Heterologous Expression and Functional Characterization of Novel CYP2C9 Variants Identified in the Alaska Native People
Heterologous Expression and Functional Characterization of Novel CYP2C9 Variants Identified in the Alaska Native People
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DOI：
10.1124/jpet.120.265850
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发表时间：
2020-08-01
影响因子：
3.5
通讯作者：
Rettie, Allan E.
中科院分区：
文献类型：
--
作者：
McDonald, Matthew G.;Henderson, Lindsay M.;Rettie, Allan E.
CYP 2C 9是人肝细胞色素P450的主要形式，负责几种广泛使用的低治疗指数药物（包括（S）-华法林和苯妥英）的氧化代谢。在阿拉斯加原住民队列中，超罕见或新型CYP 2C 9蛋白变体M1L（rs 114071557）、N2181（rs780801862）和P279 T（rs 182132442，CYP 2C 9 *29）的表达频率高于充分表征的CYP 2C 9 *2和CYP 2C 9 *3等位基因。我们在这里报告他们在慢病毒感染的HepG 2细胞的相对表达和纯化的重组酶变体在大肠杆菌中表达对（S）-华法林，苯妥英钠，氟比洛芬，（S）-萘普生的功能表征。在感染的HepG 2细胞中，野生型、N218 I和P279 T变体获得了稳健的mRNA和蛋白表达，但正如预期的那样，M1L变体蛋白在该肝源性细胞系中不翻译。His标记的野生型蛋白和N218 I和P279 T变体在E. coli，亲和层析后得到高度纯化。用细胞色素P450氧化还原酶和细胞色素b5复溶后，N218 I和P279 T蛋白变体将（S）-华法林、苯妥英、氟比洛芬和（S）-萘普生代谢为预期的单羟基化或O-脱甲基代谢物。稳态动力学分析显示，P279 T和N218 I变体对（S）-华法林代谢的相对催化效率比分别为野生型CYP 2C 9蛋白的87%和24%。观察到苯妥英、氟比洛芬和（S）-萘普生代谢的相似排序。我们的结论是变异N218 I的携带者，特别是M1L等位基因的携带者将处于依赖于CYP 2C 9代谢清除的药物的治疗效果恶化的风险中。重要性声明CYP 2C 9-M1L和N2181的新基因变异体，沿着P279 T（CYP 2C 9 - 29）-在阿拉斯加土著人中以相对高的频率表达。对其功能效应的体外表征表明，每种变体都会降低对多种底物的催化效率，包括低治疗指数药物（S）--华法林和苯妥英。这些数据提供了该未充分研究人群中新的常见CYP 2C 9变体的第一个功能信息。这些数据可能有助于指导等位基因携带者的剂量调整，从而减轻潜在的医疗差异。
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), N2181 (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 wildtype, 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 STATEMENTNovel gene variants of CYP2C9-M1L, and N2181, 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.