Carfentanil is a β‐arrestin‐biased agonist at the μ opioid receptor

Abstract

Background and PurposeThe illicit use of fentanyl‐like drugs (fentanyls), which are μ opioid receptor agonists, and the many overdose deaths that result, has become a major problem. Fentanyls are very potent in vivo, leading to respiratory depression and death. However, the efficacy and possible signalling bias of different fentanyls is not clearly known. Here, we compared the relative efficacy and bias of a series of fentanyls.Experimental ApproachFor agonist signalling bias and efficacy measurements, Bioluminescence Resonance Energy Transfer experiments were undertaken in HEK293T cells transiently transfected with μ opioid receptors, to assess Gi protein activation and β‐arrestin 2 recruitment. Agonist‐induced cell surface receptor loss was assessed using an enzyme‐linked immunosorbent assay, whilst agonist‐induced G protein‐coupled inwardly rectifying potassium channel current activation was measured electrophysiologically from rat locus coeruleus slices. Ligand poses in the μ opioid receptor were determined in silico using molecular dynamics simulations.Key ResultsRelative to the reference ligand DAMGO, carfentanil was β‐arrestin‐biased, whereas fentanyl, sufentanil and alfentanil did not display bias. Carfentanil induced potent and extensive cell surface receptor loss, whilst the marked desensitisation of G protein‐coupled inwardly rectifying potassium channel currents in the continued presence of carfentanil in neurones was prevented by a GRK2/3 inhibitor. Molecular dynamics simulations suggested unique interactions of carfentanil with the orthosteric site of the receptor that could underlie the bias.Conclusions and ImplicationsCarfentanil is a β‐arrestin‐biased opioid drug at the μ receptor. It is uncertain how such bias influences in vivo effects of carfentanil relative to other fentanyls.

Potency, dissociation kinetics and reversibility of fentanyls and nitazenes by naloxone at the μ opioid receptor

“…As we have previously reported that carfentanil shows bias for β-arrestin translocation over G protein activation (Ramos-Gonzalez, Groom et al ., 2023) we next sought to examine whether a slow rate of agonist dissociation from the μ opioid receptor was associated with bias for β-arrestin 2 translocation over G protein activation. We compared the ability of DAMGO (reference ligand), morphine, alfentanil, ohmefentanyl, carfentanil, isotonitazene and etonitazene to activate G protein or recruit β-arrestin 2 in HEK293T cells expressing the μ opioid receptor (Figure 6).…”

“…When compared to morphine as the standard opioid agonist, the rank order of potency of the fentanyls and nitazenes to produce membrane hyperpolarisation in vitro was largely as expected (Tables 1 & 3). These relative potency values agree with those reported previously using a variety of in vitro assays involving G protein activation (Emmerson, Clark et al ., 1996; Toll, Berzetei-Gurske et al ., 1998; McPherson, Rivero et al ., 2010; Åstrand, Guerrier et al ., 2020; Vandeputte, Van Uytfanghe et al ., 2021; Faouzi, Wang et al ., 2022; Malcolm, Palkovic et al ., 2023; Glatfelter, Vandeputte et al ., 2023; Ramos-Gonzalez, Groom et al, 2023; Vandeputte, Tsai et al ., 2023).…”

“…To determine the relative ability of the opioid agonists to activate Gα i G proteins and β-arrestin 2 translocation to the receptor, BRET 2 -based assays were used as described previously (Hill, Disney et al ., 2018; Ramos-Gonzalez, Groom et al ., 2023). For Gi activation the assay monitored the separation of Gα i1 and Gγ 2 whereas for arrestin translocation the assay measured μ opioid receptor and β-arrestin 2 association.…”

Comparison of the effects of fentanyls and other μ opioid receptor agonists on the electrical activity of respiratory muscles in the rat

“…To do this we used a BRET assay of G-protein activation in HEK-293 cells expressing rat µ opioid receptors ( Figure 6 ). G protein coupled receptors that couple to Gi/Go proteins, such as the μ opioid receptor, have been extensively studied in HEK293 cells and behave as they do in single brain neurones with respect to relative potency and efficacy of opioid agonists ( Ramos-Gonzalez et al, 2023 ). In a separate study we have examined the potency and efficacy of fentanyls on human μ opioid receptors expressed in AtT20 cells, a cell line with neuronal characteristics.…”

“…Preliminary data in freely moving mice suggest that carfentanil does not produce a marked decrease in tidal volume at doses which depress respiratory rate (Cavallo, Abdala and Henderson, unpublished observations) which correlates with the reduced ability of carfentanil to induce respiratory muscle rigidity in this study. We have recently reported that carfentanil shows significant bias in favour of arrestin recruitment over G protein signalling ( Ramos-Gonzalez et al, 2023 ). How this could affect its ability to induce respiratory muscle rigidity is not clear; enhanced μ opioid receptor desensitization is unlikely as, at the concentration studied in the plethysmography experiments, bradypnea was maintained throughout the period of carfentanil exposure.…”