P4502C9--ACTIVE SITE STRUCTURE AND GENETIC POLYMORPHISM

细胞色素P4502 C9在华法林药物的病因学中起核心作用 具有抑制基础的相互作用。 它是唯一的单加氧酶 负责（s）-华法林（对映异构体）的代谢清除 所有的抗凝血作用都存在于药物中。 因此，与P4502 C9共同给药的药物对P4502 C9的抑制作用可能与P4502 C9的抑制作用有关。 抗凝剂可能是许多药物的主要原因 这是临床上经常遇到的问题。 识别 P4502 C9的许多（等位基因）变体是一个值得注意的并发症 因为这些变异体中的一些似乎缺乏催化作用， 它们代谢（S）-华法林和其他底物的能力， 苯妥英和甲苯磺丁脲。 这些突变等位基因的表达可能 作为某些P4502 C9慢代谢表型的基础 印刷受体. 因此，我们建议评估代谢后果 P4502 C9结构变异体在人群中的表达， 并定义结构特征，P4502 C9和相互作用 药物，控制酶底物和酶的形成， 抑制剂复合物。 这将通过以下方式实现： 1)确定两者的共同结构和构象元素 底物和抑制剂使用分子力学， 2)使用光亲和探针共价修饰， 轰击/质谱法鉴定存在的关键氨基酸 在P4502 C9的活性位点， 3)表达该酶的定点突变体，并比较它们的 代谢能力与野生型酶相同， 4)对200名受试者进行药物遗传学研究，使用 亚治疗剂量的（s）-华法林的代谢作为报告者， 个体P4502 C9表型， 5)使用等位基因特异性聚合酶对这些个体进行基因分型 链式反应分析， 6)研究（S）-华法林和 预先选择的杂合子和纯合子中的苯妥英，以 评估该现象的基底依赖性。 项目2的长期目标是详细了解 由CYP 2C 9基因编码的蛋白质的活性位点地形图。 上述 研究将提供有关结构特征的补充数据， 控制底物和抑制剂与酶的相互作用， 其结构变体。 在本提案的所有阶段， 信息将被整合到我们的同源性模型中， 它足以允许，最终，一个前瞻性的评估 联合给药药物可能激发抑制药物的程度 与P4502 C9底物的相互作用。

Cytochrome P4502C9 plays a central role in the etiology of warfarin drug interactions which have an inhibitory basis. It is the sole monoxygenase responsible for the metabolic clearance of (s)-warfarin, the enantiomer of the drug in which essentially all of the anticoagulant effect resides. Therefore, inhibition of P4502C9 by drugs which are co-administered with the anticoagulant is likely to be a primary cause of many of the drug interactions frequently encountered in the clinic. The identification of numerous (allelic) variants of P4502C9 is a notable complication because some of these variants appear to be catalytically deficient in their ability to metabolize (S)-warfarin and other substrates such as phenytoin and tolbutamide. Expression of these mutant alleles may underlie the slow metabolizer phenotype identified for some P4502C9 substrates. Therefore, we propose to evaluate the metabolic consequences of expression of structural variants of P4502C9 in the human population, and to define the structural features, both P4502C9 and of interacting drugs, which govern the formation of enzyme-substrate and enzyme- inhibitor complexes. This will be accomplished by; 1) determining common structural and conformational elements for both substrates and inhibitors using molecular mechanics, 2) using photoaffinity probes to covalently modify, and fast atom bombardment/mass spectrometry to identify, critical amino acids present in the active site of P4502C9, 3) expressing site-directed mutants of the enzyme and comparing their metabolic capabilities with that of the wild-type enzyme, 4) undertaking a pharmacogenetic study with 200 subjects, using the metabolism of sub-therapeutic doses of (s)-warfarin as a reporter of individual P4502C9 phenotype, 5) genotyping these same individuals using an allele-specific polymerase chain-reaction assay, 6) investigating the extent of co-segregation of (S)-warfarin and phenytoin in pre-selected heterozygotes and homozygotes, in order to assess the substrate-dependency of the phenomenon. The long-term aim of project 2 is to gain a detailed understanding of the active-site topography of proteins encoded by the CYP2C9 gene. The above studies will provide complementary data on the structural features which govern the interactions of substrates and inhibitors with the enzyme and its structural variants. At all stages of this proposal the accrued information will be integrated into our homology model in order to refine it sufficiently to permit, ultimately, a prospective assessment of the extent to which a co-administered agent might provoke inhibitory drug interactions with P4502C9 substrates.