Inhibition of Cytochrome P450 2D6:  Structure−Activity Studies Using a Series of Quinidine and Quinine Analogues

Several pharmacophore models have suggested that substrates and inhibitors of cytochrome P450 2D6 (P450 2D6) possess a nitrogen with a positive charge that participates in a charge−pair interaction with the aspartate 301 residue. In an effort to investigate this paradigm for P450 2D6 binding, an analogue series of the stereoisomers quinidine and quinine were synthesized and screened for binding affinity as measured by inhibition. Results revealed that bulky substituents added to the quinuclidine nitrogen (quaternary salts) did not affect the inhibitory potency of quinidine (IC50 = 0.02 μM), suggesting minimal contribution to binding affinity of this inhibitor by the purported ionic-binding interaction. Meanwhile, substantial decreases in inhibitory potency were observed for the N-methyl, N-ethyl, and N-benzyl quininium salts, suggesting that the quaternary nitrogen of this antipode interacts with a distinct region of the P450 2D6 active site as compared to the corresponding nitrogen of quinidine. Interestingly, esterification of quinidine resulted in a substantial loss of inhibitory potency, likely due to disruption of a hydrogen-bonding interaction of the hydroxyl group. This suggests that hydrogen bonding contributes more to the tight binding of quinidine than does the charge−pair interaction of the positively charged nitrogen. Moreover, benzoyl ester formation of quinine caused the binding orientation to switch from type II to type I, with concomitant restoration of P450 2D6 inhibitory potency. Thus, it appears that both hydrogen bonding and the ionic interaction of the basic nitrogen of quinine contribute to inhibitory potency, while the hydroxyl group also apparently contributes to directing type II binding. Overall, results suggest that when analyzing a series of compounds that include stereoisomers for development of predictive pharmacophore/protein models describing P450 2D6 binding, it may be inappropriate to assume that the ionic interaction of the basic nitrogen with aspartate 301 represents the primary binding interaction.