Pharmacogenetic study of Cytochrome P450 2D6 and 2C19 (CYP2D6 and CYP2C19): determining the functional roles of the recombinant variants using in vitro kinetic assays and in silico modelling
Creators: name: Min, Amelia Nathania Dong Hui
Publisher: name: Monash University
Year: 2017
Subjects: Pharmacology

description: Cytochrome P450 (CYP) is a superfamily of haemoproteins. Various isoforms of this enzyme superfamily have broad and overlapping substrate specificities which usually provide for a robust elimination of xenobiotics from human body. However, their extremely variable expressions and functions lead to unforeseen drug responses such as over-reaction, toxicity or lack of response in the treated patients. One major source of inter-individual and intra- individual variability in CYP activity is genetic polymorphism of the CYP genes. These polymorphisms, particularly affect CYP2D6 and CYP2C19 to a functionally relevant extent. Despite numerous work done in this area, there remains some gap in the knowledge of CYP2D6 and CYP2C19 genetic polymorphism and their functional and clinical impact on drug therapy. Therefore, the aims of this study were to elucidate the enzyme kinetic and molecular basis for the altered activity in CYP2D6 and CYP2C19 alleles using substrate and inhibitor probes that have not been investigated. Site-directed mutagenesis to generate mutant CYP2D6*2, CYP2D6*10, CYP2D6*17, CYP2C19*23, CYP2C19*24 and CYP2C19*25 cDNAs were carried out successfully. Enzymatically active CYP proteins were produced from successful heterologous co-expression of each of the CYPs and OxR in E. coli. Two HPLC-based (venlafaxine O-demethylase and omeprazole 5-hydroxylase assays for CYP2D6 and CYP2C19 respectively) and one common fluorescence-based in vitro assays (3-cyano-7-ethoxycoumarin deethylase assay) were established and validated. CYP2D6*2 allele was found to have higher enzymatic activity and enhanced ligand binding as compared to the wild type. CYP2D6*10 and *17, on the other hand, have exhibited decreased enzymatic activity and deleterious effect on ligand binding. When the inhibition by quinidine, fluoxetine, paroxetine and terbinafine was examined for different CYP2D6 variants, CYP2D6*10 and CYP2D6*17 were generally less sensitive to inhibition whereas CYP2D6*2 tended to show similar or higher inhibition susceptibility when compared to the wild type. For CYP2C19, only CYP2C19*23 enzyme activity was measurable in omeprazole 5-hydroxylase assay whereas both CYP2C19*24 and *25 have extremely low enzyme activity, making it impossible to determine their kinetic parameters. Data from this assay indicated that CYP2C19*23 exhibited much lower activity as compared to CYP2C19 wild type. The enzymatic data obtained from the CYP2D6 and CYP2C19 fluorescence-base assay complemented the results of both HPLC-based assays where reduced intrinsic clearance was noted in all allelic variants (with the exception of CYP2D6*2). Furthermore, the inhibition of CYP2C19 allelic variants, determined using ketoconazole, fluoxetine, sertraline and loratadine, demonstrated increased inhibition susceptibility when compared to the wild type. In silico molecular docking of inhibitors to each CYP2D6 and CYP2C19 alleles have generated evidences that generally supported their catalytic and inhibition potencies from in vitro kinetic study. These in silico data have provided insight into the structural permutations that have occurred as a result of the mutations. There were changes in the size and architecture of the substrate access channel and active site cavity that have resulted in differential binding interaction energy values in the various models examined. By and large, the multi-dimension approaches of this study have allowed comprehensive examination of the functional consequences of genetic mutations in CYP2D6 and CYP2C19 naturally occurring alleles in terms of inhibition potency, ligand specificity and molecular binding mechanism. Data derived from the present project have contributed to the understanding of the biochemistry and the structure-activity relationship of the two important CYP isoforms.