In Vitro Functional Characterisation of Cytochrome P450 (CYP) 2C19 Allelic Variants CYP2C19*23 and CYP2C19*24

详细信息    查看全文

• 作者：Pui Shen Lau ; Kenny Voon Gah Leong ; Chin Eng Ong…
• 关键词：CYP2C19 ; Polymorphism ; HPLC ; Functional characterisation
• 刊名：Biochemical Genetics
• 出版年：2017
• 出版时间：February 2017
• 年：2017
• 卷：55
• 期：1
• 页码：48-62
• 全文大小：
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Human Genetics; Biochemistry, general; Zoology; Medical Microbiology;
• 出版者：Springer US
• ISSN：1573-4927
• 卷排序：55

文摘

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 (V_max 111.5 ± 16.0 pmol/min/mg, K_m 158.3 ± 88.0 μM) protein relative to CYP2C19 WT (V_max 101.6 + 12.4 pmol/min/mg, K_m 123.0 ± 19.2 μM) protein had no significant difference. In contrast, the K_m of CYP2C19*24 (270.1 ± 57.2 μM) increased significantly as compared to CYP2C19 WT (123.0 ± 19.2 μM) and V_max of 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 (CL_int = V_max/K_m) for CYP2C19*23 protein was 85.4 % of that of CYP2C19 WT protein. The corresponding CL_int value 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, K_m of CYP2C19*23 (1911 ± 244.73 μM) was at least 100 times higher than that of CYP2C19 WT (18.37 ± 1.64 μM) and V_max of 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 V_max/K_m, the CL_int value of CYP2C19 WT was 785 folds of CYP2C19*23. K_m and V_max values 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.