A novel nanobody-based bio-assay using functional complementation of a split nanoluciferase to monitor Mu- opioid receptor activation

The Mu opioid receptor (MOR) has been the subject of intense research over the past decades, especially in the field of analgesic therapeutics. It is the primary target for both clinical and recreational opioids. Recently, camelid-derived nanobodies have received significant attention due to their applicability in stabilizing the crystal structure of activated MOR, via specific recognition of and binding to the active receptor conformation. In the present study, we developed and applied a novel bio-assay to monitor MOR activation, utilizing intracellular expression of one such nanobody, Nb39. The principle of functional complementation of a split nanoluciferase was used to assess recruitment of Nb39 to MOR, following activation by a set of five synthetic opioids. The obtained pharmacological parameters—negative logarithm of EC 50 (pEC 50 , as a measure of potency) and maximal response provoked by a ligand ( E max , as a measure of efficacy; relative to hydromorphone)—were compared with those obtained using a G protein recruitment assay, in which a mini-Gi protein (engineered GTPase domain of Gαi subunit) is recruited to activated MOR. Similar EC 50 but distinct E max values were obtained with both bio-assays, with lower E max values for the Nb-based bio-assay. Both bio-assays may assist to gain better insight into activation of the MOR. Graphical abstract