Laboratory of Toxicology, Department of Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent University, Campus Heymans, Ottergemsesteenweg 460, 9000, Ghent, Belgium.
Arch Toxicol. 2020 Oct;94(10):3449-3460. doi: 10.1007/s00204-020-02836-w. Epub 2020 Jul 5.
Serotonergic psychedelics, substances exerting their effects primarily through the serotonin 2A receptor (5-HTR), 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-HTR, 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.
血清素能迷幻剂，主要通过 5-羟色胺 2A 受体（5-HTR）发挥作用的物质，继续构成报告的新精神活性物质（NPS）的很大一部分。迷幻剂的确切作用机制仍有待进一步阐明，并且对于该类化合物，某些途径在分子水平上仍在很大程度上未被探索。缺乏对属于不同子类别的物质进行系统比较，监测受体近端β-arrestin 2 募集的情况。基于先前报道的使用分裂纳米荧光素酶的体外生物测定来监测 5-HTR 中β-arrestin 2 的募集情况，我们在此报告了一种稳定的 HEK 293 T 细胞基于生物测定的设置。在验证了与基于瞬时转染的系统相比，该新稳定细胞系统的性能之后，认为稳定表达系统适合迷幻 NPS 的药理学特征。随后，将其应用于一组 30 种物质的体外评估，这些物质代表了苯乙胺迷幻剂的不同子类，其中 12 种苯乙胺衍生物（2C-X），7 种苯异丙胺（DOx）和 11 N-苄基衍生物（25X-NB）。得到的效力和功效值提供了对测试化合物的结构活性关系的深入了解，总体上证实了其他报道的体外测定中观察到的发现，甚至与估计的常见剂量显示出显著相关性。这种方法，其中使用相同的测定设置，对属于不同子类别的大量迷幻 NPS 进行比较测试，监测受体近端事件，不仅提供了药理学见解，而且还可以对与最有效（并且可能是危险的）化合物相关的法律行动进行优先级排序。
