The G protein-biased κ-opioid receptor agonist RB-64 is analgesic with a unique spectrum of activities in vivo

Abstract

The hypothesis that functionally selective G protein-coupled receptor (GPCR) agonists may have enhanced therapeutic benefits has revitalized interest for many GPCR targets. In particular, although κ-opioid receptor (KOR) agonists are analgesic with a low risk of dependence and abuse, their use is limited by a propensity to induce sedation, motor incoordination, hallucinations, and dysphoria-like states. Several laboratories have produced a body of work suggesting that G protein-biased KOR agonists might be analgesic with fewer side effects. Although that has been an intriguing hypothesis, suitable KOR-selective and G protein-biased agonists have not been available to test this idea. Here we provide data using a G protein-biased agonist, RB-64 (22-thiocyanatosalvinorin A), which suggests that KOR-mediated G protein signaling induces analgesia and aversion, whereas β-arrestin-2 signaling may be associated with motor incoordination. Additionally, unlike unbiased KOR agonists, the G protein-biased ligand RB-64 does not induce sedation and does not have anhedonia-like actions, suggesting that a mechanism other than G protein signaling mediates these effects. Our findings provide the first evidence for a highly selective and G protein-biased tool compound for which many, but not all, of the negative side effects of KOR agonists can be minimized by creating G protein-biased KOR agonists.

Fig. 1.

In vitro effects of agonists on signaling and internalization of the mouse KOR. Dose-response curves in HEK cells for (A) arrestin mobilization and (B) G protein signaling–induced by U69593, sal A, and RB-64. Error bars represent S.E.M. (C) Internalization of mouse KOR on stimulation with agonists (arrows indicate internalized KOR). The percent of internalization was quantified in an unbiased manner for the 15-minute study as previously described (Bhatnagar et al., 2001) as follows: control = 36 ± 9%; U69593 = 83 ± 5% (P < 0.05 versus control); salvinorin A = 82 ± 7% (P < 0.05 versus control); RB-64 = 32.7 ± 6% (NS versus control).

Fig. 2.

KOR agonist–induced G protein signaling causes analgesia-like effects in the hotplate assay. (A) U69593 caused an analgesia-like effect in WT (n = 8) and β-arrestin-2 KO mice (n = 10) 10 and 20 minutes after treatment. (B) Sal A produced analgesia-like effects in WT mice (n = 11) and β-arrestin-2 KO mice (n = 16) 10 minutes after treatment. (C) RB-64 induced analgesia-like effects in WT (n = 9) and β-arrestin-2 KO mice (n = 9) 20 and 30 minutes after treatment. The vehicle-treated WT mice (n = 12) and β-arrestin-2 KO mice (n = 9) showed no differences in response. (D) U69593, sal A, and RB-64 showed a KOR-selective effect when comparing KOR KO mice (n = 8 for all drug treatments) and WT mice (n = 9, 7, and 6, for U69593, sal A, and RB-64). Mice were tested 20 minutes after treatment of U69593 and sal A and 10 minutes after treatment with sal A. (E) There was an increased baseline performance in the hotplate assay for β-arrestin-2 KO mice compared with WT mice, but no difference was seen in baseline between KOR KO and WT mice (n = 12 for all genotypes). (F) A higher dose of RB-64 (n = 10) or sal A (n = 9) does not increase the analgesic response in WT mice. Data are plotted as the percent of baseline latency to respond for each animal, and baseline was established on the day of testing before drug treatment ± S.E.M. (A–D) *P < 0.05; **P < 0.01; ***P < 0.001. BArr2, β-arrestin-2.

Fig. 3.

KOR agonist–induced G protein signaling causes conditioned place aversion. (A) U69593 (1 mg/kg)-induced aversion in WT (n = 8) and β-arrestin-2 KO mice (n = 7). 1 mg/kg sal A had no effect on WT (n = 6) or β-arrestin-2 KO mice (n = 8), but 3 mg/kg sal A did cause aversion in WT (n = 9) and β-arrestin-2 KO mice (n = 6). RB-64 (1 mg/kg) did not cause aversion in WT (n = 8) or β-arrestin-2 KO mice (n = 7), but 3 mg/kg RB-64 did induce aversion in both WT (n = 8) and β-arrestin-2 KO mice (n = 7). All P values were generated in comparison with vehicle-treated WT (n = 8) or β-arrestin-2 KO mice (n = 8). (B) Using a dimethylsulfoxide (DMSO) vehicle instead of 10% Tween-80 did not cause an increased aversion in U69593-treated mice (n = 6) relative to RB-64 (n = 6). This was done to determine whether 10% Tween-80 causes a ceiling effect in U69593 mice. Using either vehicle (DMSO or 10% Tween-80), no difference is seen in the aversion induced by U69593 and 3 mg/kg RB64. Data are plotted as amount of time spent in drug-paired room during after the test compared with pretest. Data are plotted as amount of time spent in drug-paired room during posttest compared with pretest ± S.E.M. *P < 0.05; **P < 0.01. BArr2, β-arrestin-2.

Fig. 4.

β-arrestin 2 signaling contributes to KOR agonist–induced rotarod deficit. (A) U69593 induced a rotarod deficit in both WT (n = 23) β-arrestin-2 KO mice (n = 23) but produced a larger deficit in performance for WT mice compared with β-arrestin-2 KO mice (P < 0.0003). (B) Sal A also produced a larger deficit in rotarod performance in WT mice (n = 25) than in β-arrestin-2 KO mice (n = 25) (P < 0.0006). (C) RB-64 (3 mg/kg) had no effect on performance in either WT (n = 5) or β-arrestin-2 KO mice (n = 5). (D) 3 mg/kg U69593 had a strong effect in both WT (n = 18) and β-arrestin-2 KO mice (n = 17). (E) sal A (10 mg/kg) produced a larger deficit in rotarod performance in WT mice (n = 10) than in β-arrestin-2 KO mice (n = 10). (F) Ten milligrams per kilogram RB-64 had no effect on either WT (n = 8) or β-arrestin-2 KO mice (n = 10). (G) U69593 (n = 5) and sal A (n = 5) had no effect on rotarod performance in KOR KO mice. (H) No difference was seen in rotarod baseline performances between genotypes. Data are plotted as the percent of baseline performance (A–G) or time spent on rod (H) ± S.E.M. BArr2, β-arrestin-2.

Fig. 5.

Effect of KOR agonists on novelty induced locomotion. (A) The novelty induced locomotion for vehicle-treated WT (n = 7) and β-arrestin-2 KO mice (n = 8). Both genotypes habituated and decreased activity after approximately 30 minutes. (B) U69593 did not differentially affect WT (n = 6) and β-arrestin-2 KO mice (n = 8). (C) No difference was seen in locomotion for sal A treatment between WT (n = 6) and β-arrestin-2 KO mice (n = 6). (D) RB-64 induced similar effects in both WT (n = 6) and β-arrestin-2 KO mice (n = 7). (E) The total distance traveled in the first 30 minutes (time before habituation). U69593 and sal A caused a strong decrease in activity, whereas RB-64 had no effect relative to vehicle-treated mice. Variance is represented as S.E.M. ***P < 0.001. BArr2, β-arrestin-2.

Fig. 6.

A G protein–biased KOR agonist does not display anhedonia-like effects. (A) The response for different frequencies of brain stimulation reward by C57BL/6J mice treated with 1 mg/kg drug. All treatments showed a rightward shift in the average rate-frequency curves compared with vehicle; U69593 had the largest effect, followed by sal A, and then RB-64 (n = 13 for all conditions). (B) Dose-response relationship for the effects of U69593, sal A, and RB-64 on the BSR. Results are presented as mean percentages of preinjection baseline during the four 15-minute postinjection response series ± S.E.M. *Significance (P < 0.05) of drug versus vehicle. ^#Significance (P < 0.05) of drug compared with another drug treatment. (C) Dose-response relationship for the effects of U69593, sal A, and RB-64 on the maximum response rate (MAX) in C57BL/6J mice. Results are presented as mean percentage of preinjection baseline during the four 15-minute postinjection response periods ± S.E.M. *Significance (P < 0.05) versus vehicle. ^#Significance (P < 0.05) versus a separate drug treatment. n = 13 for all treatment conditions.