Do frequently used opioids exhibit biased signaling at the µ opioid receptor?

B Schmid1,3, S Mayer1, K van Unen4, J Goedhart4, C Hoffmann1,2. 1Institute of Pharmacology, University of Würzburg, Würzburg, Germany, 2Rudolf-Virchow-Center, University of Würzburg, Würzburg, Germany, 3Interdisciplinary Center for Clinical Research, University Hospital Würzburg, Würzburg, Germany, 4Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands

Opioids currently represent the gold standard in the treatment of severe acute and chronic pain. Despite the opioids’ huge clinical importance, little data are available regarding the downstream signaling properties of each of those substances used in countless patients every day. The desired anesthetic effect of opioid drugs is mainly ascribed to the μ subtype of opioid receptors (OPRM). The first wave of signaling triggers the activation of an inhibitory G-protein heterotrimer. A second signal wave manifests in the phosphorylation dependent recruitment of β-arrestin2 to the receptor and is often followed by receptor internalization. To study the signaling properties of opioid based drugs, we assessed both ligand-induced Gi- activation and subsequent β-arrestin2 recruitment to the receptor in living transfected HEK293 cells. We tested a total of 13 clinically relevant opioid agonists, two antagonists, one physiologically occurring metabolites, and utilized the synthetic full agonist DAMGO as reference compound. Gi activation was quantitatively assessed using a previously published fluorescence resonance energy transfer (FRET) assay for which Gαi and Gγsubunits were tagged with yellow (YFP) or cyan fluorescent protein (CFP), respectively. Upon receptor activation, the Gi complex undergoes conformational changes which lead to a change in the FRET ratio. Full concentration-response curves are given for all substances. This assay allowed to clearly differentiate between full and partial agonists (e.g. fentanyl, sufentanil and tramadol), or antagonists (e.g. naloxone). β-arrestin2 recruitment was assessed by dynamic confocal microscopy of CFP-tagged OPRM and β-arrestin2-YFP. Maximum β- arrestin2 recruitment was quantitatively determined as the percentage of YFP-tagged β- arrestin2 translocating to the membrane 10 min after application of saturating opioid ligand concentrations. Since the OPRM was fluorescently tagged, we were able to measure receptor internalization subsequent to β-arrestin2 recruitment. Taken together, our data provide evidence that some clinically used opioids exhibit preference for one signaling pathway or the other. Therefore, our data hint towards biased agonism of some opioids for either Gi activation or β-arrestin2 recruitment (Pharmacological parameters for selected opioids given in the table).