β-Arrestin-Biased Allosteric Modulator of NTSR1 Selectively Attenuates Addictive Behaviors

Abstract

Small molecule neurotensin receptor 1 (NTSR1) agonists have been pursued for more than 40 years as potential therapeutics for psychiatric disorders, including drug addiction. Clinical development of NTSR1 agonists has, however, been precluded by their severe side effects. NTSR1, a G protein-coupled receptor (GPCR), signals through the canonical activation of G proteins and engages β-arrestins to mediate distinct cellular signaling events. Here, we characterize the allosteric NTSR1 modulator SBI-553. This small molecule not only acts as a β-arrestin-biased agonist but also extends profound β-arrestin bias to the endogenous ligand by selectively antagonizing G protein signaling. SBI-553 shows efficacy in animal models of psychostimulant abuse, including cocaine self-administration, without the side effects characteristic of balanced NTSR1 agonism. These findings indicate that NTSR1 G protein and β-arrestin activation produce discrete and separable physiological effects, thus providing a strategy to develop safer GPCR-targeting therapeutics with more directed pharmacological action.

Keywords: G protein-coupled recpetor; GPCR; NTSR1; PET; addiction; allosteric modulator; cocaine; dopamine; methamphetamine; neurotensin receptor 1; positron emission tomography; self-administration; β-arrestin.

Figure 1.. SBI-553 activates NTSR1 to stimulate β-arrestin-associated cellular responses.

(A) [³H]SBI-553 saturation binding to NTSR1-containing HEK293T cell membranes. (B) [³H]NTS saturation binding to NTSR1-containing HEK293T cell membranes in the absence (vehicle) and presence of SBI-553 (0.01 – 10 μM). (C) Western blot analysis of NTSR1 phosphorylation. NTSR1-expressing HEK293T cells were treated with vehicle, NTS (10 nM), SBI-553 (10 μM) or SR142948A (10 μM). Phosphorylation of NTSR1 at threonine residues (p-NTSR1) was assessed by anti-phosphothreonine antibody. (D) Confocal imaging of β-arrestin2 translocation to NTSR1. U2OS cells expressing NTSR1 and β-arrestin2-GFP without stimulation (Basal) and following treatment with vehicle or 30 μM SR142948A in combination with 10 nM NTS or 10 μM SBI-553. Scale bar = 10 μm. (E-G) BRET-based assay of β-arrestin2 recruitment to NTSR1 in HEK293T cells expressing NTSR1-Rluc and β-arrestin2-Venus. (E) β-arrestin2 recruitment to NTSR1 induced by co-treatment with NTS and Vehicle or 0.03, 0.3, 1, 3, or 30 μM SBI-553. (F) NTS-induced β-arrestin2 recruitment to NTSR1 in the presence of Vehicle or 0.001, 0.01, 0.1, 1, or 10 μM SR142948A. (G) SBI-553-induced β-arrestin2 recruitment to NTSR1 in the presence of Vehicle or 0.01, 0.1, 1, 10 or 100 μM SR142948A. Increasing concentrations are indicated by increasing color intensity. NTS + Vehicle -- Red line. (H-J) Confocal microscopy-based assay of β-arrestin2 translocation to NTSR1 in U2OS cells expressing NTSR1 and β-arrestin2-GFP. (H) Ligand induced-β-arrestin2 translocation to NTSR1 with NTS, SBI-553, or SR1425948A. (I) β-arrestin2 translocation to NTSR1 induced by co-treatment with NTS and SBI-553. (J) β-arrestin2 translocation to NTSR1 induced by NTS (5 nM) or SBI-553 (10 μM) in the presence of SR142948A. (K-L) U2OS cells transfected with a fluorogen-activated protein (FAP, MarsCy1) tagged-NTSR1 and imaged after fluorogen treatment. (K) Dose-response of ligand-induced NTSR1 internalization. (L) NTSR1 internalization induced by co-treatment with NTS and SBI-553. Cells were pretreated with SBI-553 (0, 1 or 10 μM) prior to addition of NTS. (M) NTSR1 internalization induced by NTS (10 nM) or SBI-553 (10 μM) in the presence of SR142948A. For curve parameters and details on statistical comparisons, see Table S1. Also see Figure S1, Figure S2.

Figure 2.. SBI-553 does not stimulate NTSR1 Gq activation and antagonizes NT-induced Gq signaling.

(A) Gq protein activation in the TGFα shedding assay induced by individual NTSR1 ligands. (B) Gq activation induced by NTS in the presence of Vehicle, SBI-553, or SR142948A. (C) Inositol 1,4,5-triphosphate (IP3) generation in an intramolecular BRET assay induced by individual NTSR1 ligands. (D) IP3 generation induced by NTS in the presence of Vehicle, SR142948A or SBI-553. (E) Calcium (Ca²⁺) mobilization in an aequorin assay induced by individual NTSR1 ligands. (F) Ca²⁺ mobilization induced by NTS in the presence of Vehicle, SBI-553, or SR142948A. For curve parameters and details on statistical comparisons, see Table S2.

Figure 3.. SBI-553 antagonizes NTS-induced, Gq-mediated pERK, but permits NTS-induced, β-arrestin-mediated pERK generation.

Cells expressing NTSR1 were stimulated with NTSR1 ligands. Phosphorylated ERK (pERK) and total ERK expression were assessed in whole cell lysates by Western blot analysis. (A-B) Time course for NTS-stimulated pERK in HEK293T cells. NTSR1-expressing cells were treated with NTS in the presence or absence of the NTSR1/2 antagonist SR142948A. Controls cells lacking NTSR1 were stimulated only with NTS. (C-D) Time course for SBI-553-stimulated pERK in HEK293T cells. NTSR1-expressing cells were treated with SBI-553 or vehicle. (E-F) Time course for NTS and Vehicle or SBI-553 co-treatment-stimulated pERK in HEK293T cells. NTSR1-expressing cells were treated concurrently with NTS and vehicle or SBI-553. (G-H) Time course for NTS and Vehicle or SBI-553 co-treatment-stimulated pERK in Gq-null cells. HEK293 cells genetically engineered to lack the Gq protein were treated concurrently with NTS and vehicle or SBI-553. (I-J) Time course for NTS and Vehicle or SBI-553 co-treatment-stimulated pERK in β-arrestin1/2-null cells. HEK293 cells genetically engineered to lack β-arrestin1 and β-arrestin2 were treated concurrently with NTS and vehicle or SBI-553. For details on statistical comparisons, see Table S3.

Figure 4.. SBI-553 attenuates L-DOPA-induced changes in regional brain glucose utilization.

The ability of SBI-553 to modulate L-DOPA-induced changes in [¹⁸F]-FDG uptake was evaluated in dopamine-depleted dopamine transporter knockout (DAT KO) mice by PET/CT. (A) Open field activity of DAT KO in the model of dopamine-induced locomotion used for imaging studies. DAT KO mice received the tyrosine hydroxylase inhibitor α-methyl-p-tyrosine (AMPT, 12 mg/kg, i.p.) 60 min prior to co-administration of L-DOPA (25 mg/kg, i.p.) and vehicle (saline, i.p.) or L-DOPA and SBI-553 (12 mg/kg, i.p.). (B) Timeline indicating the schedule of treatments and scan acquisitions. (C) Representative images showing registration of a PET to a CT scan and a CT scan to the 3-dimensional brain atlas with 332 regional labels. (D) [¹⁸F]-FDG PET/CT scans from a representative animal acquired at baseline (top), after L-DOPA and vehicle co-treatment (middle), or after L-DOPA and SBI-553 co-treatment (bottom) are shown in coronal (left), transverse (middle) and sagittal (right) sections. Position relative to bregma: anteroposterior, −1.31 mm; mediolateral +0.05 mm; dorsoventral, +3.37 mm. (E,F) [¹⁸F]-FDG uptake was compared among groups using statistical parametric mapping (SPM). This voxel-based analysis revealed a single cluster of significantly reduced ¹⁸F-FDG standardized uptake values (SUVs) in the L-DOPA + Vehicle versus (vs.) Baseline comparison (left; kE = 330,117 voxels, pcluster-level = 0.049; peak-level T = 7.40, peak-level Z = 3.60, peak-level <0.0001). No significant clusters were identified in the L-DOPA + SBI-553 vs. Baseline comparison (middle). A single cluster of significantly elevated SUVs was identified in the L-DOPA + SBI-553 vs. L-DOPA + Vehicle comparison (right; k_E = 419,788 voxels, p_{cluster-level} = 0.042; peak-level T = 7.74, peak-level Z = 3.67, p_peak-level <0.0001). T Maps of significantly reduced (blue) and increased (orange) voxels are shown in the brain atlas as (E) rendered 3-dimensional surfaces and (F) coronal sections. Position of coronal sections relative to bregma along anteroposterior axis: +1.51, +0.61, +0.21, −0.19, −0.63, −1.03, −1.43, −1.83, −2.23, −3.23, −3.83 mm. For details on statistical comparisons, see Table S4. Also see Figure S3.

Figure 5.. SBI-553 attenuates behavioral evidence of acute and chronic psychostimulant exposure.

The effect of SBI-553 on cocaine and methamphetamine (meth)-associated behaviors in C57BL/6J mice. (A,B) Cocaine-induced hyperlocomotion. Animals were acclimated to an open field for 30 min (indicated by grey box) prior to concurrent administration of cocaine (30 mg/kg, i.p.) and SBI-553 (12 mg/kg, i.p.) or vehicle (saline, i.p.). (A) Time course. (B) Cumulative distance traveled in the 30 min post-treatment period. (C) Methamphetamine conditioned place preference (CPP). After conditioning, animals received vehicle (saline, i.p.) or SBI-553 (12 mg/kg, i.p.) 15 min prior to placement into the center chamber. Time spent in the methamphetamine- and vehicle-paired end chambers was recorded for 30 min. (Left) Pre- vs. post-conditioning chamber preference. (Right) Conditioning score. (D-L) Cocaine self-administration. Mice were trained to self-administer cocaine intravenously via active lever responding at a fixed lever response to reinforcement schedule of 4. Once stable performance was achieved, self-administration was assessed once daily in 60 min sessions. (D-I) Results of daily 0.1 mg/kg/infusion cocaine self-administration sessions following vehicle (saline, i.p., day 1), SBI-553 (12 mg/kg, i.p., day 2) or no treatment (day 3) 5 min prior to placement into the operant chamber. (D) Cumulative cocaine reinforcement time-courses. (E) Lever responding. (F) Total cocaine reinforcements received and cocaine intake. (G) Latency to initiate the first lever response. (H) Percent of responses occurring in the post-reinforcement cue period in the absence of cocaine reinforcement. (I) Lever accuracy across treatment days. (J-L) Results of cocaine and SBI-553 dose-response studies. Animals received vehicle (saline, i.p.) or SBI-553 (2, 6 or 12 mg/kg, i.p.) immediately prior to placement into self-administration chambers with access to cocaine at doses of 0.1, 0.3, 0.5 or 1 mg/kg/infusion. (J) Cocaine reinforcement dose-response curves. (K) Latency to initiate the first lever response. (L) Lever accuracy. For details on statistical comparisons, see Table S5.

Figure 6.. SBI-553 treatment, unlike that of peptide NTSR1 ligands, is not associated with hypothermia, motor impairment or hypotension.

The effects of the peptide NTSR1 ligand PD149163 and SBI-553 in C57BL/6J mice. (A, B) Core body temperature. (A) Animals received vehicle (0.2% DMSO in saline) or PD149163 (i.p.) at Time 0. (B) Animals received vehicle (saline) or SBI-553 (i.p.) at Time 0. (C, D) Wire hang. Latency to grasp wire with hind-limbs was measured in trained animals hanging by their forepaws. (C) Animals received vehicle or PD149163 (i.p.) 120 min prior to testing. (D) Animals received vehicle or SBI-553 (i.p.) 30 min prior to testing. (E, F) Righting reflex. Contact righting deficits were scored on a six-point scale. (E) Animals received vehicle or PD149163 (i.p.) 120 min prior to testing. (F) Animals received vehicle or SBI-553 (i.p.) 30 min prior to testing. (G) Blood pressure. Systolic ventricular pressure was continuously recorded in anesthetized mice after treatment with vehicle (0.6% DMSO in saline), SBI-553, or PD149163. Baseline was set to 0. (H) Side-effect dose-response curves. Data from the time of peak drug effect, 120 and 30 min post-treatment for PD149163 and SBI-553, respectively, are represented as percent PD149163 at the 1 mg/kg dose for the wire hang, contact righting, and body temperature tests. (I,J,K) Effects of orally administered SBI-553 on PD149163-induced reductions in body temperature and motor activity. (I) Animals received vehicle (10% DMSO, 0.05% Tween-80) or 10, 30, or 100 mg/kg SBI-553 (p.o.) 30 min prior to the study (−30 min, blue arrow). Animals received vehicle (saline) or PD149163 (i.p.) at Time 0 (red arrow) and core body temperature was monitored at multiple time-points. The same administration schedule was used for quantitation of (J) horizontal motor activity (for group comparisons to Table S6) and (K) cumulative motor activity over a 45 min period starting 120 min post PD149163 or vehicle treatment. For details on statistical comparisons, see Table S6.

Figure 7.. SBI-553 attenuates cocaine and methamphetamine-induced locomotion via a β-arrestin2-dependent mechanism.

The effect of SBI-553 on cocaine and methamphetamine-induced hyperlocomotion was assessed in global and neuron-specific β-arrestin2 KO mice. Animals were acclimated to the open field for 30 min (indicated by grey box) prior to concurrent administration of either cocaine (30 mg/kg, i.p.) or methamphetamine (2 mg/kg, i.p.) and SBI-553 (12 mg/kg, i.p.) or vehicle (saline, i.p.). (A) Cocaine-induced hyperlocomotion in WT and global β-arrestin2 KO mice. (B) Methamphetamine-induced hyperlocomotion in WT and global β-arrestin2 KO mice. (C) Diagram of the Cre-Lox breeding system used to develop region- and neuron subtype-specific β-arrestin2 KO mouse lines. (D) Methamphetamine-induced hyperlocomotion in non-Cre expressing β-arrestin2^f/f littermate control mice. (E) Methamphetamine-induced hyperlocomotion in mice with selective deletion of β-arrestin2 in D1R-expressing neurons (D1RCre/β-arrestin2^f/f). (F) Methamphetamine-induced hyperlocomotion in mice with selective deletion of β-arrestin2 in D2R-expressing neurons (D2RCre/β-arrestin2^f/f). (G) Methamphetamine-induced hyperlocomotion in mice with selective deletion of β-arrestin2 in midbrain D2R-expressing neurons (DATCre/β-arrestin2^f/f). (H) Methamphetamine-induced hyperlocomotion in mice with selective deletion of β-arrestin2 in striatal D2R-expressing neurons (A2aCre/β-arrestin2^f/f mice). (I) Cumulative distance traveled in methamphetamine-treated mice. Distance traveled in the 50 min post-treatment in Cre negative β-arrestin2^f/f control and neuron-specific β-arrestin2 KO mice. For details on statistical comparisons, see Table S7.