Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: Investigation of D2R internalization in non-neuronal cell systems. (A) HEK293 cells were transiently transfected with D2R-Rluc8 and a modified GFP10, which together produce constitutive bystander BRET. On the day of assay, the cells were stimulated with either dopamine (DA) or MLS1547 for 30 min at 37°C before BRET measurement. The data are graphed as mean dose-response curves from three independent experiments. DA exhibited an logEC50 of −7.00 ± 0.36 (EC50, 0.1 μM) for BRET reduction/D2R internalization, while MLS1547 treatment failed to alter the BRET signal. (B) Maximum BRET signal obtained after treatment with vehicle (control, 0.81 ± 0.02), 10 μM DA (0.65 ± 0.02), or 30 μM MLS1547 (0.79 ± 0.03). All data are expressed as mean ± SEM of the BRET ratio from three independent experiments. * P < 0.05, unpaired Student's t -test compared to untreated controls. (C) HA-tagged D2R was transiently transfected into HEK293 cells and surface D2R was biotinylated as described in Methods. After washing, cells were treated with either vehicle (control), 10 μM quinpirole, or 10 μM MLS1547 or for 30 min at 37°C. Biotin was cleaved from D2R that remained on the cell surface, followed by cell lysis and isolation of biotinylated (internalized) D2R using avidin beads. Western blotting was used to detect D2R and also transferrin receptor (TfR) (as a gel loading control). A single experiment, representative of three, is shown (the original full blot is displayed in the ). (D) Densitometric analyses were performed and the data expressed as D2R/TfR ratios, relative to the untreated controls (1.0). Treatment values are provided as mean ± S.E.M: quinpirole = 2.8 ± 0.2 and MLS1547 = 1.5 ± 0.03. One-way ANOVA followed by Bonferroni's post-test was performed: * p < 0.05, and ** p < 0.01.

Article Snippet: Notably, Chen et al. ( ) have replicated our results showing that MLS1547 is a G protein-biased agonist using cAMP inhibition assays and the DiscoverX β-arrestin recruitment assay.

Techniques: Transfection, Modification, Control, Lysis, Isolation, Avidin-Biotin Assay, Western Blot

Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: Investigation of D2R internalization in neuronal cell systems. (A) Striatal neurons were prepared and transfected with the D2R-pHluorin construct (pH-DRD2) as described in Methods. 48 h later, the neurons were treated with vehicle (control) or 10 μM dopamine (DA), 30 μM MLS1547, or 30 μM sulpiride for 45 min. Neurons were washed and then stained with a polyclonal GFP antibody to detect surface D2R followed by fixation, permeabilization and re-staining with a monoclonal GFP antibody to detect total cellular D2R. The ratios between surface to total pH-DRD2 signal were normalized to the control group in each experiment and are expressed as mean ± S.E.M of the indicated number of cells ( n ): control = 1.00 ± 0.02, n = 746; DA = 0.72 ± 0.02, n = 627; MLS1547 = 0.90 ± 0.03, n = 812, sulpiride = 1.05 ± 0.08, n = 171. One-way ANOVA followed by Bonferroni's post-test was performed: * p < 0.0001 and ** p < 0.0262. (B) Striatal neurons expressing the D2R-pHluorin construct were exposed to vehicle (control) or 10 μM DA, 30 μM MLS1547, or 30 μM sulpiride for 20 min and the number of reinsertion events/(min × μm 2 ) were visualized in real time as described in Methods. The average reinsertion frequencies for the drug treatments were normalized to the control group in each experiment and are expressed as mean ± S.E.M of the indicated number of cells ( n ): control = 1.00 ± 0.20, n = 51; DA = 5.70 ± 0.54, n = 49; MLS1547 = 1.73 ± 0.22, n = 50, sulpiride = 1.56 ± 0.34, n = 15. One-way ANOVA followed by Bonferroni's post-test was performed, * p < 0.0001.

Article Snippet: Notably, Chen et al. ( ) have replicated our results showing that MLS1547 is a G protein-biased agonist using cAMP inhibition assays and the DiscoverX β-arrestin recruitment assay.

Techniques: Transfection, Construct, Control, Staining, Expressing

Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: Structure of MLS1547. (A) Pharmacophore model depicting four required features for agonist activity and G protein bias. Green represents a hydrophobic component, orange represents two aromatic components, and purple represents a positively charged component. (B) The four main areas of the scaffold that were modified are indicated.

Article Snippet: Notably, Chen et al. ( ) have replicated our results showing that MLS1547 is a G protein-biased agonist using cAMP inhibition assays and the DiscoverX β-arrestin recruitment assay.

Techniques: Activity Assay, Modification

Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: Modification and replacement of the hydroxyquinoline group (R 1 ).

Article Snippet: Notably, Chen et al. ( ) have replicated our results showing that MLS1547 is a G protein-biased agonist using cAMP inhibition assays and the DiscoverX β-arrestin recruitment assay.

Techniques: Modification, Inhibition, Control

Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: Modification and replacement of the 2-pyridyl moiety (R 2 ).

Article Snippet: Notably, Chen et al. ( ) have replicated our results showing that MLS1547 is a G protein-biased agonist using cAMP inhibition assays and the DiscoverX β-arrestin recruitment assay.

Techniques: Modification, Inhibition, Control

Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: Modification or replacement of both the hydroxyquinoline group (R 1 ) and 2-pyridyl moiety (R 2 ).

Article Snippet: Notably, Chen et al. ( ) have replicated our results showing that MLS1547 is a G protein-biased agonist using cAMP inhibition assays and the DiscoverX β-arrestin recruitment assay.

Techniques: Modification, Inhibition, Control

Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: A halogen moiety in area 1 on ring B of MLS1547 enables G protein-bias. CHO cells stably expressing the D2R were assayed for agonist-mediated inhibition of forskolin-stimulated cAMP accumulation (A) or recruitment of β-arrestin to the D2R (B) , as described in the Materials and Methods. Representative experiments are shown with averaged curve parameters derived from at least three separate experiments displayed in Table . Data are expressed as a percentage of the maximum response to dopamine, which was assessed in each experiment.

Article Snippet: Notably, Chen et al. ( ) have replicated our results showing that MLS1547 is a G protein-biased agonist using cAMP inhibition assays and the DiscoverX β-arrestin recruitment assay.

Techniques: Stable Transfection, Expressing, Inhibition, Derivative Assay

Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: A hydroxyl group in area 2 on ring B of MLS1547 is required for both potency and efficacy of G protein signaling. CHO cells stably expressing the D2R were assayed for agonist-mediated inhibition of forskolin-stimulated cAMP accumulation (A) or recruitment of β-arrestin to the D2R (B) , as described in the Materials and Methods. Representative experiments are shown with averaged curve parameters derived from at least three separate experiments displayed in Table . Data are expressed as a percentage of the maximum response to dopamine, which was assessed in each experiment.

Article Snippet: Notably, Chen et al. ( ) have replicated our results showing that MLS1547 is a G protein-biased agonist using cAMP inhibition assays and the DiscoverX β-arrestin recruitment assay.

Techniques: Stable Transfection, Expressing, Inhibition, Derivative Assay

Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: Effects of combined modifications of areas 1 and 2 of ring B of MLS1547 on signaling bias. CHO cells stably expressing the D2R were assayed for agonist-mediated inhibition of forskolin-stimulated cAMP accumulation (A) or recruitment of β-arrestin to the D2R (B) , as described in the Materials and Methods. Representative experiments are shown with averaged curve parameters derived from at least three separate experiments displayed in Table . Data are expressed as a percentage of the maximum response to dopamine, which was assessed in each experiment.

Article Snippet: Notably, Chen et al. ( ) have replicated our results showing that MLS1547 is a G protein-biased agonist using cAMP inhibition assays and the DiscoverX β-arrestin recruitment assay.

Techniques: Stable Transfection, Expressing, Inhibition, Derivative Assay

Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: A hydrophobic moiety in the correct orientation is integral to G protein biased signaling activity of the MLS1547 scaffold. CHO cells stably expressing the D2R were assayed for agonist-mediated inhibition of forskolin-stimulated cAMP accumulation (A) or recruitment of β-arrestin to the D2R (B) , as described in the Materials and Methods. Representative experiments are shown with averaged curve parameters derived from at least three separate experiments displayed in Table . Data are expressed as a percentage of the maximum response to dopamine, which was assessed in each experiment.

Article Snippet: Notably, Chen et al. ( ) have replicated our results showing that MLS1547 is a G protein-biased agonist using cAMP inhibition assays and the DiscoverX β-arrestin recruitment assay.

Techniques: Activity Assay, Stable Transfection, Expressing, Inhibition, Derivative Assay

Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: Investigation of D2R internalization in non-neuronal cell systems. (A) HEK293 cells were transiently transfected with D2R-Rluc8 and a modified GFP10, which together produce constitutive bystander BRET. On the day of assay, the cells were stimulated with either dopamine (DA) or MLS1547 for 30 min at 37°C before BRET measurement. The data are graphed as mean dose-response curves from three independent experiments. DA exhibited an logEC50 of −7.00 ± 0.36 (EC50, 0.1 μM) for BRET reduction/D2R internalization, while MLS1547 treatment failed to alter the BRET signal. (B) Maximum BRET signal obtained after treatment with vehicle (control, 0.81 ± 0.02), 10 μM DA (0.65 ± 0.02), or 30 μM MLS1547 (0.79 ± 0.03). All data are expressed as mean ± SEM of the BRET ratio from three independent experiments. * P < 0.05, unpaired Student's t -test compared to untreated controls. (C) HA-tagged D2R was transiently transfected into HEK293 cells and surface D2R was biotinylated as described in Methods. After washing, cells were treated with either vehicle (control), 10 μM quinpirole, or 10 μM MLS1547 or for 30 min at 37°C. Biotin was cleaved from D2R that remained on the cell surface, followed by cell lysis and isolation of biotinylated (internalized) D2R using avidin beads. Western blotting was used to detect D2R and also transferrin receptor (TfR) (as a gel loading control). A single experiment, representative of three, is shown (the original full blot is displayed in the ). (D) Densitometric analyses were performed and the data expressed as D2R/TfR ratios, relative to the untreated controls (1.0). Treatment values are provided as mean ± S.E.M: quinpirole = 2.8 ± 0.2 and MLS1547 = 1.5 ± 0.03. One-way ANOVA followed by Bonferroni's post-test was performed: * p < 0.05, and ** p < 0.01.

Article Snippet: MLS1547 was originally obtained from the NIH Molecular Libraries Screening Center Network Library and subsequently purchased from MolPort (Riga, Latvija) for follow-up triage studies.

Techniques: Transfection, Modification, Control, Lysis, Isolation, Avidin-Biotin Assay, Western Blot

Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: Investigation of D2R internalization in neuronal cell systems. (A) Striatal neurons were prepared and transfected with the D2R-pHluorin construct (pH-DRD2) as described in Methods. 48 h later, the neurons were treated with vehicle (control) or 10 μM dopamine (DA), 30 μM MLS1547, or 30 μM sulpiride for 45 min. Neurons were washed and then stained with a polyclonal GFP antibody to detect surface D2R followed by fixation, permeabilization and re-staining with a monoclonal GFP antibody to detect total cellular D2R. The ratios between surface to total pH-DRD2 signal were normalized to the control group in each experiment and are expressed as mean ± S.E.M of the indicated number of cells ( n ): control = 1.00 ± 0.02, n = 746; DA = 0.72 ± 0.02, n = 627; MLS1547 = 0.90 ± 0.03, n = 812, sulpiride = 1.05 ± 0.08, n = 171. One-way ANOVA followed by Bonferroni's post-test was performed: * p < 0.0001 and ** p < 0.0262. (B) Striatal neurons expressing the D2R-pHluorin construct were exposed to vehicle (control) or 10 μM DA, 30 μM MLS1547, or 30 μM sulpiride for 20 min and the number of reinsertion events/(min × μm 2 ) were visualized in real time as described in Methods. The average reinsertion frequencies for the drug treatments were normalized to the control group in each experiment and are expressed as mean ± S.E.M of the indicated number of cells ( n ): control = 1.00 ± 0.20, n = 51; DA = 5.70 ± 0.54, n = 49; MLS1547 = 1.73 ± 0.22, n = 50, sulpiride = 1.56 ± 0.34, n = 15. One-way ANOVA followed by Bonferroni's post-test was performed, * p < 0.0001.

Article Snippet: MLS1547 was originally obtained from the NIH Molecular Libraries Screening Center Network Library and subsequently purchased from MolPort (Riga, Latvija) for follow-up triage studies.

Techniques: Transfection, Construct, Control, Staining, Expressing

Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: Structure of MLS1547. (A) Pharmacophore model depicting four required features for agonist activity and G protein bias. Green represents a hydrophobic component, orange represents two aromatic components, and purple represents a positively charged component. (B) The four main areas of the scaffold that were modified are indicated.

Article Snippet: MLS1547 was originally obtained from the NIH Molecular Libraries Screening Center Network Library and subsequently purchased from MolPort (Riga, Latvija) for follow-up triage studies.

Techniques: Activity Assay, Modification

Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: Modification and replacement of the hydroxyquinoline group (R 1 ).

Article Snippet: MLS1547 was originally obtained from the NIH Molecular Libraries Screening Center Network Library and subsequently purchased from MolPort (Riga, Latvija) for follow-up triage studies.

Techniques: Modification, Inhibition, Control

Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: Modification and replacement of the 2-pyridyl moiety (R 2 ).

Article Snippet: MLS1547 was originally obtained from the NIH Molecular Libraries Screening Center Network Library and subsequently purchased from MolPort (Riga, Latvija) for follow-up triage studies.

Techniques: Modification, Inhibition, Control

Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: Modification or replacement of both the hydroxyquinoline group (R 1 ) and 2-pyridyl moiety (R 2 ).

Article Snippet: MLS1547 was originally obtained from the NIH Molecular Libraries Screening Center Network Library and subsequently purchased from MolPort (Riga, Latvija) for follow-up triage studies.

Techniques: Modification, Inhibition, Control

Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: A halogen moiety in area 1 on ring B of MLS1547 enables G protein-bias. CHO cells stably expressing the D2R were assayed for agonist-mediated inhibition of forskolin-stimulated cAMP accumulation (A) or recruitment of β-arrestin to the D2R (B) , as described in the Materials and Methods. Representative experiments are shown with averaged curve parameters derived from at least three separate experiments displayed in Table . Data are expressed as a percentage of the maximum response to dopamine, which was assessed in each experiment.

Article Snippet: MLS1547 was originally obtained from the NIH Molecular Libraries Screening Center Network Library and subsequently purchased from MolPort (Riga, Latvija) for follow-up triage studies.

Techniques: Stable Transfection, Expressing, Inhibition, Derivative Assay

Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: A hydroxyl group in area 2 on ring B of MLS1547 is required for both potency and efficacy of G protein signaling. CHO cells stably expressing the D2R were assayed for agonist-mediated inhibition of forskolin-stimulated cAMP accumulation (A) or recruitment of β-arrestin to the D2R (B) , as described in the Materials and Methods. Representative experiments are shown with averaged curve parameters derived from at least three separate experiments displayed in Table . Data are expressed as a percentage of the maximum response to dopamine, which was assessed in each experiment.

Article Snippet: MLS1547 was originally obtained from the NIH Molecular Libraries Screening Center Network Library and subsequently purchased from MolPort (Riga, Latvija) for follow-up triage studies.

Techniques: Stable Transfection, Expressing, Inhibition, Derivative Assay

Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: Effects of combined modifications of areas 1 and 2 of ring B of MLS1547 on signaling bias. CHO cells stably expressing the D2R were assayed for agonist-mediated inhibition of forskolin-stimulated cAMP accumulation (A) or recruitment of β-arrestin to the D2R (B) , as described in the Materials and Methods. Representative experiments are shown with averaged curve parameters derived from at least three separate experiments displayed in Table . Data are expressed as a percentage of the maximum response to dopamine, which was assessed in each experiment.

Article Snippet: MLS1547 was originally obtained from the NIH Molecular Libraries Screening Center Network Library and subsequently purchased from MolPort (Riga, Latvija) for follow-up triage studies.

Techniques: Stable Transfection, Expressing, Inhibition, Derivative Assay

Journal: Frontiers in Synaptic Neuroscience

Article Title: Structure-Activity Investigation of a G Protein-Biased Agonist Reveals Molecular Determinants for Biased Signaling of the D 2 Dopamine Receptor

doi: 10.3389/fnsyn.2018.00002

Figure Lengend Snippet: A hydrophobic moiety in the correct orientation is integral to G protein biased signaling activity of the MLS1547 scaffold. CHO cells stably expressing the D2R were assayed for agonist-mediated inhibition of forskolin-stimulated cAMP accumulation (A) or recruitment of β-arrestin to the D2R (B) , as described in the Materials and Methods. Representative experiments are shown with averaged curve parameters derived from at least three separate experiments displayed in Table . Data are expressed as a percentage of the maximum response to dopamine, which was assessed in each experiment.

Article Snippet: MLS1547 was originally obtained from the NIH Molecular Libraries Screening Center Network Library and subsequently purchased from MolPort (Riga, Latvija) for follow-up triage studies.

Techniques: Activity Assay, Stable Transfection, Expressing, Inhibition, Derivative Assay