Skip to main content
. Author manuscript; available in PMC: 2017 Dec 8.
Published in final edited form as: J Med Chem. 2016 Nov 16;59(23):10601–10618. doi: 10.1021/acs.jmedchem.6b01208

Figure 3. Compounds 1, 24, 46, 49, 53, and 54 do not activate β-arrestin recruitment in the D2R-mediated BRET assay while compound 59 is a partial agonist in the D2R β-arrestin-2 recruitment Tango assay and D2R Gi/o-mediated cAMP inhibition assay.

Figure 3

(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.