In Vitro Functional Profiling of Fentanyl and Nitazene Analogs at the μ-Opioid Receptor Reveals High Efficacy for Gi Protein Signaling

Novel synthetic opioids (NSOs), including both fentanyl and non-fentanyl analogs that act as mu-opioid receptor (MOR) agonists, are associated with serious intoxication and fatal overdose. Previous studies proposed that G-protein-biased MOR agonists are safer pain medications, while other evidence indicates that low intrinsic efficacy at MOR better explains the reduced opioid side effects. Here, we characterized the in vitro functional profiles of various NSOs at the MOR using adenylate cyclase inhibition and beta-arrestin2 recruitment assays, in conjunction with the application of the receptor depletion approach. By fitting the concentration-response data to the operational model of agonism, we deduced the intrinsic efficacy and affinity for each opioid in the Gi protein signaling and beta-arrestin2 recruitment pathways. Compared to the reference agonist [d-Ala(2),N-MePhe(4),Gly-ol(5)]enkephalin, we found that several fentanyl analogs were more efficacious at inhibiting cAMP production, whereas all fentanyl analogs were less efficacious at recruiting beta-arrestin2. In contrast, the non-fentanyl 2-benzylbenzimidazole (i.e., nitazene) analogs were highly efficacious and potent in both the cAMP and beta-arrestin2 assays. Our findings suggest that the high intrinsic efficacy of the NSOs in Gi protein signaling is a common property that may underlie their high risk of intoxication and overdose, highlighting the limitation of using in vitro functional bias to predict the adverse effects of opioids. In addition, the extremely high potency of many NSOs now infiltrating illicit drug markets further contributes to the danger posed to public health.