CYP2C9--ACTIVE SITE STRUCTURE--MOLECULAR MODELING ASPECTS

CYP2C9--活性位点结构--分子建模方面

• 批准号: 6481915
• 负责人: WILLIAM F TRAGER
• 金额: $ 25.41万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6481915
• 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.

该提案中描述的研究的长期目的是 了解独特的基板的结构基础 人CYP2C蛋白的特异性。这对 避免抑制性药物相互作用，涉及这一重要的 P450子家庭，尤其是CYP2C9，在新的开发过程中 治疗剂。我们的初步数据来自COMFA建模， 同源性建模和定向诱变研究，表明 香豆素抗凝剂和抗惊厥药与CYP2C9的结合 由b'-螺旋中的芳族结合相互作用支配， F，G和 酶的螺旋。这项研究的主要目的是测试 假设为了完善我们的CYP2C9的主动位点模型。 CYP2C19， 与CYP2C9共享超过90％的序列同源性 代谢香豆素和羟基素，但会产生代谢物概况 与CYP2C9完全不同。因此，CYP2C9和 CYP2C19可能具有一些共同的约束决定因素 促进开发三维主动站点的相关目标 CYP2C19的模型。该提案的具体目的是： 1。基于Ki的CYP2C9和CYP2C19的新COMFA型号的构建 按I型和II型配体抑制两种同工型的值。 2。鉴定CYP2C9和CYP2C19中的活动点残基 通过电喷雾的照片亲和力标记和分析加合物残留物 和MALDI质谱法。 3。鉴定CYP2C9中亲电结合位点的决定因素 通过构建混合CYP2C9/CYP2C19蛋白质和点 突变体以及它们与丙戊酸的相互作用的分析 和势力。 这种三层方法涉及使用互补技术 这将允许三维结构的迭代完善 CYP2C9和CYP2C19的表示。这种方法提供了强大的 通过确保我们正在使用的过程的内部控制 离散的CYP2C9和CYP2C19的结构表示相当相当 比低分辨率“全局” CYP2C模型。最后，如果我们能发展 与密切相关的CYP2C9和CYP2C19同工型的离散模型，那里 应该对未来的活跃站点模型几乎没有障碍 对于其他人类CYP2C同工型。