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. 2011 Nov 8;108(45):18488-93.
doi: 10.1073/pnas.1104807108. Epub 2011 Oct 24.

Discovery of β-arrestin-biased dopamine D2 ligands for probing signal transduction pathways essential for antipsychotic efficacy

John A Allen  1 , Julianne M Yost, Vincent Setola, Xin Chen, Maria F Sassano, Meng Chen, Sean Peterson, Prem N Yadav, Xi-ping Huang, Bo Feng, Niels H Jensen, Xin Che, Xu Bai, Stephen V Frye, William C Wetsel, Marc G Caron, Jonathan A Javitch, Bryan L Roth, Jian Jin
Affiliations

Discovery of β-arrestin-biased dopamine D2 ligands for probing signal transduction pathways essential for antipsychotic efficacy

John A Allen et al. Proc Natl Acad Sci U S A. .

Abstract

Elucidating the key signal transduction pathways essential for both antipsychotic efficacy and side-effect profiles is essential for developing safer and more effective therapies. Recent work has highlighted noncanonical modes of dopamine D(2) receptor (D(2)R) signaling via β-arrestins as being important for the therapeutic actions of both antipsychotic and antimanic agents. We thus sought to create unique D(2)R agonists that display signaling bias via β-arrestin-ergic signaling. Through a robust diversity-oriented modification of the scaffold represented by aripiprazole (1), we discovered UNC9975 (2), UNC0006 (3), and UNC9994 (4) as unprecedented β-arrestin-biased D(2)R ligands. These compounds also represent unprecedented β-arrestin-biased ligands for a G(i)-coupled G protein-coupled receptor (GPCR). Significantly, UNC9975, UNC0006, and UNC9994 are simultaneously antagonists of G(i)-regulated cAMP production and partial agonists for D(2)R/β-arrestin-2 interactions. Importantly, UNC9975 displayed potent antipsychotic-like activity without inducing motoric side effects in inbred C57BL/6 mice in vivo. Genetic deletion of β-arrestin-2 simultaneously attenuated the antipsychotic actions of UNC9975 and transformed it into a typical antipsychotic drug with a high propensity to induce catalepsy. Similarly, the antipsychotic-like activity displayed by UNC9994, an extremely β-arrestin-biased D(2)R agonist, in wild-type mice was completely abolished in β-arrestin-2 knockout mice. Taken together, our results suggest that β-arrestin signaling and recruitment can be simultaneously a significant contributor to antipsychotic efficacy and protective against motoric side effects. These functionally selective, β-arrestin-biased D(2)R ligands represent valuable chemical probes for further investigations of D(2)R signaling in health and disease.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Discovery of unprecedented β-arrestin–biased D2R ligands UNC9975, UNC0006, and UNC9994 via exploring multiple regions of the aripiprazole scaffold.
Fig. 2.
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.
Fig. 3.
Fig. 3.
UNC9975 exhibits potent antipsychotic-like activity in mouse hyperlocomotion studies that is attenuated in β-arrestin-2 knockout mice. (A) Locomotor responses in inbred C57BL/6 mice are shown as 5-min binned intervals to vehicle or different doses (i.p.) of UNC9975 followed 30 min later by 3 mg/kg d-amphetamine (AMPH, i.p.). (B) Bar graph of distance traveled after AMPH administration (30- to 70-min time interval). C57BL/6 mice were given vehicle or different doses of UNC9975 or aripiprazole 30 min before AMPH treatment. n = 8 animals/group. *P < 0.05, vs. vehicle + 3 mg/kg AMPH group. (C and D) Locomotor activities shown as 5-min binned intervals of wild-type (WT) or β-arrestin-2 knockout (β-ARR2 KO) littermate mice given vehicle or different doses of UNC9975 followed 30 min later with 6 mg/kg phencyclidine (PCP, i.p.). (E) Bar graph of distance traveled by WT and β-ARR2 KO mice after PCP administration (30- to 70-min time interval) as shown in C and D. n = 14 WT and β-ARR2 KO pairs/group. *P < 0.05, vs. vehicle + 6 mg/kg PCP group. (F) Bar graph of distance traveled by WT and β-ARR2 KO mice after aripiprazole injection followed by PCP treatment (30- to 70-min time interval). n = 8 littermate WT and β-ARR2 KO pairs/group. *P < 0.05, vs. vehicle + 6 mg/kg PCP group.
Fig. 4.
Fig. 4.
UNC9994 exhibits potent antipsychotic-like activity in mouse hyperlocomotion studies that is completely abolished in β-arrestin-2 knockout mice. (A and B) Locomotor activities shown as 5-min binned intervals for wild-type (WT) or β-arrestin-2 knockout (β-ARR2 KO) littermate mice given vehicle or 2.0 mg/kg UNC9994 (i.p.) followed 30 min later with 6 mg/kg phencyclidine (PCP, i.p.). n = 10–13 WT and β-ARR2 KO pairs/group.
Fig. 5.
Fig. 5.
UNC9975 and UNC0006 induce catalepsy in β-arrestin-2 knockout mice but not in wild-type littermates. (A and B) Wild-type (WT) and β-arrestin-2 knockout (β-ARR2 KO) littermate mice were administered (i.p.) vehicle, 5.0 mg/kg UNC9975, 5.0 mg/kg UNC0006, 5.0 mg/kg aripiprazole, or 2.0 mg/kg haloperidol. Catalepsy was assessed 30 and 60 min after drug injection using the inclined screen test where latency to move was scored. n = 8 WT and β-ARR2 KO pairs/group. *P < 0.05, vs. vehicle controls.
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