Structural Basis of Cytochrome P450 2A13 Activity

描述（由申请人提供）：拟议研究的目的是确定细胞色素P450 2A13（一种人类肺特异性酶）选择性代谢的相互作用。已知2A13对4-（甲基亚硝胺）-1-（3-吡啶基）-1-丁酮（NNK）的激活活性最高，NNK是烟草中的一种主要致癌物。体内2A13活性的降低与人类肺腺癌的显著减少相关，表明选择性抑制2A13是一种潜在的化学预防策略。选择性抑制剂的设计需要结构信息，这是参与致癌物原激活的p450所缺乏的，包括2A13。早期对肝脏药物代谢p450的结构和功能的研究已经确定了一组活性位点残基，其分子特征决定了底物特异性。该建议的中心假设是，一个或多个相应的2A13活性位点残基与首选2A13底物之间的特定相互作用精确地决定了2A13的代谢。这一假设将通过一系列实验方法进行验证，包括定点诱变、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射线晶体学。个体特异性目的是：1)确定2A13的x射线晶体结构，2)确定个体活性位点和天然多态性残基对2A13蛋白结构和功能的贡献，以及3)评估已知和疑似家族2A抑制剂对2A13与2A6的选择性。本应用中提出的研究具有重要意义，因为它有望为肝外细胞色素P450与其配体的特定相互作用提供新的重要信息。这些结果可能为了解肺和肝脏中相关p450的差异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。最终目标是开发和评估通过抑制2A13的化学预防策略。这项拟议中的研究有望阐明激活烟草致癌物的肺酶与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。可以利用这些差异来了解个体之间癌症风险的差异，并制定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.