The psychedelic 25C-NBF displays 5-HT2A receptor selectivity and enables in silico prediction of ligand selectivity

In recent years, psychedelics have emerged as promising candidates for the treatment of various psychiatric ailments, such as post-traumatic stress disorder (PTSD), major depressive disorder or end-of-life anxiety. Drugs such as MDMA and psilocybin are in the late stages of the drug development process. Nevertheless, there is still room for discovery of new molecular entities which present an improved safety profile.

Among the drugs being studied by our research group, N-(2-fluorobenzyl)-4-chloro-2,5-dimethoxyphenethylamine (25C-NBF) stands out as a psychoplastogen with antidepressant properties. Moreover, it shows selectivity towards the 5-HT_2A receptor, especially when compared to the 5-HT_2B receptor, which is associated with valvular heart disease upon sustained activation. In the case of 25C-NBF the 2A/2B selectivity ratio is 180.2, whereas the 2A/2C selectivity ratio is 59.48 [1].

In order to understand this selectivity, the binding mechanism of compound 25C-NBF against the three 5-HT₂ receptors was predicted by molecular docking. Afterwards, molecular dynamics simulations were carried out to assess the stability of the predicted interaction mechanism over time. Finally, the binding free energy of 25C-NBF was calculated by Umbrella Sampling, for the three receptors, by studying the energy profile of the molecule leaving stepwise the receptor.

While the docking studies did not reveal any major differences that could explain the selectivity of 25C-NBF, the molecular dynamics simulations already showed some differences. When analyzing the RMSD values, the lowest corresponded to the 5-HT_2A receptor (RMSD = 1.2 Å), followed by the 5-HT_2B receptor (RMSD = 1.4 Å), and finally the 5-HT_2C receptor (RMSD = 2.0 Å). Given that these results did not match the experimental findings, the Umbrella Sampling methodology was applied.

In order to make use of this methodology, an adequate amino acid from each receptor must be selected as a reference. At first, Ser159 was chosen for the 5-HT_2A receptor (Ser139 for 5-HT_2B and Ser138 for 5-HT_2C). Nevertheless, the energy profile revealed that choosing this amino acid implied that the 25C-NBF molecule had to leave between two intermembrane loops in the case of the 5-HT_2C receptor. This led to a colossal energy difference when compared to the other two receptors.

Thus, the reference amino acid was changed to Leu166 for the 5-HT_2A receptor (Leu146 for 5-HT_2B and Leu145 for 5-HT_2C), which yielded an energy profile which more accurately represented the experimental values.

Removing 25C-NBF from the 5-HT_2B receptor in a stepwise fashion requires the least amount of energy, whereas the most amount of energy is needed for the 5-HT_2A receptor. The energy necessary for the 5-HT_2C receptor is close to the one for 5-HT_2A.

In summary, employing the Umbrella Sampling methodology, we could develop a procedure which explains the selectivity of 25C-NBF towards the 5-HT_2A receptor. Moreover, we have employed this approach to predict the selectivity of novel 5-HT_2A receptor agonists.

References

1. Nadal-Gratacós, N., Puigseslloses, P., Guzmán, L., Weiss, N., Pottie, E., Riera-Colomer, C., Lardeux, V., Thiriet, N., Wang, F., Källsten, L., Pérez-Esteban, I., Ketsela, G., Margall, J., Berzosa, X., Pubill, D., Rodríguez-Arias, M., Ettcheto, M., Kehr, J., Stove, C., Solinas, M., Sitte, H. H., Escubedo, E., López-Arnau, R., 2026. The psychedelic phenethylamine 25C-NBF, a selective 5-HT2A agonist, shows psychoplastogenic properties and rapid antidepressant effects in male rodents. Mol. Psychiatry 31, 1909-1922.