HUMAN CYP2C MODELS

本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。多药治疗期间的酶抑制，特别是CYP2C9，是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费用的主要消耗，但有可能是可以避免的。然而，由于缺乏这些P450酶的高分辨率结构和强大的药效团模型，以及观察非典型Michaelis-Menten动力学和II型抑制剂复合物形成所带来的并发症，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限制。本项目旨在解决这些限制，具体目标如下：

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.