Bioassays to study biased signalling of novel synthetic opioids

Objective For hundreds of years, opioids have been used in the management of pain, mediating their analgesic effect via binding to the Mu opioid receptor (MOR). Fentanyl and morphine are examples of MOR agonists whose analgesic effects are associated with unwanted side effects such as tolerance and dependence. While analgesia is mainly realized through the G protein-signalling pathway, the undesirable effects of opioids have been linked to the β-arrestin pathway. Little is known about a potential “bias” of the synthetic opioids – including fentanyl analogs – that have emerged on the illegal drug market. To develop a novel, robust platform to study the activity of synthetic opioids through both the G protein and β-arrestin pathways. Methods We developed bioassays that are based on functional complementation of two split fragments of Nanoluciferase (LgBiT and SmBiT), either fused to the receptor or to the cytosolic proteins: mini G protein and β-arrestin. These assays were performed in 96-well plates using transiently transfected HEK293 T cells, using pure reference standards. Results Assays were set up in which MOR, fused to LgBiT, was combined with cytosolic proteins, fused to SmBiT. Both the mini G protein and the β-arrestin bioassays demonstrated concentration-dependent responses at MOR, using hydromorphone, DAMGO, fentanyl and carfentanil, with activation as low as 10−13M still being detectable for carfentanil. A wide range of fentanyl analogs is currently being tested–the results of which will be discussed. Discussion The present study provides an in vitro platform to study functional activation of Mu opioid receptor by NPS by means of their capacity to couple to G protein and recruit β-arrestin. Conclusion The developed bioassays not only allow insight into the signalling of synthetic opioids, which may help to better understand why certain opioids are associated with higher toxicity but may also be used to screen biofluids for opioid activity.