Dataset Information

Functional Characterization of Human CYP2C9 Allelic Variants in COS-7 Cells.

ABSTRACT: Variability in activity of CYP2C9, which is involved in the metabolism of approximately 15% of current therapeutic drugs, is an important contributor to interindividual differences in drug response. To evaluate the functional alternations of CYP2C9(*)2, CYP2C9(*)3, CYP2C9(*)8, CYP2C9(*)11 and CYP2C9(*)31, identified in our previous study in Chinese Han population, allelic variants as well as the wild-type CYP2C9 were transiently expressed in COS-7 cells. Kinetic parameters (Km, Vmax, and Clint) for S-warfarin 7-hydroxylation by these recombinant CYP2C9s were determined. Relative to CYP2C9.1, recombinant CYP2C9.3 and CYP2C9.11 exhibited significantly higher Km values, and all allelic variants showed significantly decreased Vmax and Clint values. Among all allelic variants, catalytic activity of CYP2C9.3 and CYP2C9.11 reduced the most (8.2% and 9.8% of Clint ratio, respectively; P < 0.001). These findings should be useful for predicting the phenotype profiles of CYP2C9 in Chinese Han population, comparing the functional results of these alleles accurately, and finally optimizing pharmacotherapy of drug treatment.

SUBMITTER: Du H

PROVIDER: S-EPMC4843492 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Functional Characterization of Human CYP2C9 Allelic Variants in COS-7 Cells.

Du Huihui H Wei Zhiyun Z Yan Yucai Y Xiong Yuyu Y Zhang Xiaoqing X Shen Lu L Ruan Yunfeng Y Wu Xi X Xu Qingqing Q He Lin L Qin Shengying S

Frontiers in pharmacology 20160425

Variability in activity of CYP2C9, which is involved in the metabolism of approximately 15% of current therapeutic drugs, is an important contributor to interindividual differences in drug response. To evaluate the functional alternations of CYP2C9(*)2, CYP2C9(*)3, CYP2C9(*)8, CYP2C9(*)11 and CYP2C9(*)31, identified in our previous study in Chinese Han population, allelic variants as well as the wild-type CYP2C9 were transiently expressed in COS-7 cells. Kinetic parameters (Km, Vmax, and Clint) ...[more]

PMID: 27199745

Similar Datasets

Project description:CYP2C9 is a major form of human liver cytochrome P450 that is responsible for the oxidative metabolism of several widely used low-therapeutic index drugs, including (S)-warfarin and phenytoin. In a cohort of Alaska Native people, ultrarare or novel CYP2C9 protein variants, M1L (rs114071557), N218I (rs780801862), and P279T (rs182132442, CYP2C9*29), are expressed with higher frequencies than the well characterized CYP2C9*2 and CYP2C9*3 alleles. We report here on their relative expression in lentivirus-infected HepG2 cells and the functional characterization of purified reconstituted enzyme variants expressed in Escherichia coli toward (S)-warfarin, phenytoin, flurbiprofen, and (S)-naproxen. In the infected HepG2 cells, robust mRNA and protein expression were obtained for wild-type, N218I, and P279T variants, but as expected, the M1L variant protein was not translated in this liver-derived cell line. His-tagged wild-type protein and the N218I and P279T variants, but not M1L, expressed well in E. coli and were highly purified after affinity chromatography. Upon reconstitution with cytochrome P450 oxidoreductase and cytochrome b5, the N218I and P279T protein variants metabolized (S)-warfarin, phenytoin, flurbiprofen, and (S)-naproxen to the expected monohydroxylated or O-demethylated metabolites. Steady-state kinetic analyses revealed that the relative catalytic efficiency ratios of (S)-warfarin metabolism by the P279T and N218I variants were 87% and 24%, respectively, of wild-type CYP2C9 protein. A similar rank ordering was observed for metabolism of phenytoin, flurbiprofen, and (S)-naproxen. We conclude that carriers of the variant N218I and, especially, the M1L alleles would be at risk of exacerbated therapeutic effects from drugs that rely on CYP2C9 for their metabolic clearance. SIGNIFICANCE STATEMENT: Novel gene variants of CYP2C9-M1L, and N218I, along with P279T (CYP2C9*29)-are expressed in Alaska Native people at relatively high frequencies. In vitro characterization of their functional effects revealed that each variant confers reduced catalytic efficiency toward several substrates, including the low-therapeutic index drugs (S)-warfarin and phenytoin. These data provide the first functional information for new, common CYP2C9 variants in this understudied population. The data may help guide dose adjustments in allele carriers, thus mitigating potential healthcare disparities.

Project description:BackgroundsCytochrome P450 (P450) 2E1 is one of the primary enzymes responsible for the metabolism of xenobiotics, such as drugs and environmental carcinogens. The genetic polymorphisms of the CYP2E1 gene in promoter and coding regions have been identified previously in the Han Chinese population from four different geographic areas of Mainland China.MethodsTo investigate whether genetic variants identified in the CYP2E1 coding region affect enzyme function, the enzymes of four single nucleotide polymorphism (SNP) variants in the coding region (novel c.1009C>T, causing p.Arg337X, where X represents the translational stop codon; c.227G>A, causing p.Arg76His; c.517G>A, yielding p.Gly173Ser; and c.1263C>T, presenting the highest allele frequency), two novel alleles (c.[227G>A;1263C>T] and c.[517G>A;1263C>T]), and the wild-type CYP2E1 were heterologously expressed in COS-7 cells and functionally characterized in terms of expression level and chlorzoxazone 6-hydroxylation activity. The impact of the CYP2E1 variant sequence on enzyme activity was predicted with three programs: Polyphen 2, PROVEAN and SIFT.ResultsThe prematurely terminated p.Arg337X variant enzyme was undetectable by western blotting and inactive toward chlorzoxazone 6-hydroxylation. The c.1263C>T and c.[517G>A;1263C>T] variant enzymes exhibited properties similar to those of the wild-type CYP2E1. The CYP2E1 variants c.227G>A and c.[227G>A;1263C>T] displayed significantly reduced enzyme activity relative to that of the wild-type enzyme (decreased by 42.8% and 32.8%, respectively; P < 0.01). The chlorzoxazone 6-hydroxylation activity of the c.517G>A transfectant was increased by 31% compared with the wild-type CYP2E1 enzyme (P < 0.01). Positive correlations were observed between the protein content and enzyme activity for CYP2E1 (P = 0.0005, r 2 = 0.8833). The characterization of enzyme function allelic variants in vitro was consistent with the potentially deleterious effect of the amino acid changes as determined by prediction tools.ConclusionsThese findings indicate that the genetic polymorphisms of CYP2E1, i.e., c.1009C>T (p.Arg337X), c.227G>A (p.Arg76His), and c.517G>A (p.Gly173Ser), could influence the metabolism of CYP2E1 substrates, such as chlorzoxazone.

Dataset Information

Functional Characterization of Human CYP2C9 Allelic Variants in COS-7 Cells.

Publications

Functional Characterization of Human CYP2C9 Allelic Variants in COS-7 Cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets