Structural Basis of Cytochrome P450 2A13 Activity

描述(申请人提供)：拟议研究的目标是确定负责细胞色素P450 2A13选择性新陈代谢的相互作用，细胞色素P450 2A13是一种人类肺特异性酶。2A13对烟草中的主要致癌物质4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone的激活活性最高。在体内，2A13活性的降低与人类肺腺癌的大幅减少相关，这表明选择性抑制2A13是一种潜在的化学预防策略。设计选择性抑制剂需要结构信息，而参与原癌原激活的P450缺乏结构信息，包括2A13。早期对药物代谢的肝脏P450的结构和功能的研究已经确定了一组普遍的活性部位残基，其分子特征决定了底物的特异性。这一建议的中心假设是，一个或多个相应的2A13活性位点残基与首选的2A13底物之间的特定相互作用准确地决定了2A13的代谢。这一假设将通过一系列实验方法来验证，包括定点突变、在大肠杆菌中的异源表达、各种功能分析和酶抑制研究，以及X射线结晶学。其具体目的是：1)确定2A13的X射线晶体结构；2)确定单个活性部位和自然多态残基对2A13蛋白质结构和功能的贡献；3)评估已知和可疑的2A13家族抑制剂对2A13和2A6的选择性。在这一应用中提出的研究具有重要意义，因为它有望产生关于肝外细胞色素P450与其配体的特定相互作用的新的重要信息。这些结果可能为理解相关P450在肺和肝脏中的不同底物选择性以及致癌原激活的分子结果提供更好的科学依据。最终目标是通过抑制2A13来开发和评估化学预防策略。这项拟议的研究预计将阐明激活烟草致癌物质的肺酶和清除体内外来化学物质的密切相关的肝酶之间的结构和功能差异。这些差异可以被用来了解个体之间癌症风险的差异，并开发预防烟草相关肺癌的方法。

DESCRIPTION (provided by applicant): 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-pyridyl)-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.