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-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 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.

In vitro structure–activity relationships and forensic case series of emerging 2-benzylbenzimidazole ‘nitazene’ opioids

“…For some (isotonitazene (18), N-pyrrolidino isotonitazene (19), N-piperidinyl isotonitazene (20) and the evaluated N-desethyl analogues (6, 12, 17 and 22)), the efficacy even surpassed that of DAMGO, although in most instances the confidence intervals were still overlapping with that of DAMGO. This is in line with observations by others, some of whom have used the term MOR 'superagonism' to refer to MOR activation induced by several nitazenes (Malcolm et al 2023;Tsai et al 2024). While absolute potencies differed in both our assays, the inclusion of multiple comparator opioids in this study (A-C) allowed a comparative assessment of potencies (Vandeputte et al 2022c) that was highly similar between both assays.…”

“…N-Pyrrolidino metonitazene, N-pyrrolidino protonitazene, and N-desethyl etonitazene are among the most recent nitazenes identified in the U.S. (Krotulski et al 2023). Although great strides have been made in the evaluation of nitazene structure-activity relationships (SAR) (Ujváry et al 2021;Vandeputte et al 2021Vandeputte et al , 2022bVandeputte et al , e, 2023De Luca et al 2022;Kanamori et al 2023;Walton et al 2023;Malcolm et al 2023;Glatfelter et al 2023;Kozell et al 2024;Tsai et al 2024), larger side-by-side comparisons of the effect of different systematic modifications to the nitazene core structure on MOR activity are rather scarce. Specifically, our understanding of SAR for 'ring' substituted analogues (i.e., N-pyrrolidino and N-piperidinyl analogues) remains limited (Ujváry et al 2021;Vandeputte et al 2022b, e;Kozell et al 2024;Tsai et al 2024).…”

“…Although great strides have been made in the evaluation of nitazene structure-activity relationships (SAR) (Ujváry et al 2021;Vandeputte et al 2021Vandeputte et al , 2022bVandeputte et al , e, 2023De Luca et al 2022;Kanamori et al 2023;Walton et al 2023;Malcolm et al 2023;Glatfelter et al 2023;Kozell et al 2024;Tsai et al 2024), larger side-by-side comparisons of the effect of different systematic modifications to the nitazene core structure on MOR activity are rather scarce. Specifically, our understanding of SAR for 'ring' substituted analogues (i.e., N-pyrrolidino and N-piperidinyl analogues) remains limited (Ujváry et al 2021;Vandeputte et al 2022b, e;Kozell et al 2024;Tsai et al 2024). For example, similar to N-pyrrolidino etonitazene (Vandeputte et al 2022b), N-pyrrolidino metonitazene and N-pyrrolidino protonitazene were not included in the early CIBA studies, and can thus be considered truly 'new' synthetic opioids.…”

“…Importantly, while there are clear limitations to the translatability of in vitro studies to the in vivo situation (e.g., because bioavailability, blood-brain barrier penetration, and formation of active metabolites are not taken into account), prior studies have shown that in vitro functional assays at MOR (including the MOR-βarr2 assay employed here) can serve as a tool for predicting in vivo potency trends of NSOs (including nitazenes) in rodents (Vandeputte et al 2022b(Vandeputte et al , 2023Glatfelter et al 2023). Therefore, the proposed in vitro SAR trends, indicating a (very) high MOR activating potential of many of the investigated compounds, may be cautiously extrapolated to the in vivo setting (Gillis et al 2020;Tsai et al 2024). Related to the latter, as elaborated below, the inclusion of forensic case series (although typically confounded by the co-presence of other drugs, including opioids) allowed to corroborate the in vitro findings, by demonstrating that these substances are found at low concentrations in fatal intoxications.…”

“…While various nitazenes have been characterized both in vitro and in vivo for their opioid activity (Ujváry et al 2021;Vandeputte et al 2021Vandeputte et al , 2022bVandeputte et al , e, 2023De Luca et al 2022;Kanamori et al 2023;Walton et al 2023;Malcolm et al 2023;Glatfelter et al 2023;Kozell et al 2024;Tsai et al 2024), the continuing emergence of new analogues (e.g., N-pyrrolidino metonitazene and N-pyrrolidino protonitazene) for which no or only limited pharmacology data are available, stresses the need for up-to-date reporting and evaluation of this class of NSOs. Furthermore, systematic comparisons of the impact of different structural alterations to the nitazene core structure (Fig.…”