Assessment of structure-activity relationships and biased agonism at the Mu opioid receptor of novel synthetic opioids using a novel, stable bio-assay platform

Abstract

Fentanyl and morphine are agonists of the Mu opioid receptor (MOR), which is a member of the GPCR family. Their analgesic effects are associated with unwanted side effects. On a signaling level downstream from MOR, it has been hypothesized that analgesia may be mediated through the G protein pathway, whereas the undesirable effects of opioids have been linked to the β-arrestin (βarr) pathway. Despite being an increasingly debated subject, little is known about a potential 'bias' (i.e. the preferential activation of one pathway over the other) of the novel synthetic opioids (NSO) -including fentanyl analogs-that have emerged on the illegal drug market. We have therefore developed and applied a novel, robust bio-assay platform to study the activity of 21 NSO, to evaluate to what extent these MOR agonists show biased agonism and to investigate the potential correlation with their structure. In addition, we evaluated the functional selectivity of TRV130, a purported G protein-biased agonist. We applied newly established stable bio-assays in HEK293T cells, based on the principle of functional complementation of a split nanoluciferase, to assess MOR activation via recruitment of a mini-Gi protein (GTPase domain of Gαi subunit) or βarr2. All but two of the tested NSO demonstrated a concentration-dependent response at MOR in both bio-assays. The developed bio-assays allow to gain insight into the βarr2 or G protein recruitment potential of NSO, which may eventually help to better understand why certain opioids are associated with higher toxicity. Adding to the recent discussion about the relevance of the biased agonism concept for opioids, we did not observe a significant bias for any of the evaluated compounds, including TRV130.

Activation of the μ‐opioid receptor by alicyclic fentanyls: Changes from high potency full agonists to low potency partial agonists with increasing alicyclic substructure

“…Cyclobutylfentanyl was a full agonist 3.1‐fold less potent than fentanyl in this study, while in the study by Hassanien et al 23 it was 5.0‐fold less potent with an efficacy of 69% compared with fentanyl. Similarly, in our study cyclopentylfentanyl was a full agonist 7.6‐fold less potent than fentanyl, while in other studies it was 2.8 to 19‐fold less potent with an efficacy of 46–80% compared with fentanyl 12,23 . Finally, TMCPF behaved as a partial agonist (65% efficacy) 880‐fold less potent than fentanyl in our study and Vasudevan et al 12 were unable to obtain a signal with either the mini‐Gi or the β‐arrestin assay.…”

“…Similarly, in our study cyclopentylfentanyl was a full agonist 7.6‐fold less potent than fentanyl, while in other studies it was 2.8 to 19‐fold less potent with an efficacy of 46–80% compared with fentanyl 12,23 . Finally, TMCPF behaved as a partial agonist (65% efficacy) 880‐fold less potent than fentanyl in our study and Vasudevan et al 12 were unable to obtain a signal with either the mini‐Gi or the β‐arrestin assay. In general our EC 50 ‐values compared with fentanyl are similar to those reported in previous studies, even though cyclopropylfentanyl appears somewhat less potent than previously reported.…”

“…In our study, cyclopropylfentanyl was a full agonist 2.5‐fold less potent than fentanyl. In other studies the EC 50 varied from 2.9‐fold more potent to 1.7‐fold less potent than fentanyl with efficacies between 84% and 107% compared with fentanyl 5,12,23,24 . Cyclobutylfentanyl was a full agonist 3.1‐fold less potent than fentanyl in this study, while in the study by Hassanien et al 23 it was 5.0‐fold less potent with an efficacy of 69% compared with fentanyl.…”

“…Receptor activation assays instead measure proximal or downstream activation of the receptor, allowing the potency and efficacy values for a certain agonist to be determined, making them more informative than binding assays. Receptor activation can be measured via different assays based on, for example, aequorin, 7 GTPγS binding, 8,9 cAMP, 10,11 and β‐arrestin recruitment 12 . The interpretation of the data is complicated by the fact that potency is substantially affected by the assay conditions such as the reagent concentration, which reporting system was used, the cell type, and if a human or murine receptor was used, making it difficult to compare the results from different assays.…”

“…This can be compared with an EC 50 for cyclopropylfentanyl of 11 nM reported by the Drug Enforcement Administration, 21,24 also from a GTPγS binding assay. Finally, to study biased agonism, Vasudevan et al 12 used two similar assays based on the recruitment of a G‐protein (mini‐G i ) or β‐arrestin to study cyclopropyl‐ and cyclopentylfentanyl, and TMCPF. For the mini‐G i assay they reported EC 50 values of 42 and 190 nM, with efficacies of 280% and 159% (compared with hydromorphone), for cyclopropyl‐ and cyclopentylfentanyl, respectively.…”