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Research ArticleArticle

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
Angela Arsova
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (A.A., T.C.M., L.V., S.R.F., H.B.-O.); Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, VIC, Australia (S.D.H., K.J.G.); and Cardiovascular Research, Novo Nordisk A/S, Novo Nordisk Park 1, Måløv, Denmark (J.L.H.)
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Thor C. Møller
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (A.A., T.C.M., L.V., S.R.F., H.B.-O.); Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, VIC, Australia (S.D.H., K.J.G.); and Cardiovascular Research, Novo Nordisk A/S, Novo Nordisk Park 1, Måløv, Denmark (J.L.H.)
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  • ORCID record for Thor C. Møller
Shane D. Hellyer
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (A.A., T.C.M., L.V., S.R.F., H.B.-O.); Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, VIC, Australia (S.D.H., K.J.G.); and Cardiovascular Research, Novo Nordisk A/S, Novo Nordisk Park 1, Måløv, Denmark (J.L.H.)
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Line Vedel
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (A.A., T.C.M., L.V., S.R.F., H.B.-O.); Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, VIC, Australia (S.D.H., K.J.G.); and Cardiovascular Research, Novo Nordisk A/S, Novo Nordisk Park 1, Måløv, Denmark (J.L.H.)
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Simon R. Foster
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (A.A., T.C.M., L.V., S.R.F., H.B.-O.); Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, VIC, Australia (S.D.H., K.J.G.); and Cardiovascular Research, Novo Nordisk A/S, Novo Nordisk Park 1, Måløv, Denmark (J.L.H.)
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Jakob L. Hansen
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (A.A., T.C.M., L.V., S.R.F., H.B.-O.); Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, VIC, Australia (S.D.H., K.J.G.); and Cardiovascular Research, Novo Nordisk A/S, Novo Nordisk Park 1, Måløv, Denmark (J.L.H.)
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Hans Bräuner-Osborne
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (A.A., T.C.M., L.V., S.R.F., H.B.-O.); Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, VIC, Australia (S.D.H., K.J.G.); and Cardiovascular Research, Novo Nordisk A/S, Novo Nordisk Park 1, Måløv, Denmark (J.L.H.)
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  • For correspondence: hbo@sund.ku.dk
Karen J. Gregory
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark (A.A., T.C.M., L.V., S.R.F., H.B.-O.); Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences and Department of Pharmacology, Monash University, Parkville, VIC, Australia (S.D.H., K.J.G.); and Cardiovascular Research, Novo Nordisk A/S, Novo Nordisk Park 1, Måløv, Denmark (J.L.H.)
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  • For correspondence: karen.gregory@monash.edu
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    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.

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    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.

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    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).

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    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%.

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    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.

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    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.

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    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.

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    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.

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    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.

    Equilibrium Radioligand DisplacementCompetition Association Binding
    LigandpKIa (95% CI)pKBb (95% CI)Log αc (95% CI)nkon [×106 (M−1min−1)]d (95% CI)koff (min−1)e (95% CI)RTfpKDgn
    min
    MPPA6.51 (6.25–6.77)n.d.n.d.411.0 (3.7–18.2)1n.d.n.d.6
    CDPPBn.d.7.27 (6.79–7.74)−0.64 (−1.10 to −0.24)41.91 (−0.3 to 4.1)0.211 (0.067–0.356)4.76.966
    Compound 2c5.17 (5.08–5.27)h5.36 (5.19–5.53)−0.96 (−1.17 to −0.75)60.16 (−0.01 to 0.34)1n.d.n.d.3
    • n.d., not determined due to no fit with the given model.

    • ↵a Negative logarithm of the equilibrium dissociation constant determined with a competitive binding model.

    • ↵b Negative logarithm of the equilibrium dissociation constant determined with an allosteric binding model.

    • ↵c Logarithm of the cooperativity factor.

    • ↵d Association rate constant.

    • ↵e Dissociation rate constant. For ligands with fast koff, global analyses could not derive koff; therefore, the value was constrained to 1 to enable estimation of kon.

    • ↵f RT, residence time defined as 1/koff.

    • ↵g Negative logarithm of the equilibrium dissociation constant determined from kinetic parameters (koff/kon).

    • ↵h Assumed full displacement (=constrained minimum to 0%).

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    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).

    Ca2+ MobilizationIP1 AccumulationpERK1/2Internalization
    LigandpKAa (95% CI)Log(τ/KA)b (95% CI)npKA (95% CI)Log(τ/KA) (95% CI)npKA (95% CI)Log(τ/KA) (95% CI)npKA (95% CI)Log(τ/KA) (95% CI)n
    DHPGn.d.6.78 (6.64–6.93)3n.d.5.89 (5.80–5.98)5n.d.6.06 (5.99–6.13)11n.d.5.43 (5.36–5.50)3
    MPPAn.r.n.r.4n.r.n.r.3n.r.n.r.3n.r.n.r.3
    CDPPB7.20 (6.36–7.94)6.71 (5.88–7.55)4n.d.6.18 (5.89–6.47)57.14 (6.58–7.61)7.03 (6.56–7.58)55.57 (5.22–5.96)c5.55 (5.29–5.83)3
    Compound 2cn.r.n.r.4n.r.n.r.3n.r.n.r.3n.r.n.r.3
    • n.d., not determined; n.r., no response.

    • ↵a pKA, negative logarithm of the equilibrium dissociation binding constant determined with the operational model of agonism.

    • ↵b Log(τ/KA), transduction coefficient; τ, intrinsic efficacy.

    • ↵c P < 0.05 when compared with estimates from Ca2+ mobilization or IP1 accumulation by one-way ANOVA with Tukey’s multiple comparisons test.

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    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.

    Ca2+ MobilizationIP1 AccumulationpERK1/2Internalizationa
    LigandpKBb (95% CI)npKB (95% CI)npKB (95% CI)npKB (95% CI)n
    l-Glutamate
     MPPA7.11 (6.70–7.52)47.23 (6.78–7.67)36.90 (6.31–7.30)37.19 (6.85–7.51)3
     CDPPB6.49 (6.00–6.98)4n.a.c36.57 (6.32–6.81)35.63 (5.35–6.07)3
     Compound 2c5.40 (4.89–5.91)4n.a.35.88 (5.58–6.19)34.86 (4.62–5.13)3
    DHPG
     MPPA6.72 (6.34–7.10)46.22 (5.76–6.67)36.60 (6.12–7.07)36.55 (6.20–6.90)3
     CDPPB6.67 (6.20–7.08)4n.a.36.37 (6.02–6.72)36.34 (6.12–6.57)3
     Compound 2c5.30 (4.90–6.03)4n.a.35.40 (4.59–6.22)34.50 (4.32–4.69)3
    • n.a., not applicable.

    • ↵a Select PAMs potentiated orthosteric agonist-induced internalization above the maximum achieved by orthosteric agonist alone. To fit the operational model, the Em was constrained to the maximum level of potentiation observed in the presence of PAM (143% for glutamate and 210% for DHPG).

    • ↵b pKB, negative logarithm of the dissociation binding constant determined with the operational model of allosterism, for each modulator. None of the pKB estimates determined from interactions with the same orthosteric agonist had P < 0.05 (one-way ANOVA with Tukey’s multiple comparisons test).

    • ↵c Because of the full agonist activity of CDPPB in IP1 accumulation assays, it was not possible to fit the operational model of allosterism to PAM titration curves in the presence of EC20 orthosteric agonist. Compound 2c potentiated orthosteric agonist activity for IP1 accumulation above the maximal agonist response, prohibiting accurate estimation of pKB, as there was no independent means to determine the maximal system response (Em).

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    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.

    Ca2+ MobilizationIP1 AccumulationpERK1/2Internalizationa
    LigandLog βb (95% CI)nLog β (95% CI)nLog β (95% CI)nLog β (95% CI)n
    l-Glutamate
     MPPA0.73 (0.57–0.89)40.19 (0.15–0.24)c30.34 (0.28–0.41)c30.18 (0.16–0.21)c3
     CDPPB0.39 (0.20–0.58)4n.a.d30.16 (0.04–0.28)30.44 (0.29–0.75)3
     Compound 2c0.87 (0.63–1.11)4n.a.30.48 (0.42–0.55)30.48 (0.41–0.59)3
    DHPG
     MPPA1.04 (0.88–1.24)40.45 (0.28–0.62)c,e30.94 (0.80–1.09)30.40 (0.35–0.45)c,e3
     CDPPB0.71f (0.54–0.91)4n.a.30g30g3
     Compound 2c1.40 (1.14–1.99)4n.a.31.23 (0.76–1.70)30.98 (0.89–1.08)3
    • n.a., not applicable.

    • ↵a Select PAMs potentiated orthosteric agonist-induced internalization above the maximum achieved by orthosteric agonist alone. To fit the operational model, the Em was constrained to the maximum level of potentiation observed in the presence of PAM (143% for glutamate and 210% for DHPG).

    • ↵b Log β, logarithm of the efficacy cooperativity factor.

    • ↵c P < 0.05 when compared with estimate from Ca2+ mobilization by one-way ANOVA with Tukey’s multiple comparisons test.

    • ↵d Because of the full agonist activity of CDPPB in IP1 accumulation assays, it was not possible to fit the operational model of allosterism to PAM titration curves in the presence of EC20 orthosteric agonist. Compound 2c potentiated orthosteric agonist activity for IP1 accumulation above the maximal agonist response, prohibiting accurate estimation of log β, as there was no independent means to determine the maximal system response (Em).

    • ↵e P < 0.05 when compared with estimate from pERK1/2 by one-way ANOVA with Tukey’s multiple comparisons test.

    • ↵f P < 0.05 when compared with 0 (one-sample t test).

    • ↵g An F-test revealed that log β was not different from 0, and log β was therefore constrained to 0. As such, the observed response is assumed to be a combination of the orthosteric ligand and the intrinsic agonist activity of CDPPB, where log(τB) was equal to −0.49 (Ca2+), −0.11 (pERK1/2), and −0.02 (internalization) based on intrinsic agonism in the presence of LY341495.

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    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.

    Ca2+ MobilizationIP1 Accumulation
    LigandpKAa (95% CI)Log(τ/KA)b (95% CI)npKBc (95% CI)Log βd (95% CI)npKA (95% CI)Log(τ/KA) (95% CI)npKB (95% CI)Logβ (95% CI)n
    DHPGn.d.6.42 (6.34–6.49)9———n.d.5.70 (5.47–5.95)8———
    MPPAn.r.n.r.66.74 (5.74–7.70)0.45e (0.32–0.58)56.38 (5.81–6.88)6.47 (6.12–6.96)56.53 (6.01–7.06)0f4
    CDPPBn.r.n.r.67.22 (6.70–7.76)0.53e (0.41–0.67)56.48 (6.02–6.90)6.74 (6.48–7.11)66.43 (6.00–6.86)04
    Compound 2c5.84 (4.42–6.73)5.49 (4.73–6.80)76.26 (5.84–7.29)0.94e (0.67–1.81)75.23 (4.59–6.00)5.25 (4.80–5.81)66.03 (5.43–6.78)06
    • n.d. not determined; n.r. no response.

    • ↵a pKA, negative logarithm of the equilibrium dissociation binding constant determined with the operational model of agonism. The pKA values for compound 2c in the two assays had P > 0.05 (two-tailed unpaired t test).

    • ↵b Log(τ/KA), transduction coefficient; τ, intrinsic efficacy. The ∆log(τ/KA) = log(τ/KA)DHPG − log(τ/KA)PAM values for compound 2c in the two assays had P > 0.05 (two-tailed unpaired t test).

    • ↵c pKB, negative logarithm of the dissociation binding constant determined with the operational model of allosterism. The pKB values determined for the same compound in the two assays had P > 0.05 (two-tailed unpaired t test).

    • ↵d Log β, logarithm of the efficacy cooperativity factor.

    • ↵e P < 0.05 when compared with 0 (one-sample t test).

    • ↵f For fits where log β was not different from 0 (determined by F-test), log β was constrained to 0, assuming that the observed response is due to a combination of the orthosteric ligand and the intrinsic agonist activity of the PAM.

Additional Files

  • Figures
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  • Data Supplement

    • Data Supplement -

      Supplemental Figure 1 - Influence of LY341495 on mGlu5 activity and responses to CDPPB in HEK293A cells.

      Supplemental Figure 2 - DHPG-induced real-time internalization of mGlu5.

      Supplemental Figure 3 - Concentration-response relationships for mGlu5 internalization kinetics induced by PAMs in the presence of orthosteric agonists (DHPG or L-glutamate) or antagonists (LY341495).

      Supplemental Figure 4 - Comparison of mGlu5 PAM potencies and maximal responses in HEK293A cells across the four functional assays.

      Supplemental Figure 5 - Assessment of probe dependence of mGlu5 PAMs. (A, B and D) No probe dependence was evident when comparing the Emax, pEC50 and pKB values obtained from PAM potentiation of L-glutamate and DHPG.

      Supplemental Figure 6 - Comparison of pKI estimates from radioligand inhibition binding and pKB values from functional assays for mGlu5 PAMs.

      Supplemental Table 1 -  pEC50 values from functional assays for orthosteric ligands in HEK293A cells or cortical neurons.

      Supplemental Table 2 - : CDPPB pEC50 and Emax values from functional assays in HEK293A cells in the presence of 300 µM LY341495.

      Supplemental Table 3 - Potency and maximal potentiation estimates for mGlu5 PAM potentiation of the indicated orthosteric agonist at ~EC20 in HEK293A cells.

      supplemental Table 4 -  Potency and maximal response of mGlu5 PAMs as agonists or potentiators of DHPG in cortical neurons.

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Molecular Pharmacology: 99 (5)
Molecular Pharmacology
Vol. 99, Issue 5
1 May 2021
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Research ArticleArticle

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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Research ArticleArticle

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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