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

Abstract

Novel synthetic opioids (NSOs), including both fentanyl and non-fentanyl analogs that act as μ-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 β-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 β-arrestin2 recruitment pathways. Compared to the reference agonist [d-Ala²,N-MePhe⁴,Gly-ol⁵]enkephalin, we found that several fentanyl analogs were more efficacious at inhibiting cAMP production, whereas all fentanyl analogs were less efficacious at recruiting β-arrestin2. In contrast, the non-fentanyl 2-benzylbenzimidazole (i.e., nitazene) analogs were highly efficacious and potent in both the cAMP and β-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.

Keywords: fentanyl; functional assay; intrinsic efficacy; nitazene; transduction coefficient; μ-opioid receptor.

Figure 1.

Chemical structures of the representative opioids investigated in this study.

Figure 2.. Activities of the tested opioids at MOR in the HTRF-based cAMP inhibition and β-arrestin2 recruitment assays.

Concentration-response curves were determined at 10 min for cAMP inhibition (A) and 30 min for β-arrestin2 recruitment (B). The curves shown are the averages of all corresponding experiments. The averaged E_max (% of DAMGO) versus pEC₅₀ for each opioid (see Table 1) are plotted for these two assays in panels C and D, respectively. The nitazene and fentanyl analogs are colored in orange and blue, respectively. The reference agonists DAMGO and morphine are in black, while three forms of methadone and buprenorphine are in green. Data in the curves are shown as mean ± SEM with n ≥ 4 for each experiment.

Figure 3.. Representative dose-response curves in the HTRF-based cAMP inhibition assays.

The results of global fitting of all four dose-response curves pretreated with the indicated concentrations of M-CAM are shown. The representative examples of the tested opioids include those having high (fentanyl and cyclopropylfentanyl), DAMGO-like (etonitazene and N-pyrrolidino etonitazene), and low (buprenorphine) τ. Note that we used the efficacy of etonitazene, which had exhibited the highest %E_max in Table 1, as the system E_max in the operational model fitting (defined as 100% in all the panels in this figure).

Figure 4.. Intrinsic efficacies and potencies of the tested opioids at MOR in the G protein and β-arrestin2 pathways.

Panels A and B are intrinsic efficacy (τ) versus intrinsic potency (K_A) profiles in the HTRF-based cAMP inhibition and β-arrestin2 recruitment assays. For the cAMP inhibition, intrinsic efficacy (τ) and potency (K_A) for each opioid were determined by the depletion approach and fitting the data to the “operational model depletion”; for the β-arrestin2 recruitment, τ and K_A were determined by fitting the data shown in Fig. 2B to the “operational model partial agonist” (Table 2). Note that the depletion approach was also applied for three selected nitazene analogs for the β-arrestin2 recruitment assays. The results showed that this β-arrestin2 recruitment assay has very limited signal amplification, while the derived τ and K_A are similar to those derived from the “operational model partial agonist”. Values shown for the cAMP inhibition experiments are the averages of n ≥ 7 for the cAMP inhibition and n ≥ 5 for the β-arrestin2 recruitment experiments.

Figure 5.. Distinct in vitro pharmacological profiles of nitazene and fentanyl analogs.

The transduction coefficient (log(τ/K_A)) of each opioid in each assay was normalized by subtracting that of DAMGO, resulting in Δlog(τ/K_A) (see Table 3). The Δlog(τ/K_A) of cAMP inhibition (x-axis) is plotted against that of β-arrestin2 recruitment (y-axis). Thus, DAMGO is located at (0,0) on this plot. The dotted lines enclose the area with |log(bias factor)| <0.7 in which the opioids with balanced profiles are located (see text for the rationale). Among the tested opioids, valerylfentanyl and furanylfentanyl showed obvious β-arrestin and G protein bias, respectively.