Structural Basis of Cytochrome P450 2A13 Activity

细胞色素 P450 2A13 活性的结构基础

• 批准号: 7336834
• 负责人: Emily E Scott
• 金额: $ 27.07万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7336834
• 关键词: Active Sites; Address; Area; Binding; Biochemistry; Biological; Biological Assay; Biological Availability; Butanones; Carcinogens; Characteristics; Chemicals; Chemoprevention; Cytochrome P450; Data; Development; Enzymatic Biochemistry; Enzyme Inhibition; Enzymes; Escherichia coli; Extrahepatic; Family; Genetic Polymorphism; Goals; Health; Hepatic; Holoenzymes; Human; Individual; Investigation; Knowledge; Lead; Libraries; Ligand Binding; Ligands; Link; Liver; Lung; Lung Adenocarcinoma; Malignant neoplasm of lung; Measures; Mediating; Membrane; Membrane Proteins; Metabolic; Metabolic Biotransformation; Metabolism; Methods; Molecular; Molecular Conformation; Molecular Structure; Numbers; Outcome; Physiological; Play; Positioning Attribute; Postdoctoral Individual National Research Service Award; Proteins; Research; Research Personnel; Roentgen Rays; Role; Series; Site; Site-Directed Mutagenesis; Structure; Substrate Specificity; System; Testing; Therapeutic Agents; Tissues; Tobacco; Tobacco smoke; Tobacco-Associated Carcinogen; Toxicology; Toxin; Training; X-Ray Crystallography; Xenobiotic Metabolism; Xenobiotics; base; cancer chemoprevention; cancer risk; cancer therapy; concept; defined contribution; design; drug metabolism; drug structure function; experience; improved; in vivo; inhibitor/antagonist; innovation; insight; interest; mutant; prevent; programs; protein structure function; small molecule

The objective of the proposed studies is to identify interactions responsible for selective metabolism by cytochrome P450 2A13, a human lung-specific enzyme. 2A13 has the highest known activity for activation of 4-(methylnitrosamino)-1-(3-pyridy!)-1-butanone (NNK), a primary carcinogen in tobacco. In vivo decreases in 2A13 activity have been correlated with substantial reductions in human lung adenocarcinoma, suggesting selective inhibition of 2A13 as a potential chemoprevention strategy. Design of selective inhibitors requires structural information that is lacking for P450s involved in procarcinogen activation, including 2A13. Earlier studies on the structure and function of drug-metabolizing hepatic P450s have identified a general set of active site residues whose molecular characteristics define substrate specificity. The central hypothesis of this proposal is that specific interactions between one or more of the corresponding 2A13 active site residues and preferred 2A13 substrates precisely dictate metabolism by 2A13. This hypothesis will be tested by a combination of experimental approaches, including site-directed mutagenesis, heterologous expression in E. coli, a variety of functional assays and enzyme inhibition studies, and X-ray crystallography. The individual specific aims are to: 1) determine X-ray crystal structures of 2A13, 2) define the contributions of individual active site and naturally polymorphic residues to 2A13 protein structure and function, and 3) evaluate the selectivity of known and suspected family 2A inhibitors for 2A13 versus 2A6. The research proposed in this application is significant because it is expected to generate new and important information on the specific interactions of extrahepatic cytochromes P450 with their ligands. These results may provide an improved scientific basis for understanding the differential substrate selectivity of related P450s in lung and liver and the molecular results of procarcinogen activation. The ultimate goal is to develop and evaluate chemoprevention strategies via 2A13 inhibition. The proposed research is expected to elucidate structural and functional differences between a lung enzyme that activates tobacco carcinogens and a closely-related liver enzyme that removes foreign chemicals from the body. These differences could be exploited to understand differences in cancer risk between individuals and to develop methods to prevent tobacco-associated lung cancer.

拟议研究的目的是确定负责选择性代谢的相互作用 细胞色素P450 2A13，一种人类肺特异性酶。 2A13的激活活动最高 4-（甲基硝基氨基氨基）-1-（3吡啶！） - 1-丁酮（NNK），一种烟草中的主要致癌物。体内 2A13活性的降低与人类肺腺癌的大幅降低相关， 建议选择性抑制2A13作为潜在的化学预防策略。选择性设计 抑制剂需要参与procarcinogen激活的P450缺少的结构信息， 包括2A13。关于药物代谢肝P450的结构和功能的早期研究具有 确定了一组一组活性位点残基，其分子特性定义了底物特异性。 该提议的核心假设是一个或多个之间的特定相互作用 相应的2A13活性位点残基和首选2A13底物精确决定了代谢 2A13。该假设将通过实验方法的组合来检验 诱变，大肠杆菌中异源表达，多种功能测定和酶抑制 研究和X射线晶体学。单个具体目的是：1）确定X射线晶体结构 2A13，2）定义单个活性位点和自然多态残基对2A13的贡献 蛋白质结构和功能，以及3）评估已知和可疑家族2A抑制剂的选择性 2A13与2A6。本应用程序中提出的研究很重要，因为它有望产生 有关肝外细胞色素P450与他们的特定相互作用的新的重要信息 配体。这些结果可能为理解差异基材提供了改进的科学基础 肺和肝脏中相关P450的选择性以及procarcinogen激活的分子结果。这 最终目标是通过2A13抑制制定和评估化学预防策略。 预计拟议的研究将阐明肺之间的结构和功能差异 激活烟草致癌物的酶和与外国去除的密切相关的肝脏酶 体内的化学物质。这些差异可以被利用以了解癌症风险的差异 在个体之间并开发预防烟草相关的肺癌的方法。