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

Psychedelic new psychoactive substances (NPS), compounds exerting their main pharmacological effects through the activation of the serotonin 2A receptor (5-HT2AR), continuously comprise a substantial portion of the reported NPS. However, these substances and their exact mechanism of action, differentiating them from non psychedelic 5-HT2AR agonists, require further characterization. One potentially relevant phenomenon is the occurrence of biased agonism, in which (a) certain signaling pathway(s) is preferentially activated over the other (s). To this end, a new bioassay was developed, monitoring the recruitment of an engineered miniG alpha(q) protein to the activated 5-HT2AR. The setup was designed to be analogous to that of a previously developed bioassay monitoring beta-arrestin 2 recruitment through the NanoBiT system, enabling estimation of the potential preference of a substance to trigger recruitment of one protein over the other. This approach yielded several statistically significantly biased agonists within the group of phenylalkylamine psychedelics, more specifically the N-benzyl substituted 25H analogues 25H-NBF, 25H-NBMD, 25H-NBOH and 25H-NBOMe. All four compounds show a statistically significant preference towards the recruitment of beta-arrestin 2 over miniG alpha(q), as compared to the reference psychedelic substance LSD. We identified markedly different responses for Bromo-DragonFLY in the two bioassays, suggesting biased agonism, though the calculated bias factor equalled out to approximately 0. This demonstrates that the accurate assessment of biased agonism requires both the consideration of the observed trends in addition to the numerical value of the bias factor. A second panel of structural (I-substituted) analogues of the former group of phenylalkylamines showed a similar trend in the ranking order of the bias factors, resulting in one additional compound (25I-NBF) being statistically significantly biased.