Setup of a Serotonin 2A Receptor (5-HT2AR) Bioassay: Demonstration of Its Applicability To Functionally Characterize Hallucinogenic New Psychoactive Substances and an Explanation Why 5-HT2AR Bioassays Are Not Suited for Universal Activity-Based Screening of Biofluids for New Psychoactive Substances

Classic hallucinogens, which activate the serotonin 2A receptor (5-HT2AR), represent the third largest category of new psychoactive substances. A new bioassay was developed that measures receptor activation by monitoring β-arrestin2 recruitment using a split-luciferase system. The assay determined potency and efficacy for various hallucinogens, including LSD, 5-MeO-DALT, mescaline, and several 2C compounds and their NBOMe derivatives, with EC50 values ranging from subnanomolar (NBOMes) to micromolar (mescaline) levels. When applied to plasma screening, blank samples showed pronounced receptor activation due to endogenous serotonin, confirmed by its elimination with a 5-HT2AR antagonist or MAO-A treatment, and by LC-HRMS analysis. The bioassay's main application is characterizing poorly understood serotonergic hallucinogens, as MAO-A metabolism of some compounds could bias detection in biofluids.

Study at a glance

Abstract

Classic or serotonergic hallucinogens comprise the third largest number of reported new psychoactive substances (NPS), according to the United Nations Office on Drugs and Crime. While being structurally very divergent, they share activation of the serotonin 2A receptor (5-HT2AR), a G protein-coupled receptor, as their main pharmacological mechanism. Here, we report on the development of a 5-HT2AR bioassay, which monitors β-arrestin2 recruitment to the receptor via a split-luciferase system, as a measure of receptor activation. Possible applications of the bioassay would lie in the characterization of serotonergic hallucinogens and the screening of these compounds in biofluids based on their serotonergic activity, rather than on their specific structures. The developed bioassay allowed the determination of the potency and efficacy of representatives of different classes of hallucinogens (LSD, 5-MeO-DALT, mescaline) and of a selected group of 2C hallucinogens and their corresponding NBOMes, with EC50 values from the subnanomolar (NBOMes) to micromolar (mescaline) range. When implementing the bioassay for the screening of plasma, a pronounced receptor activation was already observed in blank samples, which could be ascribed to endogenous serotonin, as suggested by annihilation of this activity by a 5-HT2AR antagonist or after incubation with MAO-A (monoamine oxidase-A). The presence and degradability by MAO-A of serotonin in plasma extracts were confirmed by LC-HRMS. Due to the possible metabolism of certain hallucinogens by MAO-A, which would cause a bias in the detectability of compounds in biofluids, the main application potential of this bioassay lies in the characterization of these scarcely characterized serotonergic hallucinogens.