Drug Metabolizing Enzymes In Humans

人体药物代谢酶

• 批准号: 8929701
• 负责人: JOYCE GOLDSTEIN
• 金额: $ 139.1万
• 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/8929701
• 关键词: Acetylation; Adverse effects; Affect; Alleles; Amino Acids; Anticoagulants; Antidiabetic Drugs; Antiplatelet Drugs; Binding; Binding Proteins; Binding Sites; Biological Models; CYP2C19 gene; CYP3A4 gene; Cell Line; Cells; Cessation of life; Chemicals; Chromatin; Chromatin Structure; Clinical; Clinical Research; Complex; Confocal Microscopy; Data; Death Rate; Drug Interactions; Enzymes; Epidemiologist; Epigenetic Process; Estrogen Receptor alpha; Estrogen Receptors; Estrogens; Exposure to; Failure; Formaldehyde; Gene Activation; Gene Silencing; Gene Targeting; Genes; Genetic Polymorphism; Genetic Transcription; Genetic Variation; Genetic screening method; Genotype; Hemorrhage; Hepatic; Hepatocyte; Histones; Hospitalization; Human; Hybrids; Immunoprecipitation; Impairment; In Vitro; Indium; Laboratories; Legal patent; Ligands; Liver; Lysine; Mediator of activation protein; Metabolism; Methylation; Molecular Conformation; Mutation; N-terminal; Nuclear; Nuclear Receptors; Outcome; Oxidative Stress; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Platelet aggregation; Plavix; Polycomb; Post-Translational Protein Processing; Process; Prodrugs; Proteins; RNA Interference; RNA Polymerase II; Recombinants; Recruitment Activity; Regulation; Regulatory Element; Role; Site; Stents; Stress; System; Tail; Techniques; Testing; Therapeutic; Tissues; Tolbutamide; Toxic effect; Transcription Factor AP-1; Up-Regulation; Warfarin; Xenobiotics; Yeasts; arginyllysine; chromatin immunoprecipitation; clopidogrel; constitutive active receptor; cytochrome P-450 CYP2C subfamily; dosage; drug clearance; environmental change; environmental chemical; histone modification; human CYP2C9 protein; in vivo; inhibitor/antagonist; member; mutant; pregnane X receptor; prevent; promoter; prototype; receptor; response; yeast two hybrid system

In a new project, yeast and mammalian two-hybrid screens showed that Med25, a variable member of the mediator complex, is an HNF4a-binding protein. We have shown that Med25 is important for the recruitment of RNA Polymerase II to select sets of HNF4a-activated promoters such as the important drug-metabolizing gene cytochrome P450 2C9 (CYP2C9) and CYP3A4. We hypothesized that this involves direct interaction between Med25 and HNF4 to alter chromatin conformation of the CYP2C9 gene to a transcriptionally active state. Conformational change requires the modification of histones by enzymes that are recruited to target genes. Histone modifications include methylation or acetylation of lysine and arginine amino acids on histone N-terminal tails. For example, histone 3 lysine 4 dimethylation (H3K4me2) is associated with gene activation, while histone 3 lysine 27 trimethylation (H3K27me3) is a marker of gene silencing. In this study, we used HepG2 cells to determine the role of Med25 in the epigenetic regulation of HNF4a-dependent CYP2C9 expression. We performed chromatin immunoprecipitation to identify histone modifications at the HNF4a binding site in relation to Med25 protein levels. Our results indicate that altering Med25 expression modified acetylation and methylation of certain lysine 27 on histone 3. When Med 25 was expressed, this lysine was acetylated. However, when Med25 was silenced with small-hairpin looped RNAi, H3K27 was trimethylated which is prototypical in gene-silencing. These results indicate that Med25 induces a permissive chromatin state at the CYP2C9 proximal HNF4a binding site. Similarly, confocal microscopy revealed that Med25 colocalized with key histone modification markers. We have also determined levels of open CYP2C9 chromatin under activating conditions using formaldehyde-assisted isolation of regulatory elements (FAIRE). FAIRE data indicated that the chromatin around the HNF4a sites of the CYP2C9 proximal promoter was open in the presence of activating nuclear receptors CAR and HNF4a and Med25 but closed when Med25 was silenced. These studies will be extended globally to more HNF4a induced genes. A new study showed that the estrogen receptor alpha (ERa) induced CYP2C9 promoter activity in the presence of ligand and exogenous Med25. Immunoprecipitation studies showed interaction between ERa and Med25 in the presence or absence of ligand. Chromatin immunoprecipitation studies showed that Med25 bound to the ERE (estrogen responsive element in the presence of ERa. CYP2C9 catalytic activity was increased in primary hepatocytes by Med25 and ERa but silenced with siMed25. Recently we found that electrophiles and oxidative stress induce CYP2C9 and CYP2C19 in human primary hepatocytes through AP-1 proteins which interact with two AP-1 sites. There is looping between the two sites when occupied by cJun and JunD respectively. Many drugs are electrophilic or known to be activated to electrophiles. This is a new mechanism of activation of CYP2C9 and CYP2C19 not involving the xenosensing receptors CAR or PXR. In collaborative studies we showed that Plavix (clopidogrel) a drug which prevents platelet aggregation is metabolized with a 75% lower clearance in vitro using recombinant CYP2C19*10 allele (discovered in our laboratory) than normal CYP2C19. Thus, although it is not an inactive allele, its activity is greatly impaired. Studies with different CYP2C19 substrates showed different degrees of impairment. CYP2C19 activates clopidogrel from a prodrug to the active drug. The CYP2C19*10 allele also interferes with a number of genotyping tests for the null CYP2C19*2 allele. This suggests clinical studies should include genotyping of the CYP2C19*10 allele.

在一个新的项目中，酵母和哺乳动物的双杂交筛选显示，Med25是中介复合体的一个可变成员，是一种HNF4a结合蛋白。我们已经证明Med25对于RNA聚合酶II的招募是重要的，以选择HNF4a激活的启动子集，例如重要的药物代谢基因细胞色素P450 2C9(CYP2C9)和CYP3A4。我们假设这涉及到Med25和HNF4之间的直接相互作用，以改变CYP2C9基因的染色质构象，使其处于转录活跃状态。构象变化需要被招募到靶基因的酶来修饰组蛋白。组蛋白修饰包括组蛋白N末端赖氨酸和精氨酸氨基酸的甲基化或乙酰化。例如，组蛋白3赖氨酸4二甲基化(H3K4me2)与基因激活有关，而组蛋白3赖氨酸27三甲基化(H3K27me3)是基因沉默的标志。在这项研究中，我们使用HepG2细胞来确定Med25在HNF4a依赖的CYP2C9表达的表观遗传调控中的作用。我们进行了染色质免疫沉淀，以确定HNF4a结合部位的组蛋白修饰与Med25蛋白水平的关系。我们的结果表明，改变Med25的表达可以改变组蛋白3上某些赖氨酸27的乙酰化和甲基化。当表达Med25时，该赖氨酸被乙酰化。然而，当Med25被小发夹环RNAi沉默时，H3K27被三甲基化，这是基因沉默的原型。这些结果表明，Med25在CYP2C9近端HNF4a结合部位诱导了一种允许的染色质状态。同样，共聚焦显微镜显示Med25与关键的组蛋白修饰标记共定位。我们还使用甲醛辅助分离调节元件(FAIRE)测定了激活条件下开放的CYP2C9染色质的水平。FIIRE数据表明，在激活核受体CAR、HNF4a和Med25的情况下，CYP2C9近端启动子HNF4a位点周围的染色质是开放的，而当Med25被沉默时，染色质是关闭的。这些研究将在全球范围内扩展到更多HNF4a诱导的基因。一项新的研究表明，在配体和外源Med25存在的情况下，雌激素受体α(ERA)诱导了CYP2C9启动子的活性。免疫沉淀研究表明，在存在或不存在配体的情况下，ERA和Med25之间存在相互作用。染色质免疫沉淀研究表明，在ERA存在的情况下，Med25与ERE(雌激素反应元件)结合。在原代培养的肝细胞中，MED25和ERA可增强细胞色素P450 2 C9的催化活性，而siMED25则抑制其催化活性。最近我们发现，亲电体和氧化应激通过AP-1蛋白与两个AP-1位点相互作用，诱导人原代肝细胞中的细胞色素P450受体C9和细胞色素P450 2 C19表达。当分别被cjun和jund占用时，两个站点之间存在环路。许多药物是亲电性的，或已知对亲电性有激活作用。这是一种不涉及异种感觉受体CAR或PXR的新的激活机制。在合作研究中，我们发现，抗血小板聚集的药物Plavix(氯吡格雷)在使用重组的CYP2C19*10等位基因(在我们实验室发现)的体外代谢清除率比正常的CYP2C19低75%。因此，虽然它不是一个不活跃的等位基因，但它的活性受到了极大的损害。使用不同底物的研究显示了不同程度的损伤。细胞色素P450 2 C19激活氯吡格雷从前药到活性药物。CYP2C19*10等位基因也干扰了许多无效的CYP2C19*2等位基因的基因分型试验。这提示临床研究应包括CYP2C19*10等位基因的基因分型。