CYP2C9--ACTIVE SITE STRUCTURE--MOLECULAR MODELING ASPECTS

本提案所述研究的长期目标是 了解独特基质的结构基础 人CYP 2C蛋白的特异性。这对于 避免涉及这一重要的抑制性药物相互作用 P450亚家族，特别是CYP 2C 9，在新的 治疗剂。我们的初步数据来自CoMFA建模， 同源性建模和定点诱变研究，表明， 香豆素类抗凝剂和抗惊厥剂乙内酰脲与CYP 2C 9的结合 由B ′-螺旋内的芳香族结合相互作用控制， F、G和G的元件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测试这一点， 假设，以完善我们的CYP 2C 9活性位点模型。CYP2C19， 与CYP 2C 9具有大于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CYP 2C 9不同。因此，CYP 2C 9的活性位点和 CYP 2C 19可能具有一些共同的结合决定簇， 促进开发三维活动网站的相关目标 CYP 2C 19的模型。该提案的具体目标是： 1.基于Ki的CYP 2C 9和CYP 2C 19新CoMFA模型的构建 I型和II型配体对两种亚型的抑制值。 2. CYP 2C 9和CYP 2C 19中活性位点残基的鉴定 通过电喷雾的加合物残留物的光亲和标记和分析 和MALDI质谱法。 3. CYP 2C 9亲电子结合位点决定簇的鉴定 通过构建CYP 2C 9/CYP 2C 19杂合蛋白， 突变体，并分析其与丙戊酸、苯妥英的相互作用 和苯丙香豆素。 这三个层次的方法涉及使用互补的技术 这将允许三维结构的迭代细化， 代表CYP 2C 9和CYP 2C 19。这种方法提供了一个强大的 我们正在使用的程序的内部控制，通过确保 创建离散的CYP 2C 9和CYP 2C 19结构表示， 而不是低分辨率的“全局”CYP 2C模型。最后，如果我们能发展 密切相关的CYP 2C 9和CYP 2C 19亚型的离散模型， 应该不会对下一代的活性部位模型造成什么阻碍 其他人类CYP 2C亚型。

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.