In vitro assays for the functional characterization of (psychedelic) substances at the serotonin receptor 5-HT2AR

Abstract

Serotonergic psychedelics are substances that induce alterations in mood, perception, and thought, and have the activation of serotonin (5-HT) 2A receptors (5-HT2ARs) as a main pharmacological mechanism. Besides their appearance on the (illicit) drug market, e.g. as new psychoactive substances, their potential therapeutic application is increasingly explored. This group of substances demonstrates a broad structural variety, leading to insufficiently described structure-activity relationships, hence illustrating the need for better functional characterization. This review therefore elaborates on the in vitro molecular techniques that have been used the most abundantly for the characterization of (psychedelic) 5-HT2AR agonists. More specifically, this review covers assays to monitor the canonical G protein signaling pathway (e.g. measuring G protein recruitment/activation, inositol phosphate accumulation, or Ca2+ mobilization), assays to monitor non-canonical G protein signaling (such as arachidonic acid release), assays to monitor β-arrestin recruitment or signaling, and assays to monitor receptor conformational changes. In particular, focus lies on the mechanism behind the techniques, and the specific advantages and challenges that are associated with these. Additionally, several variables are discussed that one should consider when attempting to compare functional outcomes from different studies, both linked to the specific assay mechanism and linked to its specific execution, as these may heavily impact the assay outcome. (Figure presented.).