Metabolism of Carcinogens and Drugs by Human P450s

人体 P450 对致癌物和药物的代谢

基本信息

批准号：
7371112
负责人：
F PETER Guengerich
金额：
$ 35.36万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-05-08 至 2011-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7371112
关键词：
17p Active Sites Aminopyrine Anabolism Aromatase Binding Bispecific Antibody 2B1 Calorimetry Carcinogen Metabolism Carcinogens Catalysis Chemicals Cholesterol Clinical Collaborations Complex Coumarins Cytochrome P450 Development Dialysis procedure Disease Enzymes Equilibrium Escherichia coli Estradiol Fluorescence Genes Goals Homeostasis Human Hydroxylation Individual Indoles Isotopes Kinetics Knowledge Laboratories Lanosterol Lauric Acids Ligands Malignant Neoplasms Mass Spectrum Analysis Molecular Morphine Mutagens Opioid Orphan Pharmaceutical Preparations Physical Dialysis Physiologic pulse Proteins Pulse taking Purpose Pyrenes Rate Reaction Reagent Reporter Reporting Role Site Source Steroids Structure System Techniques Testosterone Thermodynamics Time Tissue Extracts Tissues Titrations Universities Urethane Variant Work X-Ray Crystallography alpha benzopyrone base catalyst chemical carcinogen demethylation functional genomics indole mRNA Expression mutant oxidation pyrene three dimensional structure

项目摘要

Cytochrome P450 (P450) enzymes are the major catalysts involved in the metabolism of carcinogens, drugs, and steroids. Variations in the catalytic activities have a variety of effects in homeostasis and clinical practice. Continued studies on human P450s are proposed, with a focus on molecular understanding of function. (1) Of the 57 human P450 genes, 13 still have limited if any knowledge regarding function. We propose to establish sites of mRNA expression, express these "orphan" P450s in heterologous systems, and examine their abilities to activate a wide variety of chemical carcinogens. In addition, several HPLC-mass spectrometry approaches will be used for identification of products and substrates, with tissue extracts as sources of substrates and the P450s as reagents. This part of the project is an effort towards understanding the functional genomics of human P450s. A related aspect is establishment of the roles of individual human P450s in morphine biosynthesis, for which strong evidence has been recently presented by others. (2) Comparisons of the kinetics of human P450s already studied in detail (1A2, 2A6, 2D6, 2E1, 3A4) will be done with several other P450s reported to have much higher rates of catalysis, with the goal of understanding which steps limit the (human P450) reactions. These studies will involve a variety of steady-state, pre-steady-state, and isotope effect approaches. (3) Kinetic analysis of multi-reaction P450s will be done, including P450s 51A1 (lanosterol 14a-demethylation), 19A1 (aromatase, oxidation of testosterone to 17p-estradiol), and 2A6 (oxidation of indoles), with a goal of defining the processivity of these systems. Several pre-steady-state and analysis approaches can be readily applied to the problem, with the goal of understanding the release of intermediates. (4) P450s 3A4 and 2A6 will be analyzed regarding hypotheses about cooperativity and induced fit in substrate binding and catalysis. The P450 3A4 work will focus on pre-steady-state kinetics of substrate binding, along with thermodynamic analysis of binding. The work on P450 2A6 induced fit will involve collaborative work on X-ray crystallography of mutants that have demonstrated expansion of the active site. These results should reveal whether a P450 has a fixed structure or can "adapt" to individual substrates. Collectively, these studies have the goal of providing more understanding of the roles of human P450s in oxidation of drugs, carcinogens, and endogenous compounds and their potential contributions to cancer and other diseases and roles in disease treatments.

细胞色素P450（P450）酶是参与致癌物代谢的主要催化剂，药物和类固醇。催化活性的变化对稳态和临床有多种影响实践。建议继续对人类 P450 进行研究，重点是分子理解的功能。 (1) 在 57 个人类 P450 基因中，有 13 个基因的功能知识仍然有限。我们建议建立 mRNA 表达位点，以异源表达这些“孤儿”P450 系统，并检查它们激活多种化学致癌物质的能力。此外，多种 HPLC-质谱方法将用于鉴定产品和底物，以组织提取物作为底物来源，以 P450 作为试剂。该项目的这一部分是努力了解人类 P450 的功能基因组学。一个相关的方面是建立人类个体 P450 在吗啡生物合成中的作用，对此已有强有力的证据最近由其他人提出。 (2) 已详细研究的人类P450动力学的比较（1A2、2A6、2D6、2E1、3A4）将与据报道具有更高速率的其他几台 P450 一起完成催化，目的是了解哪些步骤限制（人类 P450）反应。这些研究将涉及各种稳态、前稳态和同位素效应方法。 (3) 动力学将进行多反应 P450 的分析，包括 P450 51A1（羊毛甾醇 14a-去甲基化）， 19A1（芳香酶，将睾酮氧化为 17p-雌二醇）和 2A6（吲哚氧化），目标是定义这些系统的过程性。几种预稳态和分析方法可以很容易地应用于问题，目的是了解中间体的释放。 (4) P450s 3A4 和 2A6 将针对有关底物结合中的协同性和诱导拟合的假设进行分析催化。 P450 3A4 工作将重点关注底物结合的前稳态动力学，以及结合的热力学分析。 P450 2A6 诱导拟合工作将涉及协作工作对已证明活性位点扩展的突变体进行 X 射线晶体学分析。这些结果应揭示 P450 是否具有固定结构或可以“适应”单独的基材。总的来说，这些研究的目标是更好地了解人类 P450 在药物氧化中的作用，致癌物和内源性化合物及其对癌症和其他疾病的潜在贡献在疾病治疗中的作用。