Serotonergic psychedelics, substances exerting their effects primarily through the serotonin 2A receptor (5-HT2AR), continue to comprise a substantial portion of reported new psychoactive substances (NPS). The exact mechanisms of action of psychedelics still remain to be elucidated further, and certain pathways remain largely unexplored on a molecular level for this group of compounds. A systematic comparison of substances belonging to different subclasses, monitoring the receptor-proximal β-arrestin 2 recruitment, is lacking. Based on a previously reported in vitro bioassay employing functional complementation of a split nanoluciferase to monitor β-arrestin 2 recruitment to the 5-HT2AR, we here report on the setup of a stable HEK 293 T cell-based bioassay. Following verification of the performance of this new stable cell system as compared to a system based on transient transfection, the stable expression system was deemed suitable for the pharmacological characterization of psychedelic NPS. Subsequently, it was applied for the in vitro assessment of the structure–activity relationship of a set of 30 substances, representing different subclasses of phenylalkylamine psychedelics, among which 12 phenethylamine derivatives (2C-X), 7 phenylisopropylamines (DOx) and 11 N-benzylderivatives (25X-NB). The resulting potency and efficacy values provide insights into the structure–activity relationship of the tested compounds, overall confirm findings observed with other reported in vitro assays, and even show a significant correlation with estimated common doses. This approach, in which a large series of psychedelic NPS belonging to different subclasses is comparatively tested, using a same assay setup, monitoring a receptor-proximal event, not only gives pharmacological insights, but may also allow prioritization of legal actions related to the most potent -and potentially dangerous- compounds.
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Alexandra Smina is acknowledged for the help during the practical implementation of the experiments. Prof. Kristof Maudens is acknowledged for kindly providing the analytical standards of 2C-B, 2C-C, 2C-I, 25B-NBOMe, 25C-NBOMe and 25I-NBOMe. The authors want to thank Chiron for the generous gift of the analytical standard of Bromo-DragonFLY. C. Stove acknowledges funding by the Research Foundation-Flanders (FWO) [G069419N] and the Ghent University–Special Research Fund (BOF) [01J15517]. A. Cannaert acknowledges funding as a postdoctoral research fellow from the Research Foundation Flanders (FWO; 12Y9520N) and the Ghent University Special Research Fund (BOF; PDO026-18).
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Pottie, E., Cannaert, A. & Stove, C.P. In vitro structure–activity relationship determination of 30 psychedelic new psychoactive substances by means of β-arrestin 2 recruitment to the serotonin 2A receptor. Arch Toxicol 94, 3449–3460 (2020). https://doi.org/10.1007/s00204-020-02836-w