CYP2C9--ACTIVE SITE STRUCTURE--MOLECULAR MODELING ASPECTS

CYP2C9--活性位点结构--分子建模方面

• 批准号: 6353021
• 负责人: WILLIAM F TRAGER
• 金额: $ 24.28万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2001-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6353021
• 关键词: active sites; affinity labeling; alpha benzopyrone; blood chemistry; chemical binding; cytochrome P450; drug interactions; drug metabolism; electrospray ionization mass spectrometry; enzyme inhibitors; enzyme substrate complex; gene expression; gene mutation; genetic polymorphism; human genetic material tag; hydantoins; matrix assisted laser desorption ionization; pharmacogenetics; phenytoin; protein isoforms; site directed mutagenesis; urinalysis; valproate

The long-term aim of the research described in this proposal is to understand the structural basis underlying the unique substrate specificities of the human CYP2C proteins. This is important for the avoidance of inhibitory drug-drug interactions involving this important P450 sub-family, and in particular CYP2C9, during the development of new therapeutic agents. Our preliminary data derived from CoMFA modeling, homology modeling and site-directed mutagenesis studies, suggest that the binding of coumarin anticoagulants and anticonvulsant hydantoins to CYP2C9 is governed by an aromatic binding interaction within the B'-helix, and two electrostatic interactions (E1 and E2) within elements of the F, G and I-helices of the enzyme. The primary goal of this research is to test this hypothesis in order to refine our active-site model for CYP2C9. CYP2C19, which share greater than 90% sequence homology with CYP2C9, also metabolizes the coumarins and hydantoins but generates metabolite profiles quite distinct from CYP2C9. Therefore, the active-sites of CYP2C9 and CYP2C19 likely share some common binding determinants which should facilitate the related goal of developing a three-dimensional active-site model for CYP2C19. The Specific Aims of this proposal are; 1. Construction of new CoMFA models for CYP2C9 and CYP2C19 based on Ki values for the inhibition of both isoforms by Type I and Type II ligands. 2. Identification of active-sites residues in CYP2C9 and CYP2C19 through photo-affinity labeling and analysis of adducted residues by electrospray and MALDI mass spectrometry. 3. Identification of electrophilic binding site determinants in CYP2C9 through the construction of hybrid CYP2C9/CYP2C19 proteins and point mutants, and analysis of their interaction with valproic acid, phenytoin and phenprocoumon. This three-tiered approach involves the use of complementary techniques which will permit the iterative refinement of three-dimensional structural representations of CYP2C9 and CYP2C19. This approach provides a powerful internal control for the procedures which we are using, by ensuring that discrete CYP2C9 and CYP2C19 structural representations are created rather than a low resolution "global" CYP2C model. Finally, if we can develop discrete models for the closely related CYP2C9 and CYP2C19 isoforms, there should be little impediment to the future generation of active-site models for the other human CYP2C isoforms.

这项提案中描述的研究的长期目标是 了解独一无二的衬底的结构基础 人类细胞色素P450 2 C蛋白的特异性。这一点对于 避免涉及这一重要的抑制药物-药物相互作用 P450亚家族，特别是CYP2C9，在新的 治疗剂。我们的初步数据来自CoMFA模型， 同源建模和定点突变研究表明， 香豆素抗凝剂和抗惊厥药物海因与细胞色素P450_2C9的结合 受B‘-螺旋内的芳香族结合相互作用控制，并且 F、G和E元素内的两个静电相互作用(E1和E2) 酶的i-螺旋。这项研究的主要目的是验证这一点。 假设，以完善我们的活性部位模型的CYP2C9。CYP2C19， 与CYP2C9有90%以上的序列同源性，也 代谢香豆素和海因，但产生代谢物特征 与CYP2C9截然不同。因此，CYP2C9和CYP2C9的活性中心 CYP2C19可能有一些共同的结合决定因素，它们应该 促进开发三维活动站点的相关目标 CYP2C19的型号。这项建议的具体目标是； 1.构建基于KI的细胞色素P450受体C9和C19的CoMFA新模型 I型和II型配体对两种异构体的抑制值。 2.通过对CYP2C9和CYP2C19中活性中心残基的鉴定 电喷雾光亲和标记分析加合物残留物 和MALDI质谱仪。 3.CYP2C9中亲电结合位点决定簇的鉴定 通过构建杂合的CYP2C9/CYP2C19蛋白和点 突变株及其与丙戊酸、苯妥英的相互作用分析 还有菲普鲁蒙。 这种三层方法涉及到互补技术的使用 这将允许对三维结构的迭代求精 细胞色素P450 2 C9和细胞色素P4 2 C19的表达。这种方法提供了一种强大的 对我们正在使用的程序进行内部控制，确保 创建的是离散的CYP2C9和CYP2C19结构表示 而不是一个低分辨率的“全球”的CYP2C模型。最后，如果我们能够发展 密切相关的CYP2C9和CYP2C19亚型的离散模型，在那里 应该不会对下一代活动站点模型造成什么障碍 对于其他人类细胞色素P450 2 C亚型。