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有显着变化，在人类和人口量表上存在于人类之间。这些个体间的许多变化归因于酶2C亚家族中的多态性。酶的CYP2C亚家族造成了许多治疗剂的代谢，例如S-甲状腺素，奥美吡唑，前甲基，某些巴比妥酸盐，地西epam，丙酰醇，丙二醇醇，二氯烯酸，tolbutamide，tolbutamide，tolbutamide，imipramine和subalol。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确定CYP酶的关键蛋白质结构，这些酶推断底物特异性和代谢活性。开发了抗癌药物紫杉醇和抗惊厥药物的代谢和动力学的方法，以鉴定cyp酶中的多态性，功能意义和底物识别位点（SRS）。 CYP3A在多种药物，类固醇和环境化学物质的代谢中很重要。 CYP3A活性的变化对人类中的新陈代谢和消除CYP3A底物具有重要影响。我们以前已经报道了单核苷酸多态性，导致三个不同种族组中的四个CYP3A4编码变体。在本研究中，使用细菌cDNA表达系统，我们通过这些重组等位基因变体检查了硝苯地平的代谢，以确定这些CYP3A4等位基因蛋白中的任何一种在代谢这种药物方面是否有缺陷。 L293P（CYP3A4*18），M445T（CYP3A4*3）和P467S（CYP3A4*19）重组等位基因蛋白的代谢与野生型CYP3A4*1相当。但是，与CYP3A4*1相比，VMAX和CLMAX的3A4 F189（CYP3A4*17）等位基因在VMAX和CLMAX的均显示> 99％。在72个种族多样的个体中，CYP3A4*17在一个单个个体中被鉴定出来，他也是CYP3A5*3的纯合子，表明此SNP是包含CYP3A5*3剪接变体的一种单倍型的一部分。由于大多数CYP3A底物在人类中被CYP3A4和CYP3A5代谢，因此开发了新的特定PCR-RFLP基因分型程序来评估CYP3A4*17等位基因和CYP3A5的主要剪接变体（CYP3A5*3）。这些新的遗传测试将在未来的临床研究中有用，以研究这种单倍型患者体内基因型/表型关联，这些单倍型将被预测代谢CYP3A底物很差。