STRUCTURE AND FUNCTION OF MAMMALIAN CYTOCHROMES P450

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中出现。列出的机构是中心的机构，不一定是研究者的机构。细胞色素 P450 是消除多种异生物质（包括药物、环境污染物和致癌物质）的第一步。来自家族 1、2 和 3 的 P450 作用于不同但重叠的不同底物组。引起代谢差异的分子相互作用在很大程度上是不确定的，但它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，并以预测方式使用。该提案的目标是阐明人类 2A 和 2E P450 不同但重叠的底物特异性的结构基础。假设是，由于共同底物和不同底物都会被代谢，因此只有少数特定的相互作用才能区分这两个 P450 亚家族的代谢能力。识别这些相互作用是预测重要候选药物和环境外源性物质代谢的第一步。首先，将鉴定 2A 和 2E P450 中定向标记底物的活性位点残基。定点诱变与常见和差异底物分析的结合将用于识别负责不同代谢与重叠代谢的相互作用。其次，我们建议生成细胞色素 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度，并对其聚集状态、稳定性和功能进行表征。有前途的候选者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。 2A 和 2E 酶之间以及与其他亚家族的 P450 之间的配体相互作用的比较预计将产生对 P450 结构功能多样性的实质性见解。这项工作将显着增强我们阐明人类 P450 之间差异代谢和抑制机制的能力，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.