title: In Vitro Functional Characterisation of Cytochrome P450 (CYP) 2C19 Allelic Variants CYP2C19*23 and CYP2C19*24.
abstract: Cytochrome P450 (CYP) 2C19 is essential for the metabolism of clinically used drugs including omeprazole, proguanil, and S-mephenytoin. This hepatic enzyme exhibits genetic polymorphism with inter-individual variability in catalytic activity. This study aimed to characterise the functional consequences of CYP2C19*23 (271 G>C, 991 A>G) and CYP2C19*24 (991 A>G, 1004 G>A) in vitro. Mutations in CYP2C19 cDNA were introduced by site-directed mutagenesis, and the CYP2C19 wild type (WT) as well as variants proteins were subsequently expressed using Escherichia coli cells. Catalytic activities of CYP2C19 WT and those of variants were determined by high performance liquid chromatography-based essay employing S-mephenytoin and omeprazole as probe substrates. Results showed that the level of S-mephenytoin 4'-hydroxylation activity of CYP2C19*23 (Vmax111.5 ± 16.0 pmol/min/mg, Km158.3 ± 88.0 μM) protein relative to CYP2C19 WT (Vmax101.6 + 12.4 pmol/min/mg, Km123.0 ± 19.2 μM) protein had no significant difference. In contrast, the Kmof CYP2C19*24 (270.1 ± 57.2 μM) increased significantly as compared to CYP2C19 WT (123.0 ± 19.2 μM) and Vmaxof CYP2C19*24 (23.6 ± 2.6 pmol/min/mg) protein was significantly lower than that of the WT protein (101.6 ± 12.4 pmol/min/mg). In vitro intrinsic clearance (CLint= Vmax/Km)for CYP2C19*23 protein was 85.4 % of that of CYP2C19 WT protein. The corresponding CLintvalue for CYP2C19*24 protein reduced to 11.0 % of that of WT protein. These findings suggested that catalytic activity of CYP2C19 was not affected by the corresponding amino acid substitutions in CYP2C19*23 protein; and the reverse was true for CYP2C19*24 protein. When omeprazole was employed as the substrate, Kmof CYP2C19*23 (1911 ± 244.73 μM) was at least 100 times higher than that of CYP2C19 WT (18.37 ± 1.64 μM) and Vmaxof CYP2C19*23 (3.87 ± 0.74 pmol/min/mg) dropped to 13.4 % of the CYP2C19 WT (28.84 ± 0.61 pmol/min/mg) level. Derived from Vmax/Km, the CLintvalue of CYP2C19 WT was 785 folds of CYP2C19*23. Kmand Vmaxvalues could not be determined for CYP2C19*24 due to its low catalytic activity towards omeprazole 5'-hydroxylation. Therefore, both CYP2C19*23 and CYP2C19*24 showed marked reduced activities of metabolising omeprazole to 5-hydroxyomeprazole. Hence, carriers of CYP2C19*23 and CYP2C19*24 allele are potentially poor metabolisers of CYP2C19-mediated substrates.
authors: School of Medical Sciences, International Medical University, No.126, Jalan Jalil Perkasa 19, 57000, Bukit Jalil, Kuala Lumpur, Malaysia.
authors: Pui Shen
authors: PS
authors: Lau
authors: School of Medical Sciences, International Medical University, No.126, Jalan Jalil Perkasa 19, 57000, Bukit Jalil, Kuala Lumpur, Malaysia.
authors: Kenny Voon Gah
authors: KV
authors: Leong
authors: Jeffery Cheah School of Medicine and Health Sciences, Monash University Sunway Campus Malaysia, Jalan Lagoon Selantan, 46150, Bandar Sunway, Selangor, Malaysia.
authors: Chin Eng
authors: CE
authors: Ong
authors: Jeffery Cheah School of Medicine and Health Sciences, Monash University Sunway Campus Malaysia, Jalan Lagoon Selantan, 46150, Bandar Sunway, Selangor, Malaysia.
authors: Amelia Nathania Hui Min
authors: AN
authors: Dong
authors: Department of Biomedical Science, The University of Nottingham Malaysia Campus, Jalan Broga, 43500, Semenyih, Selangor, Malaysia. panyan1980@hotmail.com.
authors: Yan
authors: Y
authors: Pan
mesh_terms: D000483:Alleles; D001681:Biological Assay; D065731:Cytochrome P-450 CYP2C19 / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D014644:Genetic Variation; D006801:Humans; D015151:Immunoblotting; D016297:Mutagenesis, Site-Directed; D009853:Omeprazole / Q000378:metabolism; D011110:Polymorphism, Genetic
chemical_list: D065731:Cytochrome P-450 CYP2C19; D009853:Omeprazole
keywords: CYP2C19; Functional characterisation; HPLC; Polymorphism