KINETIC AND MOLECULAR DYNAMICS CORRELATIONS IN CYTOCHROME P450 2C9 MUTANTS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 药物间相互作用改变了细胞色素P450介导的单药代谢的体外和体内动力学，并有可能降低药物治疗的疗效。某些细胞色素P450亚型表现出某些底物代谢的代谢增加或非典型动力学。我们和其他人认为，活性位点中两种底物的同时结合（双位点模型）是大多数非典型动力学特征的原因（例如，氨苯砜和结构相关底物激活氟比洛芬、萘普生和吡罗昔康的CYP2C9代谢）。动力学数据表明底物和活化剂都存在于活性位点。与野生型蛋白质相比，CYP2C9多态性导致酶催化活性降低和效应分子的更大激活，这些活性变化的机制尚未完全阐明。 我们假设激活剂通过将底物重新定位在活性位点附近，增加代谢的可能性来引起非典型动力学，并且这是由底物-激活剂、底物-活性位点和激活剂-活性位点相互作用的组合引起的，其可以通过分子建模和定点诱变实验的组合来阐明。 选定的突变涉及的关键位点，已被牵连在调节催化活性将研究分子建模方法，并与这些突变体的实验动力学数据。 这种比较将确定这些关键位点是否是导致β 2C9的各种突变体之间活性变化的原因，以及改变动力学的可能原因。 这些突变的动力学将与这些距离变化和研究的两者之间的相关性进行比较。突变体是R108I和N204I，因为它们负责活性位点内的底物结合。将研究其他突变体E300I、S209A、T304A和N474I，因为它们可能决定效应子的结合方向。这将提供有助于开发用于预测底物-效应物组合何时会引起非典型动力学的模型的信息，并提供对细胞色素P450 2C9介导的代谢的更好理解。

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.