Drug Metabolizing Enzymes In Humans And Animal Models

人类和动物模型中的药物代谢酶

• 批准号: 6672817
• 负责人: JOYCE GOLDSTEIN
• 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/6672817
• 关键词: African American; antimalarial agents; antineoplastics; antiulcer drug; arachidonate; barbiturates; caucasian American; complementary DNA; cytochrome P450; drug metabolism; environmental contamination; enzyme activity; gene expression; gene mutation; genetic polymorphism; genetic screening; genetic susceptibility; human subject; nucleic acid sequence; paclitaxel; pesticides; phenytoin; racial /ethnic difference; site directed mutagenesis; toxin metabolism

ACCOMPLISHMENTS: We have identified new genetic polymorphisms in the CYP family in humans which are responsible for introducing variability in the way humans metabolize of specific drugs and environmental chemicals and thus altered susceptibility of humans to these toxicity and disease states. CYP2C9 metabolizes numerous clinically important drugs including phenytoin, tolbutamide, warfarin, glipizide and numerous nonsteroidal antiinflammatory drugs (NSAIDs). By resequencing CYP2C9 we from the three different racial groups with ethnically diverse backgrounds we discovered 12 new alleles of CYP2C9. The recombinant human drug metabolizing enzymes were expressed in an E. coli expression system, and the catalytic activity toward tolbutamide compared with that of wild-type 2C9*1. The newly discovered CYP2C9 alleles include the amino acid changes L19I (2C9*7), R150H (2C9*8), H251R (2C9*9), E272G (2C9*10), R335W (2C9*11) and P489S (2C9*12). The CYP2C9*11 allele is markedly impaired (90% ). Last year, we discovered a homozygous null mutation of CY2C9 which decreased metabolism of the phenytoin by 85% in a clinical case study of an African-American patient hospitalized from severe clinical toxicity to the anticonvulsant phenytoin. This year we identified a patient homozygous for the same null mutation of CYP2C9 who had markedly impaired ability to metabolize the commonly used clinical anticoagulant warfarin. CYP2C19 metabolizes the common antiulcer drug omeprazole, the anticonvulsant mephenytoin, the anxiolytic valium, barbiturates, activates certain antimalarials, and sulfoxidizes the pesticide phorate. Nine new polymorphisms were discovered by resequencing CYP2C19. One P227L (2C19*10) showed a 90% decrease in catalytic activity toward a prototype substrate in vitro while a second allele, R144H (2C19*9), showed modest impairment. Almost all of the new defective alleles of CYP2C9 and CYP2C19 were prevalent in African-American or African populations, suggesting that these populations have not received sufficient study in the past. Two new important drug metabolizing genes are being resequenced. We have discovered 5 alleles of CYP3A5 which contain amino acid changes and these have been expressed in cDNA expression systems. Metabolism of testosterone is dramatically decreased by one of the alleles. We will look at metabolism of at least one clinical drug and one pesticide. We have discovered at several mutations in CY2B6. Future studies are addressing whether some of these are linked, and the degree to which they affect metabolism of testosterone and several environmental compounds. This includes the organophoshate insectide pesticide chlorpyrifos and the widely used carbamate pesticide carbaryl. Mechanistic structure-activity studies have addressed why the two similar CYP2C enzymes CYP2C9 and CYP2C19 (93% identical) have distinct substrate specificities. We have identified multiple key residues 99, 220, 221, and three residues in helix I (286, 289, 295) which appear to confer the ability to metabolize mephenytoin.

成就：我们已经确定了人类中新的基因多态性，这些基因多态性导致人类代谢特定药物和环境化学品的方式发生变化，从而改变了人类对这些毒性和疾病状态的易感性。CYP 2C 9代谢许多临床上重要的药物，包括苯妥英、甲苯磺丁脲、华法林、格列吡嗪和许多非甾体类抗炎药（NSAID）。通过对CYP 2C 9基因进行重测序，我们从三个不同种族背景的人群中发现了12个新的CYP 2C 9等位基因。重组人药物代谢酶在大肠杆菌中表达。大肠杆菌表达系统，并与野生型2C 9 *1相比，对甲苯磺丁脲的催化活性。新发现的CYP 2C 9等位基因包括氨基酸改变L19 I（2C 9 *7）、R150 H（2C 9 *8）、H251 R（2C 9 *9）、E272 G（2C 9 *10）、R335 W（2C 9 *11）和P489 S（2C 9 *12）。CYP 2C 9 *11等位基因明显受损（90%）。去年，我们在一项临床病例研究中发现了CY 2C 9的纯合无效突变，该突变使苯妥英的代谢降低了85%，该研究涉及一名因抗惊厥药苯妥英的严重临床毒性而住院的非裔美国患者。今年，我们确定了一个病人纯合子为相同的无效突变的CYP 2C 9谁有明显受损的能力，代谢常用的临床抗凝华法林。CYP 2C 19代谢常见的抗溃疡药物奥美拉唑、抗惊厥药美芬妥英、抗焦虑药安定、巴比妥酸盐，激活某些抗疟药，并使农药甲拌磷亚砜化。通过对CYP 2C 19进行重测序，发现了9个新的多态性。一个P227 L（2C 19 *10）显示出体外对原型底物的催化活性降低90%，而第二个等位基因R144 H（2C 19 *9）显示出适度的损害。几乎所有新的CYP 2C 9和CYP 2C 19缺陷等位基因在非洲裔美国人或非洲人群中普遍存在，这表明这些人群在过去没有得到足够的研究。两个新的重要药物代谢基因正在重新测序。我们发现了5个含有氨基酸变化的CYP 3A 5等位基因，并已在cDNA表达系统中表达。其中一个等位基因会显著降低睾酮的代谢。我们将研究至少一种临床药物和一种杀虫剂的代谢。我们在CY 2B 6中发现了几个突变。未来的研究正在解决其中一些是否有联系，以及它们影响睾酮和几种环境化合物代谢的程度。这包括有机磷杀虫剂毒死蜱和广泛使用的氨基甲酸酯杀虫剂西维因。机制结构-活性研究已经解决了为什么两种相似的CYP 2C酶CYP 2C 9和CYP 2C 19（93%相同）具有不同的底物特异性。我们已经确定了多个关键残基99，220，221和螺旋I中的三个残基（286，289，295），它们似乎赋予代谢美芬妥英的能力。