Dataset Information

Functional characterization of the defective CYP2C9 variant CYP2C9*18.

ABSTRACT: Cytochrome P450 2C9 (CYP2C9) is one of the most important drugs metabolizing enzymes and accounts for the metabolism of about 13%-17% of clinical drugs. Like other members in CYP2 family, CYP2C9 gene exhibits great genetic polymorphism among different races and individuals. CYP2C9*18 is one CYP2C9 allelic variant identified in a Southeast Asian population and is estimated to cause the amino acid substitutions of I359L and D397A in CYP2C9 enzyme simultaneously. Limited by the low expression level in bacteria and COS-7 cells, no valuable enzyme kinetics have been reported on this CYP2C9 variant. In this study, the baculovirus-based system was used for the high expression of recombinant CYP2C9 s in insect cells. As a result, together with I359L substitution, D397A could significantly decrease the protein expression of CYP2C9.18 in insect cells, although substitution of D397A alone had no effect on the expression of CYP2C9 in vitro. As compared with that of wild-type enzyme, both CYP2C9.18 variant and D397A variant could decrease more than 80% of the catalytic activity of CYP2C9 enzyme toward three probe substrates, suggesting that caution should be exercised when patients carrying CYP2C9*18 taking medicines metabolized by CYP2C9 enzyme with a narrow therapeutic window.

SUBMITTER: Liu J

PROVIDER: S-EPMC7842875 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Functional characterization of the defective CYP2C9 variant CYP2C9*18.

Liu Jian J Chen Hao H Wang Shuang-Hu SH Zhou Quan Q Geng Pei-Wu PW Zhou Yun-Fang YF Wu Hua-Lan HL Shi Hai-Feng HF Wang Fang F Yang Jie-Fu JF Cai Jian-Ping JP Dai Da-Peng DP

Pharmacology research & perspectives 20210201 1

Cytochrome P450 2C9 (CYP2C9) is one of the most important drugs metabolizing enzymes and accounts for the metabolism of about 13%-17% of clinical drugs. Like other members in CYP2 family, CYP2C9 gene exhibits great genetic polymorphism among different races and individuals. CYP2C9*18 is one CYP2C9 allelic variant identified in a Southeast Asian population and is estimated to cause the amino acid substitutions of I359L and D397A in CYP2C9 enzyme simultaneously. Limited by the low expression level ...[more]

PMID: 33508175

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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:Alaska Native people are under-represented in genetic research but have unique gene variation that may critically impact their response to pharmacotherapy. Full resequencing of CYP2C9 in a cross-section of this population identified CYP2C9 Met1Leu (M1L), a novel, relatively common single nucleotide polymorphism hypothesized to confer CYP2C9 poor metabolizer phenotype by disrupting the start codon. M1L is present at a minor allele frequency of 6.3% in Yup'ik Alaska Native people and thus can contribute to the risk of an adverse drug response from narrow-therapeutic-index CYP2C9 substrates such as (S)-warfarin. This study's objective was to characterize the catalytic efficiency of the Leu1 variant enzyme in vivo by evaluating the pharmacokinetic behavior of naproxen, a probe substrate for CYP2C9 activity, in genotyped Yup'ik participants. We first confirmed the selectivity of (S)-naproxen O-demethylation by CYP2C9 using activity-phenotyped human liver microsomes and selective cytochrome P450 inhibitors and then developed and validated a novel liquid chromatography mass spectrometry method for simultaneous quantification of (S)-naproxen, (S)-O-desmethylnaproxen, and naproxen acyl glucuronide in human urine. The average ratio of (S)-O-desmethylnaproxen to unchanged (S)-naproxen in urine was 18.0 ± 8.0 (n = 11) for the homozygous CYP2C9 Met1 reference group and 10.3 ± 6.6 (n = 11) for the Leu1 variant carrier group (P = 0.011). The effect of M1L variation on CYP2C9 function and its potential to alter the pharmacokinetics of drugs metabolized by the enzyme has clinical implications and should be included in a variant screening panel when pharmacogenetic testing in the Alaska Native population is warranted. SIGNIFICANCE STATEMENT: The novel CYP2C9 Met1Leu variant in Alaska Native people was recently identified. This study validated (S)-naproxen as a CYP2C9 probe substrate to characterize the in vivo functional activity of the CYP2C9 Leu1 variant. The results of this pharmacogenetic-pharmacokinetic study suggest that the CYP2C9 Leu1 variant exhibits loss of enzyme activity. This finding may be important to consider when administering narrow-therapeutic-index medications metabolized by CYP2C9 and also compels further investigation to characterize novel genetic variation in understudied populations.

Dataset Information

Functional characterization of the defective CYP2C9 variant CYP2C9*18.

Publications

Functional characterization of the defective CYP2C9 variant CYP2C9*18.

Similar Datasets

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