Abstract

Approved by the US Food and Drug Administration in 2006, varenicline was the first nicotinic-based therapy for smoking cessation, targeting the alpha 4D2 nicotinic acetylcholine receptor (nAChR). While inspired by cytisine, varenicline has distinct effects at both target and off-target receptors; however, despite being widely used clinically, the precise molecular interactions underpinning varenicline's mode of action remain unclear. Using a multidisciplinary approach, the interactions that set varenicline apart from related compounds such as nicotine and cytisine have been identified. In particular, the binding-site residues alpha 4T139, alpha 4T183, and especially D2S133 were shown to be key modulators for varenicline's function. Substituting D2S133 with valine significantly reduced efficacy, pinpointing it as a crucial determinant. Additionally, a set of novel varenicline variants showed that the positioning of the quinoxaline moiety in varenicline is essential for receptor activation. These insights reveal a unique interaction network at alpha 4D2 that underlies varenicline's function, offering a deeper understanding of the ligand's working mechanism.