This in vitro (cells) study is the first to compare the functional effects of the positioning of the methoxy groups (methyl group bound to oxygen) in the phenethylamine part of NBOMers (a subgroup of psychedelics/psychedelic-like compounds).
Abstract
“Serotonergic psychedelics are defined as compounds having serotonin 2A receptor (5-HT2AR) 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-HT2AR. Furthermore, molecular docking at the 5-HT2AR 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.“
Authors: Olga V. Kupriyanova, Asher L. Brandt, Robert B. Laprairie, Vadim A. Shevyrin & Christophe P. Stove
Summary
Serotonin 2A Receptor (5-HT2AR) Activation by 25H-NBOMe Positional Isomers: In Vitro Functional Evaluation and Molecular Docking
Serotonergic psychedelics are substances that activate the serotonin 2A receptor (5-HTfined as substances that2AR) and have a broad structural variety, including traditional known substances and new psychoactive substances (NPS).
Psychedelic substances have complex mechanisms of action, which may result in severe side effects. They are also used for the treatment of addictions, mood and anxiety disorders, and distress concerning death.
Recently, structural data became available for the 5-HT2AR interacting with different ligands, hinting at differential binding modes for the prototypical psychedelic substance LSD and the N-benzyl substituted phenethylamine 25CN-NBOH.
In this study, we compared the potential of five isomers of 25H-NBOMe to induce similar effects to the “conventional” 25X-NBOMes at a molecular level. The potential of the isomers to induce recruitment of cytosolic proteins to the activated 5-HT2AR was assessed using previously established bioassays.
To assess the functionality of the NBOMe positional isomers, the Nanoluciferase Binary Technology (NanoBiT) was employed, which involves fusing two nonfunctional parts of the nanoluciferase to potentially interacting proteins. We established an arr2 recruitment assay and a miniGq recruitment assay, and calculated the potency and efficacy of the test compounds as compared to a reference agonist, using the area under the curve (AUC) of either the first 30 min or the full 2 h activation profile.
The diversely substituted isomers of 25H-NBOMe have a range of potencies ranging from 11.4 nM to 33.6 nM. The least potent compounds are 34H-NBOMe and 35H-NBOMe, both lacking the 2-methoxy substituent, which result in a markedly reduced in vitro potency. The efficacies of the compounds tested were narrower, with 25H-NBOMe having the highest efficacy (164%), although the efficacies of 24H-, 26H-, and 34H-NBOMe were higher than those of LSD and serotonin.
The potency of LSD as a reference psychedelic substance was 7.43 nM, and the potencies of the different isomers were ranked in order of efficacies, with 24H-, 25H-, 26H-, and 34H-NBOMe being the most efficacious substances and 23H- and 35H-NBOMe being the least efficacious substances.
A different but highly analogous bioassay was taken, monitoring the recruitment of miniGq to the 5-HT2AR in the NanoBiT system, as previously described. The results showed that serotonin is a more efficacious 5-HT2AR agonist than LSD, and that the efficacy of 35H NBOMe is also trending toward LSD.
Overall, this extensive set of experiments led us to conclude that 24H-NBOMe is the most potent positional isomer, followed by 26H- and 25H-NBOMe, with a lower potency for 23H-NBOMe and even lower potencies for 34H and 35H-NBOMe.
Little pharmacological information is available on the tested positional isomers, and the results obtained in different assays can severely impact the obtained numbers. However, the order of magnitude of the obtained potency values correspond with the literature.
The most effective substitution pattern of the phenyl group of the N-unsubstituted phenylalkylamine psychedelics is 2,4,5-substitution of the phenyl ring and 3,4,5-substitution. The substituent at position 4 can be modified, and the 2,4,6-substitution pattern may involve potentially active substances. In a recent study, 2- and 5-desmethoxy analogues of 2CB and DOB were individually tested in an in vitro Ca2+ release assay and in vivo through the head twitch response in mice. The 2-methoxy group had a more severe impact on the binding affinities and functional potencies at the 5-HT2AR and 5-HT2CR.
The N-methoxybenzyl group at the N-position of the phenethylamine results in the NBOMe group of substances, with higher affinities and potencies reported for 25H-NBOMe than for the unsubstituted counterpart 2C H.2,14,17,18 Literature indicates that the 4-substitution is not as essential for the receptor activation by NBOMes.
To explain the differences in potencies/efficacies observed for the isomers, molecular docking was performed with a model based on adaptations of the recently published cryo-EM structure of the 5-HT2ARin complex with 25CN-NBOH. The calculated interaction energies between the molecules and specific 5-HT2AR residues are shown in Tables 3 and 4, with the more negative the given energy, the stronger the proposed interaction would be. The model shows that all NBOMe isomers can be docked in the same binding pocket as 25CN-NBOH, despite the change of the hydroxyl group on the N-benzyl moiety into a methoxy group. The N-benzyl moiety is stabilized in the binding pocket by salt bridge-type interactions with D1553.32. The reference compound LSD interacts stronger with S2425.46 than NBOMe compounds due to an Hbond between the indole on LSD and S2425.46, in which NBOMes cannot participate.
The interaction energies between LSD and receptor residues S2425.46 and L229ECL2 are strong, consistent with observations by Kim et al.8. Some patterns do seem to emerge in the interaction energies between receptor residues and isomers, including interactions between substances carrying a methoxy group at a certain position and the concerned receptor residue. This approach allows to propose an explanation for certain trends in interaction energies, such as the hydrophobic interaction between a methoxy group at position 2 of the phenyl ring of the phenethylamine moiety and T1603.37, S1593.36, and V1563.33, and the weaker interaction of these residues with substances lacking a 3-methoxy group. Based on the proposed interactions, it is clear that there is no trivial or “single-residue” explanation for the observed lower potencies and efficacies of 23H-, 34H-, and 35H-NBOMe.
The exact mechanism inducing psychedelic effects in humans remains elusive. Contributing factors could involve biased agonism, receptor dimerization, and activation of receptors other than the 5-HT2AR.
NBOMes are prone to a high intrinsic clearance, and the 5-methoxy group has been defined as the metabolic soft spot of the molecule. Additionally, it remains elusive how the methoxy groups on different positions of the phenyl group of the phenethylamine moiety will impact the pharmacokinetic and pharmacodynamic properties of the molecules in vivo.
We report on the functional characterization of positional isomers of 25H-NBOMe and the impact of the structural changes on the ability of the activated 5-HT2AR to recruit cytosolic proteins. The results show that the differentially substituted isomers of NBOMe are more potent and more efficacious than their unsubstituted counterparts, and that the specific residues that interact with the specific methoxy groups on the phenyl moiety of the phenethylamine part of the molecule are responsible for this differential receptor activation potential.
Promega, Sigma-Aldrich, Fetal bovine serum, poly-D-lysine hydrobromide, methanol, and 25H-NBOMe were used to synthesize positional isomers of 25H-NBOMe.
Human Embryonic Kidney (HEK) 293T cells were routinely cultured in DMEM and transfected with the 5-HT2AR and the cytosolic protein (either arr2 or miniGq) in the NanoBiT system, following the manufacturer’s protocol. After 24 h, cells were seeded in poly-D-lysine coated 96-well plates at a density of 50 000 cells per well and incubated overnight. The cells were then rinsed twice with HBSS, and 25 l of Nano-Glo Live Cell Substrate were added to each of the wells.
The data were corrected for interwell variability, fit to a sigmoidal concentration-response curve, and pooled to determine the total EC50 and Emax values per substance.
The 5-HT2AR in complex with miniGq and the psychedelic substance 25CN-NBOH was molecularly docked using induced fit docking and minimized using the OPLS3 force field in Prime.
Notes
The authors declare no competing financial interests and thank the Research Foundation-Flanders (FWO), the Ghent University Special Research Fund (BOF), GlaxoSmithKline and the Canadian Institutes of Health Research (CIHR) for financial support.