KINETIC AND MOLECULAR DYNAMICS CORRELATIONS IN CYTOCHROME P450 2C9 MUTANTS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 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。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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 substrater 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 responsiblefor 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.