STRUCTURE AND FUNCTION OF MAMMALIAN CYTOCHROMES P450

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。细胞色素P450是消除多种异生物质的第一步，包括药物、环境污染物和致癌物。来自家族1、2和3的P450作用于不同底物的不同但重叠的集合。引起差异代谢的分子相互作用在很大程度上是不确定的，但这是预测体内药物代谢的重要前提。我们的长期目标是生成能够以预测性方式使用的P450-配体相互作用预测所需的结构数据。这项建议的目标是阐明人类2A和2E P450不同但重叠的底物特异性的结构基础。假设由于共同和不同的底物都被代谢，只有少数几个特定的相互作用区分了这两个P450亚家族的代谢能力。确定这些相互作用是迈向重要候选药物和环境异生物质预测新陈代谢的第一步。首先，将确定在2A和2E P450中定位标记底物的活性位点残基。结合定点突变和对共同和不同底物的分析，将被用来确定导致不同代谢和重叠代谢的相互作用。第二，我们建议生成一个细胞色素P450 2E1的分子结构，以便与2A结构进行比较。2E1将被修饰以增加溶解性，并以其聚集状态、稳定性和功能为特征。有希望的候选人(S)将成为结晶试验的对象，目的是确定高分辨率的X射线结构。比较2A和2E酶之间的配基相互作用以及与其他亚家族的P450之间的配基相互作用有望对P450的结构-功能多样性产生实质性的见解。这项工作将极大地提高我们阐明人类P450之间差异代谢和抑制机制的能力，这些P450对药物设计、药物不良相互作用的发生率以及人类癌症的预防和干预具有潜在的影响。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Cytochromes P450 perform the first step in eliminating a wide range of xenobiotics, including drugs, environmental contaminants, and procarcinogens. P450s from families 1, 2, and 3 act on distinct yet overlapping sets of diverse substrates. The molecular interactions giving rise to differential metabolism are largely undefined but are a vital prerequisite for predicting drug metabolism in vivo. Our long-term goal is to generate the structural data required to enable prediction of P450-ligand interactions that can be used in a predictive manner. The goal of this proposal is to elucidate the structural basis for the differing but overlapping substrate specificities of human 2A and 2E P450s. The hypothesis is that since both common and distinct substrates are metabolized, only a few specific interactions distinguish the metabolic capabilities of these two P450 subfamilies. Identifying those interactions is a first step toward predictive metabolism of important drug candidates and environmental xenobiotics. First, active site residues orienting marker substrates in 2A and 2E P450s will be identified. A combination of site-directed mutagenesis and analysis with common and differential substrates will be used to identify interactions responsible for distinct vs. overlapping metabolism. Second, we propose to generate a molecular structure of cytochrome P450 2E1 for comparison with 2A structures. 2E1 will be modified to increase solubility and characterized for its aggregation state, stability, and function. Promising candidate(s) will be the subject of crystallization trials with the intent to determine a high-resolution x-ray structure. Comparison of ligand interactions both between the 2A and 2E enzymes and with P450s from other subfamilies is expected to yield substantial insights into P450 structure-function diversity. This work will significantly enhance our ability to elucidate mechanisms of differential metabolism and inhibition between human P450s with potential impact on drug design, incidence of adverse drug-drug interactions, and prevention of and intervention in human carcinoma.