Structural Basis of Cytochrome P450 2A13 Activity

拟议研究的目标是确定与选择性新陈代谢有关的相互作用 细胞色素P450 2A13，一种人肺特异的酶。2A13具有最高的激活活性 烟草素(4-(methylnitrosamino)-1-(3-pyridy！)-1-butanone)是烟草中的一种主要致癌物质。活体内 2A13活性的降低与人类肺腺癌的大幅减少有关， 提示选择性抑制2A13是一种潜在的化学预防策略。有选择的设计 抑制剂需要参与原癌原激活的P450所缺乏的结构信息， 包括2A13。早期对药物代谢的肝脏P450的结构和功能的研究有 确定了一组活性位点残基的一般集合，其分子特征决定了底物的特异性。 这一提议的中心假设是，一个或多个 相应的2A13活性中心残基和首选的2A13底物通过 2A13.这一假设将通过实验方法的组合来验证，包括现场定向 诱变、在大肠杆菌中的异源表达、多种功能检测和酶抑制 研究和X射线结晶学。个人的具体目标是：1)确定X射线晶体结构 2)定义了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)评估已知和可疑的2A族抑制剂的选择性 2A13与2A6。本申请中提出的研究具有重要意义，因为它有望生成 肝外细胞色素P450与其相互作用的新的重要信息 配基。这些结果可能为理解差异底物提供更好的科学基础。 肺和肝脏中相关P450的选择性和致癌原激活的分子结果。这个 最终目标是开发和评估通过抑制2A13的化学预防策略。 这项拟议的研究有望阐明肺在结构和功能上的差异。 激活烟草致癌物质的酶和一种与之密切相关的清除外来物质的肝酶 体内的化学物质。这些差异可以被用来理解癌症风险的差异 在个人之间，并开发预防与烟草相关的肺癌的方法。

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.