*Article* **SR-17018 Stimulates Atypical** µ**-Opioid Receptor Phosphorylation and Dephosphorylation**

**Sebastian Fritzwanker, Stefan Schulz \* and Andrea Kliewer \***

Department of Pharmacology and Toxicology, Jena University Hospital, Friedrich Schiller University Jena, Drackendorfer Straße 1, D-07747 Jena, Germany; Sebastian.Fritzwanker@med.uni-jena.de **\*** Correspondence: stefan.schulz@med.uni-jena.de (S.S.); andrea.kliewer@med.uni-jena.de (A.K.)

**Abstract:** Opioid-associated overdoses and deaths due to respiratory depression are a major public health problem in the US and other Western countries. In the past decade, much research effort has been directed towards the development of G-protein-biased µ-opioid receptor (MOP) agonists as a possible means to circumvent this problem. The bias hypothesis proposes that G-protein signaling mediates analgesia, whereas ß-arrestin signaling mediates respiratory depression. SR-17018 was initially reported as a highly biased µ-opioid with an extremely wide therapeutic window. It was later shown that SR-17018 can also reverse morphine tolerance and prevent withdrawal via a hitherto unknown mechanism of action. Here, we examined the temporal dynamics of SR-17018-induced MOP phosphorylation and dephosphorylation. Exposure of MOP to saturating concentrations of SR-17018 for extended periods of time stimulated a MOP phosphorylation pattern that was indistinguishable from that induced by the full agonist DAMGO. Unlike DAMGO-induced MOP phosphorylation, which is reversible within minutes after agonist washout, SR-17018-induced MOP phosphorylation persisted for hours under otherwise identical conditions. Such delayed MOP dephosphorylation kinetics were also found for the partial agonist buprenorphine. However, buprenorphine, SR-17018 induced MOP phosphorylation was fully reversible when naloxone was included in the washout solution. SR-17018 exhibits a qualitative and temporal MOP phosphorylation profile that is strikingly different from any other known biased, partial, or full MOP agonist. We conclude that detailed analysis of receptor phosphorylation may provide novel insights into previously unappreciated pharmacological properties of newly synthesized MOP ligands.

**Keywords:** µ-opioid receptor; DAMGO; SR-17018; buprenorphine
