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

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Abstract

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 EC50 (pEC50, as a measure of potency) and maximal response provoked by a ligand (Emax, 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 EC50 but distinct Emax values were obtained with both bio-assays, with lower Emax values for the Nb-based bio-assay. Both bio-assays may assist to gain better insight into activation of the MOR.

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Funding

This study was funded by the Bijzonder Onderzoeksfonds (BOF) from Ghent University via the PhD fellowship of L. Vasudevan (BOF.DCV.20—BOF15/DOS/021) and via project grant no. 01J15517 (for funding of C. Stove). C. Stove also received support from the Fund for Scientific Research-Flanders (G.0217.15N and G.0694.19N).

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Authors and Affiliations

  1. Laboratory of Toxicology, Department of Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent University, 9000, Ghent, Belgium

    Lakshmi Vasudevan & Christophe P. Stove

Authors
  1. Lakshmi Vasudevan
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  2. Christophe P. Stove
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Contributions

L. Vasudevan and C. Stove designed the experiments. L. Vasudevan analyzed and prepared the manuscript. C. Stove provided guidance, supervised the project, and reviewed and edited the manuscript.

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Correspondence to Christophe P. Stove.

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Vasudevan, L., Stove, C.P. A novel nanobody-based bio-assay using functional complementation of a split nanoluciferase to monitor Mu- opioid receptor activation. Anal Bioanal Chem 412, 8015–8022 (2020). https://doi.org/10.1007/s00216-020-02945-6

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  • DOI: https://doi.org/10.1007/s00216-020-02945-6

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