DAPSONE ACTIVATION OF C4P2C9: A MOLECULAR MODELING STUDY

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。细胞色素P450酶介导各种外源性和内源性化合物的代谢，并能生物激活前致癌物，如苯并[a]-芘。许多药物-药物相互作用是由其中一种药物对参与另一种药物代谢的P450亚型活性的影响引起的。某些同工异构体对某些底物的代谢表现出非典型动力学。我们和其他人已经提出，活性位点的两个底物同时结合（两个位点模型）是大多数非典型动力学特征的原因。氨苯砜和结构相关底物已被证明可激活氟比洛芬、萘普生和吡罗西康的CYP2C9代谢。动力学数据表明底物和活化剂都存在于活性部位。实验上，我们进行了动力学和核磁共振研究，并证明了氟比洛芬和氨苯砜在CYP2C9活性位点同时结合，尽管分辨率很低。在这里，我们建议建立一个分子模型，可以用来预测是否可能同时结合，因为动力学和核磁共振方法不能作为高通量筛选方法。我们拟利用对接方法和分子动力学（MD）模拟，探讨氟比洛芬和氨苯砜结合CYP2C9活性位点及氨苯砜活化CYP2C9的结构。具体来说，我们将i)对CYP2C9与氟比porfen，萘普生和吡罗西康单独进行扩展的MD模拟，并在活性位点停靠的氨苯砜存在的情况下，ii)通过分子建模方法对选定的氨苯砜类似物进行动力学筛选，并与NMR数据相关联，测试和验证计算模型。iii)比较野生型CYP2C9与F114L突变体在活性位点底物的取向；iv)比较野生型CYP2C9与F476L突变体在活性位点底物的取向。从这些研究中，我们将证实氨苯砜激活CYP2C9的两个位点模型的可能性，并将结果与核磁共振和动力学数据相关联。项目的成功完成将利用计算技术深入了解氨苯砜激活氟比洛芬、萘普生和吡罗西康的CYP2C9代谢机制。这种方法也可能是一个有用的工具，以确定是否一个两个位点的模型可以解释所有类别的非典型动力学。这些计算研究的结果将提供一种方法，通过这种方法，可以在昂贵的、耗时的体内研究之前，用计算工具预测有害和/或有益的药物-药物相互作用。它还将对药物和药物辅助设计领域产生重大影响。最后，本文开发的方法虽然仅针对CYP2C9，但也适用于其他P450亚型和底物。

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. The cytochrome P450 enzymes mediate the metabolism of various xenobiotic and endogenous compounds and can bioactivate pro-carcinogens such as benzo-[a]-pyrene. Many drug-drug interactions are caused by the effect of one of the drugs on the activity of the P450 isoforms involved in the metabolism of the second drug. Some isoforms demonstrat 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. Dapsone and structurally related substrates, have been shown to activate CYP2C9 metabolism of flurbiprofen, naproxen, and piroxicam. The kinetic data suggest both substrate and activator are present in the active site. Experimentally, we conducted kinetic and NMR studies and demonstrated the simultaneous binding of flurbiprofen and dapsone in the active site of CYP2C9, though at low resolution. Here we propose to develop a molecular model that can be used to predict whether simultaneous binding is likely as the kinetic and NMR methods can not be used as a high throughput screening method. We propose to explore the structure of the binding of flurbiprofen and dapsone to the active site of CYP2C9 and activation of CYP2C9 by dapsone utilizing docking methods and molecular dynamical (MD) simulations. Specifically, we will i) perform extended MD simulations of CYP2C9 with flurbiporfen, naproxen, and piroxicam alone and in the presence of dapsone docked in the active site, ii) test and validate the computational model by performing kinetic screens of selected dapsone analogs from a molecular modeling approach and correlate with NMR data, iii) compare wild type CYP2C9 to the F114L mutant with respect to the orientations of substrates in the active site and iv) compare wild type CYP2C9 to the F476L mutant, also with respect to the orientations of substrates in the active site. From these studies we will confirm the possibility of a two-site model for the activation of CYP2C9 by dapsone and correlate the results with NMR and kinetic data. Successful completion of the project will provide insight into the mechanism of activation of CYP2C9 metabolism of flurbiprofen, naproxen and piroxicam by dapsone using computational techniques. This method may also be a useful tool in determining if a two-site model can explain all categories of atypical kinetics. The results of these computational studies will provide a method by which harmful and/or beneficial drug-drug interactions can be predicted with computational tools prior to costly, time intensive in vivo studies. It will also have significant impact in the area of drug and drug helper design. Finally, the method develops here, though specific for CYP2C9, will be applicable to other P450 isoforms and substrates.