Dataset Information

Molecular Interaction Between Butorphanol and κ-Opioid Receptor.

ABSTRACT:

Background

The misuse of opioids stems, in part, from inadequate knowledge of molecular interactions between opioids and opioid receptors. It is still unclear why some opioids are far more addictive than others. The κ-opioid receptor (KOR) plays a critical role in modulating pain, addiction, and many other physiological and pathological processes. Butorphanol, an opioid analgesic, is a less addictive opioid with unique pharmacological profiles. In this study, we investigated the interaction between butorphanol and KOR to obtain insights into the safe usage of this medication.

Methods

We determined the binding affinity of butorphanol to KOR with a naltrexone competition study. Recombinant KORs expressed in mammalian cell membranes (Chem-1) were used for G-protein activation studies, and a human embryonic kidney-293 (HEK-293) cell line stably transfected with the human KOR was used for β-arrestin study as previously described in the literature. The effects of butorphanol on KOR internalization were investigated using mouse neuroblastoma Neuro2A cells stably transfected with mKOR-tdTomato fusion protein (N2A-mKOR-tdT) cells overexpressing KOR. The active-state KOR crystal structure was used for docking calculation of butorphanol to characterize the ligand binding site. Salvinorin A, a full KOR agonist, was used as a control for comparison.

Results

The affinity of KOR for butorphanol is characterized by Kd of 0.1 ± 0.02 nM, about 20-fold higher compared with that of the µ-opioid receptor (MOR; 2.4 ± 1.2 nM). Our data indicate that butorphanol is more potent on KOR than on MOR. In addition, butorphanol acts as a partial agonist of KOR in the G-protein activation pathway and is a full agonist on the β-arrestin recruitment pathway, similar to that of salvinorin A. The activation of the β-arrestin pathway is further confirmed by KOR internalization. The in silico docking model indicates that both salvinorin A and butorphanol share the same binding cavity with the KOR full agonist MP1104. This cavity plays an important role in determining either agonist or antagonist effects of the ligand.

Conclusions

In conclusion, butorphanol is a partial KOR agonist in the G-protein activation pathway and a potent KOR full agonist in the β-arrestin recruitment pathway. The structure analysis offers insights into the molecular mechanism of KOR interaction and activation by butorphanol.

SUBMITTER: Ji J

PROVIDER: S-EPMC7668422 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Kappa Opioid receptors（KORs） belong to family A of G-protein–coupled receptors (GPCRs), and the involvement of their ligands in analgesia and sedation has been extensively studied. KORs, which are expressed in the central and peripheral nervous systems are well known to exert potent anti-nociceptive effects by negatively modulating excitatory neurotransmission. The present study reveals that acute activation of KOR weaken SERT function via protein kinase B/Akt (Akt) and calcium calmodulin dependent kinase II (CaMKII) dependent signaling pathways. Therefore, studying phosphorylation will be helpful to elucidate the cellular mechanisms and downstream effector(s). Studies correlating the dysphoric effects of KOR agonists to the G-protein–independent activation of the p38 MAPK pathway suggest that identification of KOR agonists biased to G-protein–mediated signaling could lead to the development of therapeutics with reduced side effects. Therefore, to better understand the cellular mechanisms and downstream effector(s) by which KOR ligands produce these effects, we employed dimethyl labeling quantitative proteomic approach in this study. In this study, three monoclonal HEK293T cell lines with KOR over-expression were successfully constructed as our experimental models. Then, mass spectrometry-based quantitative approach, based on dimethyl labeling and TiO2 beads-coupled phosphopeptides enrichment, was utilized to analyze the KOR-mediated dynamic phosphorylation network in these three KOR-over-expressed monoclonal cell lines disturbed by LPK-26 or U50,488 treatments. Further Substrate-kinase prediction analysis of 212 up-regulated phosphosites indicated that 18 kinases, such as AKT1 and p70S6K, were involved in the up-regulation of KOR phosphorylation. The up-regulated phosphorylation of AKT1 and p70S6K were further confirmed by western blot, which was consistent with our mass spectrometry results. In summary, quantitative phosphoproteomic study of KOR-over-expressed cells upon agonist stimulation paves the way to systematical investigation of KOR-mediated phosphorylation network dynamics, which will be helpful to research about targeted therapy of KOR related diseases and mechanism study of analgesia addiction.