Pharmacogenomics of Human P450 Oxidoreductase

人 P450 氧化还原酶的药物基因组学

基本信息

批准号：
7727943
负责人：
WALTER L. MILLER
金额：
$ 24.87万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-02-01 至 2013-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7727943
关键词：
Accounting Adrenal Glands Affect Alleles Amino Acid Sequence Amino Acids Antley-Bixler syndrome Area Aryl Hydrocarbon Hydroxylases Biochemistry Biological Assay CYP17A1 gene CYP1A2 gene CYP2C19 gene Catalysis Cells Clinical Clinical Research Complex Congenital Abnormality Cytochrome P450 DNA Sequence Disease Dose Drug usage Electrons Endoplasmic Reticulum Environmental Pollutants Enzymatic Biochemistry Enzymes Ethnic group Exons Female infertility Flavin Mononucleotide Flavin-Adenine Dinucleotide Flavins Flavoproteins Genes Genetic Genetic Polymorphism Genetic Variation Gonadal Steroid Hormones Grant Hepatic Hepatocyte Human Human body In Vitro Individual Laboratories Location Mediating Medical Mutation Mutation Spectra NADP Oxidoreductase Oxidoreductase Gene Patients Pharmaceutical Preparations Pharmacogenetics Pharmacogenomics Phenotype Population Positioning Attribute Promoter Regions Proteins Research Design Role Toxin Variant Xenobiotics base cohort cytochrome c drug metabolism genetic variant in vitro Assay in vivo mutant promoter public health relevance receptor response steroid hormone biosynthesis

项目摘要

DESCRIPTION (provided by applicant): Most drugs, xenobiotics and environmental pollutants that enter the human body are metabolized by P450 enzymes in the endoplasmic reticulum of liver cells. Such microsomal type II P450 enzymes, which include several enzymes involved in adrenal and gonadal steroid hormone synthesis, require electron donation from P450 oxidoreductase (POR) to mediate catalysis. POR is a 680 amino-acid, 78 kDa protein that contains two flavins, a flavin adenine dinucleotide moiety (FAD), and a flavin mononucleotide (FMN). There is substantial genetic variation in the hepatic P450 enzymes. This variation, plus the genetic variation in drug transporters and receptors, accounts for some, but not all, genetic variation in drug metabolism. We propose that some of this unexplained genetic variation in drug metabolism may be explained by genetic variation in POR. Our laboratory discovered POR deficiency as a new disorder of steroid hormone biosynthesis in 2004. Through analysis of patient DNAs, and sequencing of a large cohort of normal individuals, we have identified 35 POR amino acid sequence variants. In our recent study of 842 normal individuals, we discovered that nearly 28% of human POR alleles carry the A503V sequence variant. In vitro assays show that A503V has decreased activities in assays based on cytochrome c and P450c17, but not in assays based on CYP1A2 and CYP2C19. Assays of the activities of these POR missense mutants with cytochrome c, steroidogenic P450c17 (CYP17), and drug-metabolizing CYP1A2 and CYP2C19 have shown that the activity of a particular POR mutant with one target P450 may not predict its activity with another P450 enzyme. Thus the analysis of the potential role of POR sequence variants in the pharmacogenetics of drug metabolism is both important and complicated. We hypothesize that genetic variants of POR contribute to variation in human drug metabolism and that mildly defective POR sequence variants may synergize with otherwise innocuous, mildly defective variants in hepatic P450 enzymes to cause important variations in drug metabolism. To begin to characterize the contributions of POR to human pharmacogenetics, we propose three specific aims. Aim 1 is to determine the catalytic activities of the A503V POR variant and the common disease-causing POR mutants A287P and R457H with hepatic, drug-metabolizing P450 enzymes. Aim 2 is to determine whether the A503V variant has selective loss (or gain) of activity with common variants of hepatic, drug-metabolizing P450 enzymes. Aim 3 is to characterize the activity of the POR gene promoter and determine whether it contains polymorphisms that influence the level of POR expression. These studies will establish the in vitro biochemistry of POR variants in association with the principal drug-metabolizing hepatic P450 enzymes, and will permit us to design studies to determine whether the A503V variant affects drug metabolism in clinical studies in vivo. Completion of these aims will provide critically needed information about the potential role of POR in human pharmacogenetics. PUBLIC HEALTH RELEVANCE: There is substantial variation in how people of different ethnic backgrounds metabolize drugs; understanding such variations is crucial for optimizing drug dosing in clinical settings. Much of the variation between groups is based on variations in genes encoding drug transporters and drug-metabolizing enzymes. This project explores a third possibility, a gene called POR, that is needed for the activity of drug-metabolizing enzymes.

描述（由申请人提供）：大多数进入人体的药物，异种生物和环境污染物是由P450酶在肝细胞内质网中代谢的。这种微粒体II型P450酶，其中包括参与肾上腺和性腺类固醇激素合成的几种酶，需要从P450氧化还原酶（POR）捐赠电子以介导催化。 POR是一种680个氨基酸，78 kDa蛋白，其中包含两种黄素，一个黄素腺嘌呤二核苷酸部分（FAD）和一根黄素单核苷酸（FMN）。肝P450酶有很大的遗传变异。这种差异以及药物转运蛋白和受体的遗传变异造成了药物代谢的某些但不是全部的遗传变异。我们建议，药物代谢中这种无法解释的遗传变异可以通过POR中的遗传变异来解释。我们的实验室在2004年发现了一种新型类固醇激素生物合成的疾病。通过分析患者DNA和对大量正常个体的测序，我们已经确定了35个POR氨基酸序列变体。在我们最近对842个正常人的研究中，我们发现近28％的人类POR等位基因带有A503V序列变体。体外测定表明，基于细胞色素C和P450C17的测定中A503V的活性降低，但在基于CYP1A2和CYP2C19的测定中没有降低活性。用细胞色素C，类固醇生成P450C17（CYP17）以及药物代谢的CYP1A2和CYP2C19进行的POR错义突变体的活性测定表明，特定POR突变体具有一种靶P450的活性可能无法用另一种P450酶的靶P450预测其活性。因此，对POR序列变体在药物代谢的药物遗传学中的潜在作用的分析既重要又复杂。我们假设POR的遗传变异有助于人类药物代谢的变异，而轻度缺陷的POR序列变异可能会随着肝P450酶中原本无害的轻度缺陷变异而协同作用，从而导致药物代谢的重要变异。为了开始表征POR对人类药物遗传学的贡献，我们提出了三个具体目标。目的1是确定A503V POR变体的催化活性，以及具有肝，药物代谢的P450酶的常见引起疾病的POR突变体A287p和R457H。 AIM 2是确定A503V变体是否具有肝，药物代谢P450酶的常见变体的活性损失（或增益）。 AIM 3是表征POR基因启动子的活性，并确定其是否包含影响POR表达水平的多态性。这些研究将建立与主要药物代谢肝P450酶相关的POR变体的体外生物化学，并将允许我们设计研究以确定A503V变体在体内临床研究中是否影响药物代谢。这些目标的完成将提供有关POR在人类药物遗传学中潜在作用的迫切需要的信息。公共卫生相关性：不同种族背景的人如何代谢毒品存在实质性差异；了解这种变异对于在临床环境中优化药物给药至关重要。组之间的许多变化基于编码药物转运蛋白和药物代谢酶的基因的变化。该项目探索了第三种可能性，即称为POR的基因，这是药物代谢酶的活性所需的。