Fig. 2.
UNC9975, UNC0006, and UNC9994 are functionally selective, β-arrestin–biased dopamine D2 partial agonists. (A) Activity of UNC9975, UNC0006, UNC9994, aripiprazole, and quinpirole in the D2-mediated Gi-coupled isoproterenol-stimulated cAMP production assay using HEK293T cells expressing the dopamine D2 receptor and GloSensor-22F. UNC9975, UNC0006, and UNC9994 did not activate this Gi-mediated signaling pathway whereas aripiprazole (EC50 = 38 nM, pEC50 = 7.4 ± 0.1, Emax = 51 ± 5%) was a partial agonist and quinpirole (EC50 = 3.2 nM, pEC50 = 8.49 ± 0.07, Emax = 100 ± 3%) was a full agonist. Data are representative of at least two independent experiments. (B) Activity of UNC9975, UNC0006, UNC9994, aripiprazole, quinpirole, and haloperidol in the D2-mediated β-arrestin-2 translocation Tango assay using HTLA cells transfected with a D2V2-TCS-tTA construct. UNC9975 (EC50 = 1.1 nM, pEC50 = 8.95 ± 0.03, Emax = 43 ± 0.5%), UNC0006 (EC50 = 1.2 nM, pEC50 = 8.91 ± 0.03, Emax = 47 ± 1%), UNC9994 (EC50 = 6.1 nM, pEC50 = 8.22 ± 0.09, Emax = 91 ± 3%), and aripiprazole (EC50 = 2.4 nM, pEC50 = 8.62 ± 0.03, Emax = 73 ± 1%) were partial agonists whereas haloperidol (antagonist control) had no agonist activity. Quinpirole (EC50 = 2.0 nM, pEC50 = 8.70 ± 0.05, Emax = 100 ± 2%) was used as a positive control. Data are representative of at least two independent experiments. (C) Activity of UNC9975, UNC0006, UNC9994, aripiprazole, and quinpirole in the D2-mediated β-arrestin-2 translocation DiscoveRx assay with 20 h stimulation. UNC9975 (EC50 = 5.7 nM, pEC50 = 8.24 ± 0.20, Emax = 19 ± 1%), UNC0006 (EC50 = 3.2 nM, pEC50 = 8.49 ± 0.15, Emax = 25 ± 1%), UNC9994 (EC50 = 448 nM, pEC50 = 6.35 ± 0.07, Emax = 64 ± 2%), and aripiprazole (EC50 = 3.4 nM, pEC50 = 8.47 ± 0.08, Emax = 51 ± 1%) were partial agonists. Quinpirole (EC50 = 56 nM, pEC50 = 7.25 ± 0.04, Emax = 100 ± 2%) was used as a positive control. Data are representative of at least two independent experiments. (D) Activity of UNC9975, UNC0006, UNC9994, aripiprazole, and quinpirole in the D2-mediated BRET-based β-arrestin-2 recruitment assay using HEK293 cells expressing GRK2. UNC9975 (EC50 = 6.0 nM, pEC50 = 8.22 ± 0.49, Emax = 20 ± 3%), UNC0006 (EC50 = 17 nM, pEC50 = 7.77 ± 0.38, Emax = 25 ± 4%), UNC9994 (EC50 > 1,000 nM, Emax > 50%), and aripiprazole (EC50 = 145 nM, pEC50 = 6.84 ± 0.18, Emax = 47 ± 4%) were all partial agonists that promote β-arrestin recruitment to D2 receptors. Quinpirole (EC50 = 6.7 nM, pEC50 = 8.17 ± 0.15, Emax = 100 ± 5%) was used as a positive control. Data are representative of at least three independent experiments.