In vitro structure–activity relationship determination of 30 psychedelic new psychoactive substances by means of β-arrestin 2 recruitment to the serotonin 2A receptor

Abstract

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 receptorproximal 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 set-up of a stable HEK 293T 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 set-up, monitoring a receptorproximal event, not only gives pharmacological insights, but may also allow prioritization of legal actions related to the most potent -and potentially dangerous-compounds.

Identification of psychedelic new psychoactive substances (NPS) showing biased agonism at the 5-HT2AR through simultaneous use of β-arrestin 2 and miniGαq bioassays

“…The obtained EC50 values were within the same range as those previously reported with βarr2 bioassays performed in either transiently transfected cells and cells stably expressing the bioassay components. [22,32] Therefore, LSD was selected as the reference agonist. In literature, LSD has been identified as both unbiased or biased, depending on which pathways were monitored in which receptor subtype.…”

“…Furthermore, this option employs the same receptor construct (5-HT2AR-LgBiT) as that employed in a previously developed βarr2 bioassay, [22] thereby eliminating an extra source of variability that would be introduced when using different constructs. In contrast with the successful setup of a HEK 293T system stably expressing 5-HT2AR-LgBiT with SmBiT-βarr2 [32], repeated attempts to generate a similar stable system for 5-HT2AR-LgBiT with SmBiT-miniGαq were unsuccessful, for unknown reasons. Hence, rather than using stable cell systems for the evaluation of biased agonism (as performed before for cannabinoid, opioid and adenosine receptors), we used transiently transfected cells here.…”

“…Assessing the potential occurrence of ligand bias for a given compound requires the measurement of both bioassays, and comparison of the results to a reference agonist to eliminate system bias and observational bias while also guaranteeing comparability of the experimental conditions. [19,21] LSD is a well-characterized psychedelic compound that was previously used as the reference agonist in experiments assessing the activity of compounds at the 5-HT2AR [22,32]. Figure 2C…”

Psychedelics in Psychiatry: Neuroplastic, Immunomodulatory, and Neurotransmitter Mechanisms

“…Moreover, LSD binding to the alternative (fully) edited 5-HT 2C receptor isoform results in a decreased ability to activate phosphoinositide hydrolysis signaling compared with 5-HT (Backstrom et al, 1999). Intriguingly, a recent article reported the development of a stable HEK 293 T-cell-based bioassay to monitor b-arrestin 2 recruitment to the 5-HT 2A receptor (Pottie et al, 2020). This in vitro bioassay system was used for the functional characterization of 30 phenylalkylamine psychedelics, including 12 phenethylamines (2C-X), 7 phenylisopropylamines (psychedelic amphetamines), and 11 N-benzyl derivatives, thus allowing the comprehensive assessment of the structure-activity relationship within and between the different subclasses.…”

“…This in vitro bioassay system was used for the functional characterization of 30 phenylalkylamine psychedelics, including 12 phenethylamines (2C-X), 7 phenylisopropylamines (psychedelic amphetamines), and 11 N-benzyl derivatives, thus allowing the comprehensive assessment of the structure-activity relationship within and between the different subclasses. For details, see Pottie et al (2020).…”