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.

Pharmacological Properties of δ-Opioid Receptor-Mediated Behaviors: Agonist Efficacy and Receptor Reserve.

δ-Opioid receptor (δ-receptor) agonists produce antihyperalgesia, antidepressant-like effects, and convulsions in animals. However, the role of agonist efficacy in generating different δ-receptor-mediated behaviors has not been thoroughly investigated. To this end, efficacy requirements for δ-receptor-mediated antihyperalgesia, antidepressant-like effects, and convulsions were evaluated by comparing the effects of the partial agonist BU48 and the full agonist SNC80 and changes in the potency of SNC80 after δ-receptor elimination. Antihyperalgesia was measured in a nitroglycerin-induced thermal hyperalgesia assay. An antidepressant-like effect was evaluated in the forced swim test. Mice were observed for convulsions after treatment with SNC80 or the δ-opioid receptor partial agonist BU48. Ligand-induced G protein activation was measured by [35S]guanosine 5'-O-[γ-thio]triphosphate binding in mouse forebrain tissue, and δ-receptor number was measured by [3H]D-Pen2,5-enkephalin saturation binding. BU48 produced antidepressant-like effects and convulsions but antagonized SNC80-induced antihyperalgesia and G protein activation. The potency of SNC80 was shifted to the right in δ-receptor heterozygous knockout mice and naltrindole-5'-isothiocyanate-treated mice, and the magnitude of potency shift differed across assays, with the largest shift occurring in the thermal hyperalgesia assay, followed by the forced swim test and then convulsion observation. Naltrindole antagonized these SNC80-induced behaviors with similar potencies, suggesting that these effects are mediated by the same type of δ-receptor. These data suggest that δ-receptor-mediated behaviors display a rank order of efficacy requirement, with antihyperalgesia having the highest requirement, followed by antidepressant-like effects and then convulsions. These findings further our understanding of the pharmacological mechanisms mediating the in vivo effects of δ-opioid receptor agonists. SIGNIFICANCE STATEMENT: δ-Opioid receptor (δ-receptor) agonists produce antihyperalgesia, antidepressant-like effects, and convulsions in animal models. This study evaluates pharmacological properties, specifically the role of agonist efficacy and receptor reserve, underlying these δ-receptor-mediated behaviors. These data suggest that δ-receptor-mediated behaviors display a rank order of efficacy requirement, with antihyperalgesia having the highest requirement, followed by antidepressant-like effects and then convulsions.

Alkylation of Staurosporine to Derive a Kinase Probe for Fluorescence Applications.

The natural product staurosporine is a high-affinity inhibitor of nearly all mammalian protein kinases. The labelling of staurosporine has proven effective as a means of generating protein kinase research tools. Most tools have been generated by acylation of the 4'-methylamine of the sugar moiety of staurosporine. Herein we describe the alkylation of this group as a first step to generate a fluorescently labelled staurosporine. Following alkylation, a polyethylene glycol linker was installed, allowing subsequent attachment of fluorescein. We report that this fluorescein-staurosporine conjugate binds to cAMP-dependent protein kinase in the nanomolar range. Furthermore, its binding can be antagonised with unmodified staurosporine as well as ATP, indicating it targets the ATP binding site in a similar fashion to native staurosporine. This reagent has potential application as a screening tool for protein kinases of interest.