Why is quinidine an inhibitor of cytochrome P450 2D6? The role of key active site residues in quinidine binding

We have previously shown that residues Asp301, Glu216 and Phe120 in the active site of cytochrome P450 2D6 (CYP2D6) play a key role in substrate recognition by this important drug-metabolising enzyme. We have now examined the effect of mutations of these residues on interactions of the enzyme with the prototypical CYP2D6 inhibitor, quinidine. Abolition of the negative charge on either or both residues 216 and 301 decreased quinidine inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation by at least 100-fold. The apparent dissociation constants (Kd) for quinidine binding to wild type enzyme or to the Glu216Asp and Asp301Glu mutants were 0.25-0.50 μM. The amide substitutions of Glu216 or Asp301 resulted in 30 to 64-fold increases in Kd for quinidine. The double mutant Glu216Gln/Asp301Gln showed the largest decrease in quinidine affinity with a Kd of 65 μM. Changes in the mode of quinidine binding were indicated by changes in the optical difference spectra on binding. Alanine substitution of Phe120, Phe481 or Phe483 had only a minor effect on the inhibition of bufuralol 1'-hydroxylation and dextromethorphan O-demethylation, and on binding. In contrast to the wild-type enzyme, a number of the mutants studied were found to be able to metabolise quinidine. CYP2D6 Asp301Gln and Asp301Asn produced small amounts of 3-hydroxyquinidine, Asp301Ala and Asp301Phe produced O-demethylated quinidine, and Phe120Ala and Glu216Gln/Asp301Gln produced both these metabolites. Homology modelling and molecular docking were used to predict the modes of quinidine binding to wild type and mutant enzymes; these were able to rationalise the experimental observations.