Carfentanil is a ß-arrestin-biased agonist at the µ opioid receptor.

BACKGROUND AND PURPOSE: The 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 APPROACH: For 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 ß-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 RESULTS: Relative to the reference ligand DAMGO, carfentanil was ß-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 IMPLICATIONS: Carfentanil is a ß-arrestin-biased opioid drug at the µ receptor. It is uncertain how such bias influences in vivo effects of carfentanil relative to other fentanyls.

A novel G protein-biased agonist at the µ opioid receptor induces substantial receptor desensitisation through G protein-coupled receptor kinase.

BACKGROUND AND PURPOSE: G protein-biased µ opioid receptor agonists have the potential to induce less receptor desensitisation and tolerance than balanced opioids. Here, we investigated if the cyclic endomorphin analogue Tyr-c[D-Lys-Phe-Tyr-Gly] (Compound 1) is a G protein-biased µ agonist and characterised its ability to induce rapid receptor desensitisation in mammalian neurones. EXPERIMENTAL APPROACH: The signalling and trafficking properties of opioids were characterised using bioluminescence resonance energy transfer assays, enzyme-linked immunosorbent assay and phosphosite-specific immunoblotting in human embryonic kidney 293 cells. Desensitisation of opioid-induced currents were studied in rat locus coeruleus neurones using whole-cell patch-clamp electrophysiology. The mechanism of Compound 1-induced µ receptor desensitisation was probed using kinase inhibitors. KEY RESULTS: Compound 1 has similar intrinsic activity for G protein signalling as morphine. As predicted for a G protein-biased µ agonist, Compound 1 induced minimal agonist-induced internalisation and phosphorylation at intracellular µ receptor serine/threonine residues known to be involved in G protein-coupled receptor kinase (GRK)-mediated desensitisation. However, Compound 1 induced robust rapid µ receptor desensitisation in locus coeruleus neurons, to a greater degree than morphine. The extent of Compound 1-induced desensitisation was unaffected by activation or inhibition of protein kinase C (PKC) but was significantly reduced by inhibition of GRK. CONCLUSION AND IMPLICATIONS: Compound 1 is a novel G protein-biased µ agonist that induces substantial rapid receptor desensitisation in mammalian neurons. Surprisingly, Compound 1-induced desensitisation was demonstrated to be GRK dependent despite its G protein bias. Our findings refute the assumption that G protein-biased agonists will evade receptor desensitisation and tolerance. LINKED ARTICLES: This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc.

Interaction With the Lipid Membrane Influences Fentanyl Pharmacology.

Overdose deaths from fentanyl have reached epidemic proportions in the USA and are increasing worldwide. Fentanyl is a potent opioid agonist that is less well reversed by naloxone than morphine. Due to fentanyl's high lipophilicity and elongated structure we hypothesised that its unusual pharmacology may be explained by its interactions with the lipid membrane on route to binding to the µ-opioid receptor (MOPr). Through coarse-grained molecular dynamics simulations, electrophysiological recordings and cell signalling assays, we determined how fentanyl and morphine access the orthosteric pocket of MOPr. Morphine accesses MOPr via the aqueous pathway; first binding to an extracellular vestibule, then diffusing into the orthosteric pocket. In contrast, fentanyl may take a novel route; first partitioning into the membrane, before accessing the orthosteric site by diffusing through a ligand-induced gap between the transmembrane helices. In electrophysiological recordings fentanyl-induced currents returned after washout, suggesting fentanyl deposits in the lipid membrane. However, mutation of residues forming the potential MOPr transmembrane access site did not alter fentanyl's pharmacological profile in vitro. A high local concentration of fentanyl in the lipid membrane, possibly in combination with a novel lipophilic binding route, may explain the high potency and lower susceptibility of fentanyl to reversal by naloxone.