KINETIC AND MOLECULAR DYNAMICS CORRELATIONS IN CYTOCHROME P450 2C9 MUTANTS

细胞色素 P450 2C9 突变体的动力学和分子动力学相关性

• 批准号: 8360183
• 负责人: JARRETT AGUILAR
• 金额: $ 18.16万
• 依托单位: MARSHALL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8360183
• 关键词: Active Sites; Binding; Biomedical Research; CYP2C9 gene; Cytochrome P450; Dapsone; Data; Development; Drug Interactions; Enzymes; Flurbiprofen; Funding; Genetic Polymorphism; Grant; In Vitro; Kinetics; Mediating; Metabolism; Methods; Modeling; Molecular Models; Mutation; Naproxen; National Center for Research Resources; Pharmaceutical Preparations; Piroxicam; Principal Investigator; Probability; Protein Isoforms; Proteins; Research; Research Infrastructure; Resources; Site; Site-Directed Mutagenesis; Source; United States National Institutes of Health; West Virginia; base; cost; drug efficacy; human CYP2C9 protein; improved; in vivo; molecular dynamics; molecular modeling; molecular site; mutant; research study

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Drug-drug interactions alter the in vitro and in vivo kinetics of cytochrome P450-mediated metabolism of single drugs and have the potential for reducing the efficacy of drug based therapy. Some cytochrome P450 isoforms demonstrate increased metabolism or atypical kinetics for the metabolism of certain substrates. We and others have suggested that simultaneous binding of two substrates in the active site (a two-site model) is responsible for most atypical kinetic profiles (e.g. dapsone and structurally related substrates activate CYP2C9 metabolism of flurbiprofen, naproxen, and piroxicam). The kinetic data suggest both substrate and activator are present in the active site. CYP2C9 polymorphisms have resulted in reduced enzyme catalytic activity and greater activation by effector molecules as compared to wild-type protein, with the mechanism(s) for these changes in activity not fully elucidated. We hypothesize that activators cause atypical kinetics by repositioning the substrate nearer the active site, increasing the probability of metabolism, and that this is caused by a combination of substrate-activator, substrate-active site, and activator-active site interactions that can be elucidated through a combination of molecular modeling and site-directed mutagenesis experiments. Selected mutations involving key sites that have been implicated in regulating catalytic activity will be studied by molecular modeling methods and correlated with the experimental kinetic data of these mutants. This comparison will determine if these key sites are responsible for the changes in activity between various mutants of CYP 2C9 and the likely causes of the altered kinetics. The kinetics for these mutations will be compared to these distance changes and correlations between the two studied. The mutants are R108I and N204I as they are responsible for substrate binding within the active site. Other mutants E300I, S209A, T304A, and N474I will be studied as they may determine binding orientation of the effector. This will provide information that will aid in the development of a model for predicting when substrate-effector combinations will cause atypical kinetics and provide an improved understanding of cytochrome P450 2C9 mediated metabolism.

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 药物之间的相互作用改变了细胞色素P450介导的单一药物的体外和体内代谢动力学，并有可能降低基于药物的治疗的疗效。一些细胞色素P450亚型表现出代谢增加或某些底物代谢的非典型动力学。我们和其他人认为，活性部位两种底物的同时结合(双位点模型)是导致大多数非典型动力学特征的原因(例如，氨苯砜和结构相关的底物激活了氟比洛芬、萘普生和吡罗昔康的CYP2C9代谢)。动力学数据表明，活性中心同时存在底物和活化剂。与野生型蛋白相比，CYP2C9基因的多态导致酶的催化活性降低和效应分子的激活作用增强，但这些活性变化的机制(S)尚未完全阐明。我们假设激活剂通过将底物重新定位到更接近活性部位的位置来引起非典型动力学，增加代谢的可能性，这是由底物-激活剂、底物-活性部位和激活剂-活性部位相互作用的组合引起的，这些相互作用可以通过分子建模和定点突变实验来阐明。涉及调控催化活性的关键位点的选定突变将通过分子建模方法进行研究，并与这些突变的实验动力学数据相关联。这一比较将确定这些关键位点是否对CYP 2C9的不同突变体之间的活性变化负责，以及导致动力学变化的可能原因。这些突变的动力学将与这些距离变化和两者之间的相关性进行比较。突变体是R108I和N204I，因为它们负责活性部位内的底物结合。其他突变体E300I、S209A、T304A和N474I将被研究，因为它们可能决定效应器的结合方向。这将有助于开发预测底物-效应器组合何时会导致非典型动力学的模型，并提供对细胞色素P450 2C9介导的新陈代谢的更好理解。