Journal: ACS chemical neuroscience

Article Title: Functional Characterization of a Novel Series of Biased Signaling Dopamine D3 Receptor Agonists

doi: 10.1021/acschemneuro.6b00221

Figure Lengend Snippet: SK609 and SK213 do not recruit β-arrestin-2 for signaling at D3R. Dose–response curve of PD128907, SK609, and SK213 for recruiting β-arrestin-2 at D3R was tested. PathHunter eXpress β-arrestin-2 U2OS cells stably expressing hD3R were plated at 10 000 cells/well in a 96-well plate, incubated for 48 h, and stimulated for 90 min with vehicle and indicated concentrations of compounds PD128907, SK609, and SK213 as described in Methods. Following stimulation, signal was detected using the PathHunter detection reagents following the manufacturer’s protocol. RLU (relative luminescence units) represents a measure of β-arrestin-2 recruitment in this assay, and the data is presented as net change of RLU minus vehicle control (0.027 ± 0.002) of three independent experiments performed in triplicate.

Article Snippet: Further testing in a 10-point dose–response study confirmed that neither SK609 nor its analogue SK213 had antagonizing effects on the isoproterenol-induced β 2 -adrenergic receptor activation, with an IC 50 > 10 μ M ( ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 10 caption a7 Screening for off-target effects of SK609 at high concentration (100 μ M) on a panel of 128 GPCRs using the DiscoveRx β -arrestin recruitment assay. (A) Screening at 100 μ M of SK609 in both agonist (light blue bars) and antagonist mode (dark blue bars) resulted in ~70 receptors that had an effect (nonzero value).

Techniques: Stable Transfection, Expressing, Incubation, Control

Journal: ACS chemical neuroscience

Article Title: Functional Characterization of a Novel Series of Biased Signaling Dopamine D3 Receptor Agonists

doi: 10.1021/acschemneuro.6b00221

Figure Lengend Snippet: Screening for off-target effects of SK609 at high concentration (100 μM) on a panel of 128 GPCRs using the DiscoveRx β-arrestin recruitment assay. (A) Screening at 100 μM of SK609 in both agonist (light blue bars) and antagonist mode (dark blue bars) resulted in ~70 receptors that had an effect (nonzero value). SK609 has an agonist effect at D2R and D3R and antagonist activity at adrenergic, cannabinoid receptor-1, and 5HT2A receptors at a concentration of 100 μM. Several other hits were found to be false positives. (B) Dose–response curves of SK609 and SK213 for antagonizing isoproterenol-induced β-arrestin-2 recruitment at the β2-adrenergic receptor. CHO-K1 cells stably expressing β2-adrenergic receptor were plated at 10 000 cells/well in a 96-well plate and incubated for 48 h, pretreated with vehicle or indicated concentrations of SK609 or SK213 for 30 min, and subsequently treated with vehicle or 300 nM of isoproterenol (EC80) for 90 min. Following stimulation, signal was detected using the PathHunter detection reagents following the manufacturer’s protocol. Each value represents mean ± SEM of three independent experiments performed in duplicate and represented as percent of control (300 nM of isoproterenol).

Article Snippet: Further testing in a 10-point dose–response study confirmed that neither SK609 nor its analogue SK213 had antagonizing effects on the isoproterenol-induced β 2 -adrenergic receptor activation, with an IC 50 > 10 μ M ( ). fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 10 caption a7 Screening for off-target effects of SK609 at high concentration (100 μ M) on a panel of 128 GPCRs using the DiscoveRx β -arrestin recruitment assay. (A) Screening at 100 μ M of SK609 in both agonist (light blue bars) and antagonist mode (dark blue bars) resulted in ~70 receptors that had an effect (nonzero value).

Techniques: Concentration Assay, Activity Assay, Stable Transfection, Expressing, Incubation, Control

Journal: Journal of medicinal chemistry

Article Title: Discovery of G Protein-biased D2 Dopamine Receptor Partial Agonists

doi: 10.1021/acs.jmedchem.6b01208

Figure Lengend Snippet: Benzothiazole substitution and linker modification of a β-arrestin-biased D2R agonist led to compound 1, which is a D2R Gi/o partial agonist with weak efficacy for β-arrestin recruitment.

Article Snippet: To address the discrepant partial agonist activity of 59 in the β-arrestin-2 recruitment BRET assay compared to the previously published lack of β-arrestin-2 recruitment as measured using DiscoverX, 27 we further evaluated compounds 1, 53 , and 59 relative to quinpirole using the D2R DiscoverX β-arrestin recruitment assay and confirmed lack of β-arrestin-2 recruitment activity for these compounds ( ).

Techniques: Modification

Journal: Journal of medicinal chemistry

Article Title: Discovery of G Protein-biased D2 Dopamine Receptor Partial Agonists

doi: 10.1021/acs.jmedchem.6b01208

Figure Lengend Snippet: (A) Activity of aripiprazole, compound 1, compound 59 and quinpirole in the D2R-mediated BRET-based β-arrestin-2 recruitment assay using HEK293 cells expressing GRK2. Aripiprazole and compound 59 were partial agonists that promote β-arrestin recruitment to D2R while compound 1 was not active in the BRET assay. Quinpirole (EC50 = 7.0 nM) was used as a positive control. (B) Activity of compound 59 and quinpirole in the D2R-mediated β-arrestin-2 translocation Tango assay using HTLA cells transfected with a D2V2-TCS-tTA construct. Compound 59 displayed robust β-arrestin recruitment in the Tango assay (EC50 = 46 nM, Emax = 67%) compared to the full agonist quinpirole (EC50 = 2.3 nM). (C) Activity of compound 59 and quinpirole in the D2R-mediated Gi/o coupled isoproterenol-stimulated cAMP production assay using HEK293T cells expressing D2R and GloSensor-22F. Compound 59 was a partial agonist (EC50 = 102 nM, Emax = 63%) compared to quinpirole (EC50 = 1.2 nM), which was used as a positive control. (D) D2R β-arrestin recruitment activity by compounds 1, 53, and 59 as measured by DiscoverX in D2R-expressing CHO cells. Compounds 1, 53, and 59 showed no activity up to 10 µM. Quinpirole (EC50 = 28 nM) was used as a positive control. (E) Compound 1 (IC50 = 18 nM) blocked dopamine (DA, EC50 = 11 nM) stimulated β-arrestin recruitment in the BRET assay. (F) Activity of compounds 24, 46, 49, 53 and 54 in the BRET assay. All compounds were not active in the BRET-based β-arrestin-2 recruitment assay while quinpirole (EC50 = 12 nM) was used as a positive control. (G) Activity of aripiprazole and compounds 1 and 53 in the D2R Gαi1-Gγ2 dissociation BRET assay. Aripiprazole (EC50 = 0.99 nM), compound 1 (EC50 = 3.6 nM), and 53 (EC50 = 4.6 nM), all displayed partial agonist activity (78%, 57%, and 47%, respectively) relative to quinpirole. Quinpirole (EC50 = 1.7 nM) was used as a positive control. Except for the DiscoverX assay (n=1, performed in duplicate), data are representative of at least three independent experiments performed in triplicate.

Article Snippet: To address the discrepant partial agonist activity of 59 in the β-arrestin-2 recruitment BRET assay compared to the previously published lack of β-arrestin-2 recruitment as measured using DiscoverX, 27 we further evaluated compounds 1, 53 , and 59 relative to quinpirole using the D2R DiscoverX β-arrestin recruitment assay and confirmed lack of β-arrestin-2 recruitment activity for these compounds ( ).

Techniques: Activity Assay, Expressing, Bioluminescence Resonance Energy Transfer, Positive Control, Translocation Assay, Transfection, Construct

Journal: Journal of medicinal chemistry

Article Title: Discovery of G Protein-biased D2 Dopamine Receptor Partial Agonists

doi: 10.1021/acs.jmedchem.6b01208

Figure Lengend Snippet: (A) Activity of compounds 1, 24, 46, 49, 53, 54, and quinpirole in the D2R-mediated Gi/o coupled isoproterenol-stimulated cAMP production assay using HEK293T cells expressing D2R and GloSensor-22F. All six tested compounds were partial agonists compared to quinpirole (EC50 = 1.7 nM), which was used as a positive control. (B) Activity of compounds 1, 24, 46, 49, 53, 54, and quinpirole in the D2R-mediated β-arrestin-2 translocation Tango assay using HTLA cells transfected with a D2V2-TCS-tTA construct. 1 recruited β-arrestin weakly and other five compounds did not substantially recruit β-arrestin compared to the full agonist quinpirole (EC50 = 0.8 nM). Data are representative of at least three independent experiments performed in triplicate. (C) Bias plot of compounds 1, 24, 46, 49, 53, 54, and quinpirole in the D2R-mediated cAMP inhibition GloSensor assay versus the D2R-mediated β-arrestin-2 translocation Tango assay. Quinpirole shows equal activity in both assays, but compounds 1, 24, 46, 49, 53, and 54 cluster toward G protein activation.

Article Snippet: To address the discrepant partial agonist activity of 59 in the β-arrestin-2 recruitment BRET assay compared to the previously published lack of β-arrestin-2 recruitment as measured using DiscoverX, 27 we further evaluated compounds 1, 53 , and 59 relative to quinpirole using the D2R DiscoverX β-arrestin recruitment assay and confirmed lack of β-arrestin-2 recruitment activity for these compounds ( ).

Techniques: Activity Assay, Expressing, Positive Control, Translocation Assay, Transfection, Construct, Inhibition, Activation Assay

Journal: Journal of medicinal chemistry

Article Title: Discovery of G Protein-biased D2 Dopamine Receptor Partial Agonists

doi: 10.1021/acs.jmedchem.6b01208

Figure Lengend Snippet: Radioligand binding affinity of compounds 1, 24, 46, 49, 53 and 54 at select GPCRs. a

Article Snippet: To address the discrepant partial agonist activity of 59 in the β-arrestin-2 recruitment BRET assay compared to the previously published lack of β-arrestin-2 recruitment as measured using DiscoverX, 27 we further evaluated compounds 1, 53 , and 59 relative to quinpirole using the D2R DiscoverX β-arrestin recruitment assay and confirmed lack of β-arrestin-2 recruitment activity for these compounds ( ).

Techniques: Binding Assay

Journal: British Journal of Pharmacology

Article Title: Novel approaches leading towards peptide GPCR de‐orphanisation

doi: 10.1111/bph.14950

Figure Lengend Snippet: New proposed peptide ligands for orphan GPCRs

Article Snippet: GPR1 , Gastrin‐releasing peptide , GRP (24–50) , VPLPAGGGTVLTKMYPRGNHWAVGHLM , Known (GRP) , β‐arrestin recruitment (TANGO and DiscoverX) , 5.3–6.4 , BB 1 and BB 2 receptor agonists.

Techniques: Sequencing, Variant Assay