Structural Basis of Cytochrome P450 2A13 Activity

拟议研究的目的是通过以下方式确定负责选择性代谢的相互作用： 细胞色素P450 2A 13，一种人类肺特异性酶。2A 13具有最高的已知活化活性 4-（甲基亚硝胺基）-1-（3-吡啶基）- 1-丁酮（NNK），烟草中的主要致癌物。体内 2A 13活性的降低与人肺腺癌的显著降低相关， 提示选择性抑制2A 13作为潜在的化学预防策略。选择性设计 抑制剂需要参与前致癌原活化的P450缺乏的结构信息， 包括2A 13。早期对药物代谢肝P450的结构和功能的研究， 鉴定了一组通用的活性位点残基，其分子特征定义了底物特异性。 这一建议的中心假设是，一个或多个基因之间的特定相互作用， 相应的2A 13活性位点残基和优选的2A 13底物精确地决定代谢， 2A13这一假设将通过实验方法的组合进行测试，包括定点 诱变，异源表达E.大肠杆菌，各种功能测定和酶抑制 研究和X射线晶体学。具体目标是：1）确定X射线晶体结构 2）定义单个活性位点和天然多态性残基对2A 13的贡献 蛋白质结构和功能，以及3）评估已知和疑似家族2A抑制剂的选择性， 2A 13对比2A 6。本申请中提出的研究具有重要意义，因为它有望产生 肝外细胞色素P450与其特异性相互作用的新的重要信息， 配体。这些结果可能为理解差异底物提供更好的科学依据 肺和肝脏中相关P450的选择性以及原致癌物激活的分子结果。的 最终目标是开发和评估通过2A 13抑制的化学预防策略。 这项拟议中的研究有望阐明肺与肺之间的结构和功能差异。 一种激活烟草致癌物的酶和一种密切相关的肝酶， 体内的化学物质。这些差异可以用来了解癌症风险的差异 并开发预防烟草相关肺癌的方法。

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.