Serotonin 2A Receptor (5-HT2AR) Activation by 25H-NBOMe Positional Isomers: In Vitro Functional Evaluation and Molecular Docking

Abstract

Serotonergic psychedelics are defined as compounds having serotonin 2A receptor (5-HT_2AR) activation as an important pharmacological mechanism. These compounds include the phenylalkylamine class, containing substances with e.g. 2C-X structures (phenethylamines) or their N-methoxybenzyl analogues (NBOMes). Besides their abuse potential, psychedelics are increasingly recognized for having therapeutic benefits. However, many psychedelics remain incompletely characterized, even concerning their structure-activity relationships. Here, five positional isomers of 25H-NBOMe, with two methoxy groups on the different positions of the phenyl ring of the phenethylamine moiety, were subjected to split-nanoluciferase assays assessing the in vitro recruitment of cytosolic proteins to the 5-HT_2AR. Furthermore, molecular docking at the 5-HT_2AR allowed estimation of which residues interact with the specific isomers' methoxy groups. Although the optimal substitution pattern of N-unsubstituted phenylalkylamines has been extensively studied, this is the first comparative evaluation of the functional effects of the positioning of the methoxy groups in the phenethylamine moiety of NBOMes.

Figure 1

Structures of the substances tested in the 5-HT_2AR bioassays: the reference substances LSD and serotonin and the “conventionally substituted” 25H-NBOMe, together with the five tested positional isomers. The numbers in the name of the positional isomer correspond to the methoxy group positions in the phenethylamine fragment, e.g. 25H-NBOMe has one methoxy group at position 2 and one methoxy group at position 5.

Figure 2

Concentration–response curves obtained by stimulation of the 5-HT_2AR followed by recruitment of (A and C) βarr2 or (B and D) miniGα_q constructs in the NanoBiT system. Data points are given as the mean of three independent experiments (each performed in duplicate) ± SEM (standard error of the mean). The AUC is normalized in each independent experiment for the maximum response (100%) of the reference agonist LSD (A and B) or serotonin (C and D).

Figure 3

Visual representations of the NBOMe isomers docked into the binding pocket of the 5-HT_2AR (based on PDB: 6WHA). (A) 24H-NBOMe, as seen from the perspective of the N-benzyl moiety; (B) 24H-NBOMe and (C) 35H-NBOMe bound to the 5-HT_2AR looking from the perspective of the phenyl group, with specific mentioning of the residues proposed to interact with the methoxy groups on the phenethylamine moiety.