Background and Purpose:Mitragyna speciosa extract and kratom alkaloids decrease alcohol consumption in mice at least in part through actions at the δ-opioid receptor (δOR). However, the most potent opioidergic kratom alkaloid, 7-hydroxymitragynine, exhibits rewarding properties and hyperlocomotion presumably due to preferred affinity for the mu opioid receptor (µOR). We hypothesized that opioidergic kratom alkaloids like paynantheine and speciogynine with reduced µOR potency could provide a starting point for developing opioids with an improved therapeutic window to treat alcohol use disorder.Experimental Approach: We characterized paynantheine, speciociliatine, and four novel kratom-derived analogs for their ability to bind and activate δOR, µOR, and κOR. Select opioids were assessed in behavioral assays in male C57BL/6N WT and δOR knockout mice.Key Results: Paynantheine (10 mg∙kg−1, i.p.) produced aversion in a limited conditioned place preference (CPP) paradigm but did not produce CPP with additional conditioning sessions. Paynantheine did not produce robust antinociception but did block morphine-induced antinociception and hyperlocomotion. Yet, at 10 and 30 mg∙kg−1 doses (i.p.), paynantheine did not counteract morphine CPP. 7-hydroxypaynantheine and 7-hydroxyspeciogynine displayed potency at δOR but limited µOR potency relative to 7-hydroxymitragynine in vitro, and dose-dependently decreased voluntary alcohol consumption in WT but not δOR in KO mice. 7-hydroxyspeciogynine has a maximally tolerated dose of at least 10 mg∙kg−1 (s.c.) at which it did not produce significant CPP neither alter general locomotion nor induce noticeable seizures.Conclusion and Implications: Derivatizing kratom alkaloids with the goal of enhancing δOR potency and reducing off-target effects could provide a pathway to develop novel lead compounds to treat alcohol use disorder with an improved therapeutic window.

beta-Thalassemia, a hemoglobinopathy that results in the precipitation of denatured alpha-globin chains on the membrane, is characterized by erythrocytes with significantly reduced lifespans. We have demonstrated previously that hemoglobin denaturation on the membrane can promote clustering of integral membrane proteins, and that this clustering in turn leads to autologous antibody binding, complement fixation, and rapid removal of the cell by macrophages. To evaluate whether this pathway also occurs in beta-thalassemic cells, we have isolated and characterized the immune complexes from the membranes of these cells. We observe that autologous IgG-containing complexes obtained by either immunoprecipitation or simple centrifugation of nondenaturing detergent extracts of beta-thalassemic cell membranes contain globin, band 3, IgG, and complement as major components. Absorption spectra of these complexes demonstrate that the globin is, indeed, mainly in the form of hemichromes. Immunoblotting studies further show that much of the band 3 protein in the aggregates is covalently cross-linked to a dimeric or tetrameric form, consistent with the preference of the autologous IgG for clustered band 3. Although the insoluble aggregates constitute only approximately 1.6% of the total membrane protein, they still contain 27% of the total IgG and 35% of the total complement C3 on the thalassemic cell surface. Because cell surface IgG and complement component C3 are thought to trigger removal of erythrocytes from circulation, the hemichrome-induced clustering of band 3 may contribute to the beta-thalassemic cell's shortened lifespan.

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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.

Arrestins are a family of proteins involved in regulation of G‐protein coupled receptor (GPCR) signaling. By binding to phosphorylated receptors, they uncouple the receptor from its corresponding G‐protein, terminating GPCR signaling in a process known as desensitization. Within the arrestin family, the β‐arrestin 1and β‐arrestin 2 isoforms are ubiquitously expressed throughout the body, including the central nervous system, and interact with a multitude of GPCRs. Additionally, the β‐arrestin proteins also have an important impact on behavior, with various studies indicating each isoform has unique signaling interactions and different roles in drug responses. This has prompted researchers to develop molecules that are specific towards recruitment of one isoform over the other; as well as further understand the biological role of each protein. The δ‐opioid receptor (DOR) is a promising target for the treatment of a variety of neurological disorders, including chronic pain disorders, alcohol use disorder and mood disorders. However, a major side effect that occurs when activating the DOR is the induction of seizures. Notably, mice with a genetic knockout (KO) of β‐arrestin 1 mice show a markedly increased sensitivity to agonist induced seizures compared to wild type or β‐arrestin 2 KO mice. Understanding the mechanism linking DOR agonists and seizures will allow for the use of more rational drug design to develop effective therapeutics. This project aims to 1) investigate the cellular mechanism of action for β‐arrestin's involvement in the seizurogenic effects of DOR activation 2) Assess whether DOR agonists with lower β‐arrestin recruitment efficacy are less seizurogenic and 3) Determine if β‐arrestin 1 KO mice are a novel model of status epilepticus? To address these questions, we measured seizure activity (assessed by Racine score), in male and female wild‐type, β‐arrestin 1 KO and β‐arrestin 2 KO C57BL/6 mice (n≥6 per treatment). We utilized three different DOR agonists, which we determined differed in their β‐arrestin recruitment efficacy using cellular signaling assays. When we injected these three agonists, SNC80, ADL5859 and ARM390 at equi‐analgesic doses, we noted a strong correlation between β‐arrestin 2 recruitment efficacy and seizure intensity. We did not observe any sex differences. In the hippocampus of our mice we also noted a strong increase in pERK following DOR activation. The ERK activation was more pronounced when mice were treated with a DOR agonist with strong β‐arrestin recruitment efficacy, and also was more pronounced in β‐arrestin 1 KO mice. Blocking ERK activation, with the MEK inhibitor SL327, however, did not reduce DOR induced seizure, suggesting that the ERK activation comes as a result of the seizure and is not part of the mechanism of action of DOR‐induced seizures. Thus far, our results suggest that the likelihood of seizures induced by DOR activation can be mitigated using a DOR agonist with low β‐arrestin 2 recruitment efficacy.