STRUCTURE AND FUNCTION OF MAMMALIAN CYTOCHROMES P450

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。细胞色素P450在消除广泛的外源物质，包括药物，环境污染物和致癌物质的第一步。家族1、2和3的p450作用于不同的底物，但又重叠。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先决条件。我们的长期目标是生成预测p450 -配体相互作用所需的结构数据，这些数据可用于预测方式。本提案的目的是阐明人类2A和2E p450不同但重叠的底物特异性的结构基础。假设是，由于共同和独特的底物都被代谢，只有少数特定的相互作用区分这两个P450亚家族的代谢能力。确定这些相互作用是预测重要候选药物和环境外源代谢的第一步。首先，定位2A和2E p450中标记底物的活性位点残基。位点定向诱变和共同底物和差异底物分析的结合将用于确定导致不同代谢和重叠代谢的相互作用。其次，我们提出生成细胞色素P450 2E1的分子结构与2A的结构进行比较。将对2E1进行修饰以增加溶解度，并以其聚集状态、稳定性和功能为特征。有希望的候选人将成为结晶试验的主题，目的是确定高分辨率x射线结构。比较2A和2E酶之间以及与其他亚家族的P450酶之间的配体相互作用，有望对P450结构-功能多样性产生实质性的见解。这项工作将显著增强我们阐明人类p450之间差异代谢和抑制机制的能力，并对药物设计、不良药物相互作用的发生率以及人类癌症的预防和干预产生潜在影响。

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.