Metabolic Activity Of Human Recombinant Cytochrome P450

人重组细胞色素 P450 的代谢活性

• 批准号: 7169296
• 负责人: BURHAN I GHANAYEM
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7169296
• 关键词: anticonvulsants; binding sites; chemical binding; cytochrome P450; drug metabolism; enzyme activity; genetic polymorphism; high performance liquid chromatography; human tissue; hydroxylation; molecular shape; omeprazole; paclitaxel; recombinant proteins; single nucleotide polymorphism; site directed mutagenesis; tolbutamide

Significant variations in the metabolism of drugs and environmental chemicals which are metabolized via the cytochrome P450 (CYP) enzymes exist between humans on an individual and population scale. Many of these interindividual variations are attributed to polymorphisms in the 2C subfamily of enzymes. CYP2C subfamily of enzymes are responsible for the metabolism of a number of therapeutic agents such as S-mephenytoin, omeprazole, proguanil, certain barbiturates, diazepam, propranolol, diclofenac, tolbutamide, imipramine, and taxol. The overall objectives of this work is to elucidate the molecular and metabolic basis of CYP related polymorphisms, characterization of the substrate-structure metabolism relationships, and the determination of critical protein structures of CYP enzymes that infer substrate specificity and metabolic activity. Methods to study the metabolism and kinetics of the anticancer drug taxol and the anticonvulsant drug mephenytoin were developed in order to identify the polymorphisms, their functional significance, and the substrate recognition site (SRS) in CYP enzymes. The CYP3As are important in the metabolism of a wide range of drugs, steroids, and environmental chemicals. Variation in CYP3A activity has an important influence on the metabolism and the elimination of CYP3A substrates in humans. We previously have reported single nucleotide polymorphisms resulting in four CYP3A4 coding variants in three different racial groups. In the present study, using a bacterial cDNA expression system, we examined nifedipine metabolism by these recombinant allelic variants to determine whether any of these CYP3A4 allelic proteins are defective in metabolizing this drug. Metabolism of nifedipine by the L293P (CYP3A4*18), M445T (CYP3A4*3), and P467S (CYP3A4*19) recombinant allelic proteins was comparable to that of wild type CYP3A4*1. However, the 3A4 F189S (CYP3A4*17) allele exhibited >99% decrease in both Vmax and CLmax compared to CYP3A4*1. Out of 72 racially diverse individuals, CYP3A4*17 was identified in a single individual who was also homozygous for CYP3A5*3, indicating this SNP is part of one haplotype containing the CYP3A5*3 splice variant. Since most CYP3A substrates are metabolized by both CYP3A4 and CYP3A5 in humans, new specific PCR-RFLP genotyping procedures were developed to assess both the CYP3A4*17 allele and the major splice variant of CYP3A5 (CYP3A5*3). These new genetic tests will be useful in future clinical studies to study genotype/phenotype associations in vivo in individuals with this haplotype who would be predicted to metabolize CYP3A substrates poorly.

通过细胞色素 P450 (CYP) 酶代谢的药物和环境化学物质的代谢在个体和群体规模上存在显着差异。许多这些个体差异归因于 2C 酶亚家族的多态性。 CYP2C 酶亚家族负责许多治疗药物的代谢，如 S-美芬妥英、奥美拉唑、氯胍、某些巴比妥类药物、地西泮、普萘洛尔、双氯芬酸、甲苯磺丁脲、丙咪嗪和紫杉醇。这项工作的总体目标是阐明 CYP 相关多态性的分子和代谢基础、底物-结构代谢关系的表征以及推断底物特异性和代谢活性的 CYP 酶的关键蛋白质结构的确定。开发了研究抗癌药物紫杉醇和抗惊厥药物美芬妥英的代谢和动力学的方法，以确定 CYP 酶中的多态性、其功能意义和底物识别位点 (SRS)。 CYP3As 在多种药物、类固醇和环境化学物质的代谢中发挥着重要作用。 CYP3A活性的变化对人体CYP3A底物的代谢和消除具有重要影响。我们之前报道过单核苷酸多态性导致三个不同种族群体中产生四种 CYP3A4 编码变异。在本研究中，我们使用细菌 cDNA 表达系统检查了这些重组等位基因变体对硝苯地平的代谢，以确定这些 CYP3A4 等位蛋白中的任何一个在代谢该药物时是否存在缺陷。 L293P (CYP3A4*18)、M445T (CYP3A4*3) 和 P467S (CYP3A4*19) 重组等位基因蛋白对硝苯地平的代谢与野生型 CYP3A4*1 相当。然而，与 CYP3A4*1 相比，3A4 F189S (CYP3A4*17) 等位基因的 Vmax 和 CLmax 均降低了 99% 以上。在 72 名不同种族的个体中，在一名 CYP3A5*3 纯合的个体中鉴定出 CYP3A4*17，表明该 SNP 是包含 CYP3A5*3 剪接变体的一种单倍型的一部分。由于人类中大多数 CYP3A 底物均由 CYP3A4 和 CYP3A5 代谢，因此开发了新的特异性 PCR-RFLP 基因分型程序来评估 CYP3A4*17 等位基因和 CYP3A5 (CYP3A5*3) 的主要剪接变体。这些新的基因测试将有助于未来的临床研究，以研究具有这种单倍型的个体的体内基因型/表型关联，预计这些个体对 CYP3A 底物的代谢较差。