamine hyperlocomotion assay (Gregory et al., 2019). However, whether such correlations extend to structurally diverse mGlu5 PAMs remains to be tested. Indeed, MPPA has higher positive cooperativity than CDPPB, yet a lower CDPPB dose is required for efficacy in reducing amphetamine-induced hyperlocomotion in rats relative to MPPA (Kinney et al., 2005; Sharma et al., 2009). It is unknown how the pharmacokinetics of MPPA compares with CDPPB. Additionally, differences in receptor residency times may also be linked to in vivo efficacy; further investigation is warranted.

In summary, we have determined the binding and signaling profiles of three mGlu5 PAMs from distinct scaffolds at four measures of mGlu5 function in recombinant cells. Key differences in in vitro pharmacological profiles translated to natively expressed mGlu5 in primary cortical neurons. By assessing the kinetics of PAM binding to the mGlu5 receptor, we reveal previously unappreciated differences that may contribute to observations of PAM agonist activity, as well as biased cooperativity. Improved molecular characterization provides a better basis to understand the pharmacological properties of mGlu5 PAMs, which can be implemented in the future for improved structure-activity relationship interrogation and rational drug discovery.

Acknowledgments

We thank Morten Jørgensen and Søren Møller Nielsen, H. Lundbeck A/S, for providing the PAMs tested in this study.

Authorship Contributions

Participated in research design: Arsova, Vedel, Foster, Hansen, Bräuner-Osborne, Gregory.

Conducted experiments: Arsova, Møller, Hellyer, Gregory.

Performed data analysis: Arsova, Møller, Hellyer, Gregory.

Wrote or contributed to the writing of the manuscript: Arsova, Møller, Hellyer, Vedel, Foster, Hansen, Bräuner-Osborne, Gregory.

Footnotes

    • Received October 20, 2020.
    • Accepted January 27, 2021.

  • ↵1 Current affiliation: QIMR Berghofer Medical Research Institute, Brisbane, QLD, Australia.

  • ↵2 A.A. and T.C.M. contributed equally to this work.

  • ↵3 H.B.-O. and K.J.G. contributed equally to this work.

  • A.A. acknowledges financial support from the University of Copenhagen, Oticon Foundation, and Torben and Alice Frimodts Foundation. H.B.-O. acknowledges financial support from the Augustinus Foundation, the Lundbeck Foundation, and the Independent Research Fund Denmark. This project received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Sklodowska-Curie grant agreement (797497) (T.C.M.). S.R.F. acknowledges financial support from the Lundbeck Foundation and the Independent Research Fund Denmark. This work was supported by the National Health and Medical Research Council of Australia (NHMRC): Project Grants APP1084775 (K.J.G.) and APP1127322 (K.J.G.). K.J.G. is supported by an Australian Research Council Future Fellowship: FT170100392.

  • J.L.H. was an employee and shareholder of Novo Nordisk A/S at the time of the study.

  • A prior version of the paper was included in the following PhD thesis: Arsova A (2018) Biased signaling and allosteric modulation of metabotropic glutamate receptor 5. Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

  • https://doi.org/10.1124/molpharm.120.000185.

  • ↵Embedded ImageThis article has supplemental material available at molpharm.aspetjournals.org.

Abbreviations

AM
acetoxymethyl
CDPPB
3-cyano-N-(2,5-diphenylpyrazol-3-yl)benzamide
compound 2c
1-[4-(4-chloro-2-fluoro-phenyl)piperazin-1-yl]-2-(4-pyridylmethoxy)ethenone
DHPG
(S)-3,5-dihydroxyphenylglycine
DL-TBOA
DL-threo-β-benzyloxyaspartic acid
DMEM
Dulbecco’s modified Eagle’s medium
CI
confidence interval
EAAT3
excitatory amino acid transporter 3
Em
maximum response of a system
Emax
Maximum response to an agonist in a functional assay
ERK1/2
extracellular signal–regulated kinase 1/2
GPCR
G protein–coupled receptor
GPT
glutamic-pyruvic transaminase
HA
hemagglutinin
HBSS
Hanks’ balanced salt solution
HEK293A
human embryonic kidney 293A
IP1
inositol monophosphate
LY341495
(1S,2S)-2-[(1S)-1-amino-1-carboxy-2-(9H-xanthen-9-yl)ethyl]cyclopropane-1-carboxylic acid
methoxy-PEPy
3-methoxy5-(2-pyridinylethynyl)pyridine
mGlu
metabotropic glutamate
mGlu5
metabotropic glutamate subtype 5
MPEP
2-methyl-6-(phenylethynyl)pyridine hydrochloride
MPPA
N-methyl-5-(phenylethynyl)pyrimidin-2-amine
NAM
negative allosteric modulator
PAM
positive allosteric modulator
PAMMAX
Maximal level of potentiation induced by a PAM when assessed in a modulator titration curve in the presence of orthosteric agonist
pEC50
negative logarithm of the half maximal effective concentration of an agonist
pERK1/2
phosphorylated extracellular signal-regulated kinases 1 and 2
pPAM50
negative logarithm of the half maximal effective concentration of a PAM from a modulator titration curve in the presence of orthosteric agonist;

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Positive Allosteric Modulation of the mGlu5 Receptor

Angela Arsova, Thor C. Møller, Shane D. Hellyer, Line Vedel, Simon R. Foster, Jakob L. Hansen, Hans Bräuner-Osborne and Karen J. Gregory
Molecular Pharmacology May 1, 2021, 99 (5) 328-341; DOI: https://doi.org/10.1124/molpharm.120.000185
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