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.

本提案中描述的研究的长期目标是 了解独特基材的结构基础 人类 CYP2C 蛋白的特异性。这对于 避免涉及这一重要的抑制性药物间相互作用 P450 亚家族，特别是 CYP2C9，在新的开发过程中 治疗剂。我们的初步数据来自 CoMFA 建模， 同源建模和定点突变研究表明 香豆素抗凝剂和抗惊厥剂乙内酰脲与 CYP2C9 的结合 受 B'-螺旋内芳香族结合相互作用的控制，并且 F、G 和 F 元素内的两个静电相互作用（E1 和 E2） 酶的 I-螺旋。这项研究的主要目标是测试这一点 假设以完善我们的 CYP2C9 活性位点模型。 CYP2C19, 与 CYP2C9 具有超过 90% 的序列同源性， 代谢香豆素和乙内酰脲，但产生代谢物谱 与 CYP2C9 完全不同。因此，CYP2C9和CYP2C9的活性位点 CYP2C19 可能具有一些共同的结合决定因素，这些决定因素应该 促进开发三维活动站点的相关目标 CYP2C19 的模型。该提案的具体目标是： 1. 基于Ki构建CYP2C9和CYP2C19新的CoMFA模型 I 型和 II 型配体对两种异构体的抑制值。 2. 通过以下方法鉴定 CYP2C9 和 CYP2C19 中的活性位点残基 通过电喷雾进行光亲和标记和加合残基分析 和MALDI 质谱分析。 3. CYP2C9 中亲电结合位点决定因素的鉴定 通过构建杂交 CYP2C9/CYP2C19 蛋白和点 突变体及其与丙戊酸、苯妥英的相互作用分析 和苯丙香豆素。 这种三层方法涉及互补技术的使用 这将允许三维结构的迭代细化 CYP2C9 和 CYP2C19 的表示。这种方法提供了强大的 我们正在使用的程序的内部控制，通过确保 离散的 CYP2C9 和 CYP2C19 结构表示是创建的 比低分辨率“全局”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，有 对下一代活性位点模型应该没有什么障碍 对于其他人类 CYP2C 同工型。