In Silico Investigation of Fabomotizole Binding to the Active-State Human Cannabinoid CB2 Receptor: Structural Insights into a Potential Off-Target Mechanism

Abstract

Background: Fabomotizole (Afobazole) is a widely utilized non-benzodiazepine anxiolytic and neuroprotective agent whose canonical mechanism is linked to sigma-1 (σ1) receptor agonism and monoaminergic modulation. However, its complete polypharmacological off-target interactome remains poorly explored, particularly regarding neuroinflammatory pathways. Methods: We performed molecular docking simulations of fabomotizole against the high-resolution cryo-EM structure of the human cannabinoid receptor 2 in its active signaling state (CB2, PDB ID: 6KPC) using AutoDock Vina with high search exhaustiveness (exhaustiveness = 30). Results: Fabomotizole exhibited a favorable binding free energy of ΔG = -7.850 kcal/mol within the orthosteric binding cavity. Structural analysis revealed that the ligand is anchored via a direct hydrogen bond between the morpholine nitrogen atom and the side-chain hydroxyl of Thr114 (d = 3.0 Å, TM3). The core benzimidazole scaffold is stabilized by parallel-displaced π–π stacking interactions with Phe183 (ECL2) and Phe91 (TM2), supplemented by steric contacts with surrounding hydrophobic residues. Conclusions: These computational results provide structural evidence for a potential off-target interaction between fabomotizole and human CB2 receptors. This interaction may contribute to the observed immunomodulatory and stress-protective profile of the drug.