Activation of µ receptors by SR-17018 through a distinctive mechanism

Agonists at μ opioid receptors relieve acute pain, however, their long-term use is limited by side effects, which may involve β-arrestin2. Agonists biased against β-arrestin2 recruitment may be advantageous. However, the classification of bias may be compromised by assays utilising overexpressed μ receptors which overestimate efficacy for G-protein activation. There is a need for re-evaluation with restricted receptor availability to determine accurate agonist efficacies. We depleted μ receptor availability in PathHunter CHO cells using the irreversible antagonist, β-funaltrexamine (β-FNA), and compared efficacies and apparent potencies of twelve agonists, including several previously reported as biased, in β-arrestin2 recruitment and cAMP assays. With full receptor availability all agonists had partial efficacy for stimulating β-arrestin2 recruitment relative to DAMGO, while only TRV130 and buprenorphine were partial agonists as inhibitors of cAMP accumulation. Limiting receptor availability by prior exposure to β-FNA (100 nM) revealed morphine, oxycodone, PZM21, herkinorin, U47700, tianeptine and U47931e are also partial agonists in the cAMP assay. The efficacies of all agonists, except SR-17018, correlated between β-arrestin2 recruitment and cAMP assays, with depleted receptor availability in the latter. Furthermore, naloxone and cyprodime exhibited non-competitive antagonism of SR-17018 in the β-arrestin2 recruitment assay. Limited antagonism by naloxone was also non-competitive in the cAMP assay, while cyprodime was competitive. Furthermore, SR-17018 only negligibly diminished β-arrestin2 recruitment stimulated by DAMGO (1 µM), whereas fentanyl, morphine and TRV130 all exhibited the anticipated competitive inhibition. The data suggest that SR-17018 achieves bias against β-arrestin2 recruitment through interactions with μ receptors outside the orthosteric agonist site.