OBJECTIVE: This in vitro study investigated the inhibitory effects of agarwood tea aqueous extract on the eight major human drug-metabolising cytochrome P450 (CYP) enzyme activities.
METHODS: High-throughput fluorescence-based Vivid® CYP450 screening kits were employed to obtain the enzyme activities before and after incubation with agarwood tea aqueous extract.
RESULTS: Agarwood aqueous extract potently inhibited CYP2C9, CYP2D6, and CYP3A4 activities with Ki values of 5.1, 34.5, and 20.3μg/ml, respectively. The most likely inhibition mode responsible for these inhibitions was non-competitive inhibition. On the other hand, at 1000μg/ml, agarwood tea aqueous extract negligibly inhibited CYP1A2, CYP2B6, CYP2C19, CYP2E1, and CYP3A5 activities.
CONCLUSION: These findings can be used to design additional in vitro investigations using clinical relevant drug substrates for CYP2C9, CYP2D6, and CYP3A4. Subsequently, future studies can be conducted to determine potential interactions between agarwood tea aqueous extract and CYP using in vivo models.
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.