METHODS: The three variants expressed by bacteria were investigated using substrate (omeprazole and 3- cyano-7-ethoxycoumarin[CEC]) and inhibitor (ketoconazole, fluoxetine, sertraline and loratadine) probes in enzyme assays along with molecular docking.
RESULTS: All alleles exhibited very low enzyme activity and affinity towards omeprazole and CEC (6.1% or less in intrinsic clearance). The inhibition studies with the four inhibitors, however, suggested that mutations in different variants have a tendency to cause enhanced binding (reduced IC50 values). The enhanced binding could partially be explained by the lower polar solvent accessible surface area of the inhibitors relative to the substrates. Molecular docking indicated that G91R, R335Q and F448L, the unique mutations in the alleles, have caused slight alteration in the substrate access channel morphology and a more compact active site cavity hence affecting ligand access and binding. It is likely that these structural alterations in CYP2C19 proteins have caused ligand-specific alteration in catalytic and inhibitory specificities as observed in the in vitro assays.
CONCLUSION: This study indicates that CYP2C19 variant selectivity for ligands was not solely governed by mutation-induced modifications in the active site architecture, but the intrinsic properties of the probe compounds also played a vital role.
METHODS: CYP proteins expressed in Escherichia coli were studied using the substrate 3-cyano-7- ethoxycoumarin (CEC) and inhibitor probes (quinidine, fluoxetine, paroxetine, terbinafine) in the enzyme assay. Computer modelling was additionally used to create three-dimensional structures of the CYP2D6*14 variants.
RESULTS: Kinetics data indicated significantly reduced intrinsic clearance in CYP2D6*14 variants, suggesting that P34S, G169R, R296C, and S486T substitutions worked cooperatively to alter the conformation of the active site that negatively impacted the deethylase activity of CYP2D6. For the inhibition studies, IC50 values decreased in quinidine, paroxetine, and terbinafine but increased in fluoxetine, suggesting a varied ligand-specific susceptibility to inhibition. Molecular docking further demonstrated the role of P34S and R296C in altering access channel dimensions, thereby affecting ligand access and binding and subsequently resulting in varied inhibition potencies.
CONCLUSION: In summary, the differential selectivity of CYP2D6*14 variants for the ligands (substrate and inhibitor) was governed by the alteration of the active site and access channel architecture induced by the natural mutations found in the alleles.