HUMAN CYP2C MODELS

本研究的长期目标是了解人类CYP 2C酶独特底物特异性的结构基础。多药治疗期间的酶抑制，尤其是对CYP 2C 9的酶抑制，是与华法林和苯妥英等药物发生重度药物相互作用的一般机制。在美国，这类药物不良反应是医疗保健费用的主要来源，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-药物相互作用的能力受到这些P450酶缺乏高分辨率结构和稳健药效团模型以及观察非典型Michaelis-Menten动力学和II型抑制剂复合物形成所引起的并发症的限制。该项目旨在解决这些限制因素，具体目标如下： 具体目标1：通过测定一系列苯溴马隆类似物的抑制常数构建新的CYP 2C CoMFA模型，以确定哪些特征赋予与CYP 2C 9、CYP 2C 19和CYP 2C 8的强结合相互作用。 具体目标二：通过测量一系列香豆素类配体分子与CYP 2C 9的结合亲和力，确定pKa和空间因子对II型相互作用强度的影响，这些香豆素类配体分子有和没有含氮取代基，并且在氮附近具有不同的空间特征。 具体目标3：（a）通过定点诱变确定非那西丁（和芘）与人CYP 2C 9和CYP 2C 19同向协同行为的结构基础，（B）开发互补的NMR方法来研究人CYP 2C协同性。 具体目标4：（a）阐明Arg 108在CYP 2C 9底物选择性中的作用;（B）设计超稳定、铁-氮配位的Arg 108 His突变体的可溶性单体形式，以及（c）开发组氨酸扫描诱变和铁-氮复合物的形成，作为检查CYP 2C 9 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，我们将汇集有机合成，I型和II型配体的QSAR分析，定点诱变，蛋白质灵活性的构象分析，并最终，人类CYP 2C突变体的晶体学，以开发配体与这些重要的人类酶相互作用的综合图片，使我们能够以前瞻性的方式评估抑制性药物相互作用的潜力。

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.