Role of β‐arrestin Isoforms in Delta Opioid Receptor Agonist‐Induced Seizures

Abstract

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.

Identification of a Novel Delta Opioid Receptor Agonist Chemotype with Potential Negative Allosteric Modulator Capabilities

“…AZD2327 exhibited proconvulsant effects [ 8 ], whereas Adolor was able to modify the SNC structure enough to not observe tonic-clonic seizures [ 6 , 7 ]. However, recent studies have led to a better understanding of the mechanism by which SNC80 can cause seizures, implicating β-arrestin as a critical factor [ 12 , 13 ]. The SNC compounds are super-recruiters of β-arrestin, and it appears that ADL5859 and AZD2327, recruit β-arrestin on par with the endogenous agonist Leu-enkephalin, if not stronger [ 14 , 15 , 16 ].…”