HUMAN CYP2C MODELS

人类 CYP2C 模型

• 批准号: 7551055
• 负责人: Allan Edward Rettie
• 金额: $ 40.01万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7551055
• 关键词: Active Sites; Address; Affinity; Behavior; Benzbromarone; Binding; CYP2C18 gene; CYP2C19 gene; CYP2C9 gene; Cell Nucleus; Complex; Cooperative Behavior; Coumarins; Crowding; Crystallography; Cytochrome P450; Data; Development; Drug Interactions; Electrostatics; Engineering; Enzyme Inhibition; Enzymes; Family; Health Care Costs; Heme; Histidine; Human; In Vitro; Iron; Kinetics; Ligand Binding; Ligands; Link; Measures; Modeling; Modification; Molecular Conformation; Mutagenesis; Nitrogen; Organic Synthesis; Pharmaceutical Preparations; Phenacetin; Phenytoin; Play; Pliability; Principal Investigator; Property; Protein Analysis; Protein Isoforms; Protein Region; Proteins; Pyrenes; Quantitative Structure-Activity Relationship; Reaction; Research; Resolution; Role; Scanning; Series; Shapes; Site; Site-Directed Mutagenesis; Structural Models; Structure; Substrate Specificity; Therapeutic Agents; Warfarin; alpha benzopyrone; analog; base; cytochrome P-450 CYP2C subfamily; enzyme model; inhibitor/antagonist; mutant; novel therapeutics; pharmacophore; programs; prospective; pyrene; small molecule; tool

The long-term aim of this research is to understand the structural basis underlying the unique substrate specificities of the human CYP2C enzymes. Enzyme inhibition during polytherapy, particularly for CYP2C9, is a general mechanism underlying severe drug-drug interactions with agents such as warfarin and phenytoin. Adverse drug reactions of this type are a major, but potentially avoidable, drain on health care costs in the US. However, our ability to predict inhibitory drug-drug interactions from data obtained in vitro is limited by the lack of high resolution structures and robust pharmacophore models for these P450 enzymes, as well by complications imposed by the observation of atypical Michaelis-Menten kinetics and Type II inhibitor complex formation. This project addresses these limitations with the following specific aims: Specific Aim 1: Construct new CYP2C CoMFA models by determining the inhibition constants for a series of benzbromarone analogs to determine which features confer strong binding interactions with CYP2C9, CYP2C19 and CYP2C8. Specific Aim 2: Determine the effect of pKa and steric factors on strength of type II interactions by measuring binding affinities to CYP2C9 for a series of coumarin-based ligand molecules with and without nitrogen-bearing substituents and with different steric features adjacent to the nitrogen. Specific Aim 3: (a) Determine the structural basis for homotropic cooperative behavior of phenacetin (and pyrene) with human CYP2C9 and CYP2C19 through site-directed mutagenesis, (b) develop complementary NMR approaches to the study of human CYP2C cooperativity. Specific Aim 4: (a) Delineate the role of Arg108 in CYP2C9 substrate selectivity; (b) engineer a soluble, monomeric form of the ultra-stable, iron-nitrogen coordinated Arg108His mutant, and (c) develop histidine scanning mutagenesis and formation of the iron-nitrogen complex as a tool for examining the role of CYP2C9 B-C and F-G loop region mobility in substrate specificity. In this manner, we will bring together organic synthesis, QSAR analysis of Type I and Type II ligands, site-directed mutagenesis, conformational analysis of protein flexibility and, ultimately, crystallography of human CYP2C mutants, to develop an integrated picture of ligand interactions with these important human enzymes that will enable us to assess, in a prospective manner, the potential for inhibitory drug-drug interactions.

这项研究的长期目标是了解人类CYP2C酶独特底物特异性的结构基础。在综合治疗过程中的酶抑制，特别是对CYP2C9的抑制，是导致与华法林和苯妥英等药物发生严重药物相互作用的一般机制。这类药物不良反应是美国医疗保健成本的主要消耗，但可能是可以避免的。然而，我们从体外获得的数据预测抑制性药物相互作用的能力受到以下因素的限制：缺乏这些P450酶的高分辨率结构和强大的药效团模型，以及观察到非典型的Michaelis-Menten动力学和II型抑制物复合体形成所带来的并发症。该项目旨在解决这些限制，具体目标如下： 具体目标1：通过测定一系列苯溴马龙类似物的抑制常数来构建新的CYP2C CoMFA模型，以确定哪些特征与CYP2C9、CYP2C19和CYP2C8具有较强的结合作用。 具体目标2：通过测量一系列香豆素配体分子与CYP2C9的结合亲和力来确定PKA和空间因素对第二类相互作用强度的影响。这些分子带有和不带有含氮取代基，并且在氮附近具有不同的空间特征。 具体目标3：(A)通过定点突变，确定非那西丁(和芘)与人CYP2C9和CYP2C19同质协同行为的结构基础，(B)建立互补的核磁共振方法来研究人CYP2C的协同作用。 具体目标4：(A)描述Arg108在CYP2C9底物选择性中的作用；(B)设计一种可溶的、单体形式的超稳定、铁-氮配位的Arg108His突变体，以及(C)开发组氨酸扫描突变和铁-氮复合体的形成，以此作为研究CYP2C9 B-C和F-G环区迁移率在底物特异性中的作用的工具。 通过这种方式，我们将把有机合成、I型和II型配体的QSAR分析、定点突变、蛋白质灵活性的构象分析以及最终人类CYP2C突变的结晶学结合在一起，形成配体与这些重要的人类酶相互作用的完整图景，使我们能够以前瞻性的方式评估抑制药物相互作用的可能性。