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. 2022 Aug 11:13:914651.
doi: 10.3389/fphar.2022.914651. eCollection 2022.

Exploration of beta-arrestin isoform signaling pathways in delta opioid receptor agonist-induced convulsions

Arryn T Blaine  1   2 , Yiming Miao  1 , Jinling Yuan  1 , Sophia Palant  1 , Rebecca J Liu  1 , Zhong-Yin Zhang  1   3   4 , Richard M van Rijn  1   3   4   5
Affiliations

Exploration of beta-arrestin isoform signaling pathways in delta opioid receptor agonist-induced convulsions

Arryn T Blaine et al. Front Pharmacol. .

Abstract

The δ-opioid receptor (δOR) has been considered as a therapeutic target in multiple neurological and neuropsychiatric disorders particularly as δOR agonists are deemed safer alternatives relative to the more abuse-liable µ-opioid receptor drugs. Clinical development of δOR agonists, however, has been challenging in part due to the seizure-inducing effects of certain δOR agonists. Especially agonists that resemble the δOR-selective agonist SNC80 have well-established convulsive activity. Close inspection suggests that many of those seizurogenic δOR agonists efficaciously recruit β-arrestin, yet surprisingly, SNC80 displays enhanced seizure activity in β-arrestin 1 knockout mice. This finding led us to hypothesize that perhaps β-arrestin 1 is protective against, whereas β-arrestin 2 is detrimental for δOR-agonist-induced seizures. To investigate our hypothesis, we characterized three different δOR agonists (SNC80, ADL5859, ARM390) in cellular assays and in vivo in wild-type and β-arrestin 1 and β-arrestin 2 knockout mice for seizure activity. We also investigated downstream kinases associated with β-arrestin-dependent signal transduction. We discovered that δOR agonist-induced seizure activity strongly and positively correlates with β-arrestin 2 efficacy for the agonist, but that indirect inhibition of ERK activation using the MEK inhibitor SL327 did not inhibit seizure potency and duration. Inhibition of the PI3K/AKT/mTOR signaling with honokiol but not PQR530, attenuated SNC80 seizure duration in β-arrestin 1 knockout, but honokiol did not reduce SNC80-induced seizures in wild-type mice. Ultimately, our results indicate that β-arrestin 2 is correlated with δOR agonist-induced seizure intensity, but that global β-arrestin 1 knockout mice are a poor model system to investigate their mechanism of action.

Keywords: ERK; PQR530; beta-arrestin 1; beta-arrestin 2; biased signaling; honokiol; mice; seizure.

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Conflict of interest statement

RVR is currently employed as a Principal Scientist at Septerna Inc. and holds stock options in the company. RVR holds a US patent (10,954,224) describing novel delta opioid receptor agonists. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Chemical structure of SNC80, ADL 58589 and ARM 390.
FIGURE 2
FIGURE 2
β-arrestin 1 knockout mice display enhanced seizure activity for δOR agonists relative to wild-type and β-arrestin 2 knockout mice. (A) Seizure activity over 1 h period for equi-antinociceptive doses of SNC80 (10 mg/kg i.p.), ADL 58589 (30 mg/kg, i.p.) and ARM 390 (10 mg/kg p.o.) in wild-type mice. (B) Seizure activity in β-arrestin 1 KO mice. (C) Seizure activity for SNC80 in male and female β-arrestin 1 KO mice over a 3-h period. (D) Area under the curve (AUC) for seizure activity induced by SNC80 (10 mg/kg i.p.) in male and female mice in β-arrestin 1 KO mice over 60 and 180 min. (E) seizure activity in β-arrestin 2 KO mice (F) Area under the curve for SNC80, ADL 58589 and ARM 390 in wild-type, β-arrestin1 KO, and β-arrestin 2 KO mice. Racine score data is shown 60 minutes post-injection. Equal number of males and females were tested for each drug (n = 6 total, except n = 10 for SNC80 in β-arrestin 1 KO mice). *p < 0.05. In (G) male mice are indicated with a cross whereas female mice are depicted as circles.
FIGURE 3
FIGURE 3
δOR agonist-induced seizure efficacy strongly and positively correlates with β-arrestin 2 recruitment efficacy. Concentration-dependent recruitment of β-arrestin 1 (A) or β-arrestin 2 (B) recruitment to δOR induced by SNC80, ADL5859 and ARM390. Data is normalized to the endogenous agonist peptide Leu5-enkephalin (Leu-Enk). Correlation plot for Racine score (60 min AUC) for 10 mg/kg SNC80 (n=12 for wild-type and β-arrestin 1 knockout mice, and n = 6 for β-arrestin 2 knockout mice), 30 mg/kg ADL 5859 (n = 6) and 10 mg/kg ARM 390 (n = 6) obtained in wild-type and β-arrestin 2 knockout mice (C) and in wild-type and β-arrestin 1 knockout mice (D) mice in correlation with its β-arrestin 1 (C) or β-arrestin 2 (D) recruitment Emax. *p < 0.05. Symbols and colors in (C,D) correspond with the agonists as described in (A,B). Emax values and the standard deviation represented are as depicted in Table 1.
FIGURE 4
FIGURE 4
ERK activation by SNC80 does not cause seizures. (A) Representative western blot of SNC80 (10 mg/kg, i.p.)-induced activation of ERK in hippocampal tissue from male and female β-arrestin 1 KO mice relative to vehicle (n = 3 per treatment and sex). (B) Quantification of phosphorylated ERK (pERK) over total ERK (ERK) all normalized to GAPDH as loading control. Seizure activity over time (C,E) and as area under the curve (AUC) (D,F) in wild-type (C,D) or β-arrestin 1 KO (E,F) mice exposed to SNC80 (10 mg/kg, i.p.), SL327 (50 mg/kg i.p.) or SNC80 + SL327. Equal number of males and females tested (n = 6 total). *p < 0.05. Dotted line depicts 10 mg/kg SNC80 in wild-type (C) or β-arrestin 1 KO mice (E) for reference. In (D,F) male mice are depicted with a cross, whereas female mice are depicted as solid symbols.
FIGURE 5
FIGURE 5
Honokiol dose-dependently reduces SNC80-induced seizures in β-arrestin 1 KO mice, but not in wild-type mice, and its seizure activity is attenuated by honokiol. Seizure activity over time (A) and as area under the curve (AUC) (B) in β-arrestin 1 KO or wild-type (C,D) mice exposed to SNC80 (10 mg/kg, i.p.), honokiol (40 mg/kg p.o.) or SNC80 + 40 or 80 mg/kg honokiol. [n = 5 (3 female and 2 male) for β-arrestin 1 KO mice and n = 6 female per treatment for wild-type mice]. *p < 0.05. Dotted line depicts 10 mg/kg SNC80 in β-arrestin 1 KO (A) or wild-type mice (C) for reference. In (B) male mice are depicted with a cross, whereas female mice are depicted as solid symbols.
FIGURE 6
FIGURE 6
Stronger seizures induced by 32 mg/kg SNC80 in wild-type mice are not reduced by honokiol and similar in β-arrestin 2 knockout mice. Seizure activity over time (A) and as area under the curve (AUC) (B) in β-arrestin 2 KO or wild-type mice exposed to SNC80 (32 mg/kg, i.p.), or 32 mg/kg SNC80 and 80 mg/kg honokiol. [n = 6 (3 female and 3 male) in wild-type mice]. In (B) male mice are depicted with a cross, whereas female mice are depicted as circles.
FIGURE 7
FIGURE 7
The PI3K and mTOR inhibitor PQR530 does not inhibit SNC80-induced seizures in β-arrestin 1 knockout mice. Seizure activity over time (A) and as area under the curve (AUC) (B) in β-arrestin 1 KO exposed to SNC80 (10 mg/kg, i.p.) and 25 mg/kg PQR530 [n = 6 (3 female and 3 male)]. Dotted line depicts 10 mg/kg SNC80 in β-arrestin 1 KO for reference. In (B) male mice are depicted with a cross, whereas female mice are depicted as circles.
FIGURE 8
FIGURE 8
Enhanced δOR-agonist induced seizures in β-arrestin 1 knockout mice are modulated by ERK and AKT inhibition, while those kinases are not part of δOR-induced seizures in wild-type mice. 10 mg/kg SNC80 produces more severe seizures in β-arrestin 1 knockout mice compared to wild-type mice (A). 32 mg/kg SNC80 in wild-type mice produces clonic seizures (Racine score 4), similar to 10 mg/kg SNC80 in β-arrestin 1 knockout mice, but the intensity tapers off more rapidly in wild-type mice (B). Inhibition of PI3K/AKT reduces seizure effects for 10 mg/kg SNC80 in β-arrestin 1 knockout mice to levels comparable to 32 mg/kg SNC80 in wild-type mice (C). The component of enhanced seizure effect observed for 10 mg/kg SNC80 in β-arrestin 1 knockout mice is sensitive to modulation by ERK and PI3K/AKT inhibition (D).
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