Structural insight into the mechanism of 4-aminoquinolines selectivity for the alpha2A-adrenoceptor

α2A-adrenoceptor (AR) is a potential target for the treatment of degenerative diseases of the central nervous system, and α2A-AR agonists are one of the most effective drugs for this condition. However, the lack of high selectivity for α2A-AR subtype of traditional drugs greatly limits their clinic usage. In this study, a series of homobivalent 4-aminoquinolines conjugated by two 4-aminoquinoline moieties via varying alkane linker length (C2-C12) were characterized for their affinities for each α2-AR subtype. Subsequently, docking, molecular dynamics and mutagenesis were applied to uncover the molecular mechanism. Most 4-aminoquinolines (4-aminoquinoline monomer, C2-C6, C8-C10) were selective for the α2A-AR over α2B- and α2C-ARs. Besides, the affinities are of similar linker length-dependence for each α2-AR subtype. Among all the compounds tested, C10 has the highest affinity for the α2A-AR (pKi=-7.45±0.62), which is 12-fold and 60-fold selective over α2B-AR and α2C-AR, respectively. Docking and molecular dynamics studies suggest that C10 simultaneously interacts with an orthosteric and an “allosteric site” of the α2A-AR. The mutation of F205, which is situated at the orthosteric binding pocket decreases the affinity by 2-fold. The potential allosteric residues include Ser90, Asn93, Glu94 and W99.The specificity of C10 for the α2A-AR and the potential orthosteric and allosteric binding sites proposed in this study provide valuable guidance for the development of novel α2A-AR subtype selective compounds.