In vitro assays for the functional characterization of (psychedelic) substances at the serotonin receptor 5‐HT_2AR

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.

Structure–Activity Relationships for Psilocybin, Baeocystin, Aeruginascin, and Related Analogues to Produce Pharmacological Effects in Mice

“…We found that psilocin and norpsilocin were both potent agonists in the calcium mobilization assay, and our results generally agree with those of Sherwood and colleagues . Although many assays can be used to study the functional potency and efficacy of psychedelics at 5-HT 2A , it is unclear which signaling pathway(s) are predictive for therapeutic or other effects. The usefulness of in vitro functional potencies may be limited as these values do not always relate to binding affinities at 5-HT 2A ,− or in vivo potencies in rodent behavioral models .…”

“…5-HT 2A and other 5-HT receptors couple to many canonical and noncanonical intracellular signaling pathways, depending on biological context, which could mediate distinct pharmacological effects of psychedelics. , In cells transfected with rat 5-HT 2A , drug potencies in the 5-HT 2A calcium mobilization assay associated with G αq signaling have been shown to predict potency for phenethylamine psychedelic-induced HTR in mice . However, a previous mouse study showed that genetic deletion of G αq only partially reduces drug-induced HTR, while another mouse study pointed to a possible role for G αs in producing psychedelic-like drug effects .…”

“…Whether 5-HT 2A -mediated β-arrestin recruitment underlies the psychedelic effects of tryptamine or LSD-like compounds warrants further investigation. Some data indicate that LSD and related psychedelics exhibit preferential β-arrestin 2 bias at 5-HT 2A receptors, , but other investigators found no evidence for such bias when directly comparing the effects of compounds in assays measuring 5-HT 2A -mediated β-arrestin 2 versus mini-G αq recruitment. , Unraveling which in vitro assay(s) and receptor signaling pathway(s) are predictive for pharmacological and potential clinical effects of psychedelics will be crucial for future drug development efforts.…”

“…β-Arrestin 2 recruitment at the human 5-HT 2A was assessed as previously described using Human Embryonic Kidney (HEK) 293T cells stably expressing the SmBiT-βarr2 and 5-HT 2A -LgBiT constructs. , Briefly, the dilutions for functional activity assessments were made in Hank’s buffered saline solution (HBSS) by means of serial dilution, while keeping the solvent concentration constant, yielding in-well concentration ranges from 10 μM to 10 –11 M. One day prior to the experiment, cultured cells stably expressing 5-HT 2A -LgBiT and SmBiT-β-arrestin 2 (LgBiT and SmBiT being the enzyme fragments of the NanoBiT system, initially developed by Promega) were seeded at a density of 50,000 cells per well in 96-well plates coated with poly-D-lysine for optimal cell adhesion. Plates were incubated at 37 °C and 5% CO 2 until the readout on day 2.…”

Discovery of β-Arrestin-Biased 25CN-NBOH-Derived 5-HT2AReceptor Agonists

“…The 5-HT 2A R is able to signal through members of both Gα q and Gα i/o protein families and also through beta-arrestin mediated pathways. , Several psychedelics and 5-HT 2A R agonists have been evaluated for their respective bias profiles toward the Gα q and βarr2 transducers, by means of highly analogous functional complementation assays. − Recently, the first partial agonist ( E max = 13%) with bias toward the βarr2 over Gα q‑γ9 pathway, compared to the reference 5-HT, has been disclosed (IHCH-7086) . However, no strongly biased agonist for the G-protein-mediated signaling pathway has been identified.…”