DAPSONE ACTIVATION OF C4P2C9: A MOLECULAR MODELING STUDY

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。细胞色素P450酶介导各种异生物质和内源性化合物的代谢，并可生物活化致癌物，如苯并[a]芘。许多药物相互作用是由一种药物对参与第二种药物代谢的P450亚型活性的影响引起的。某些异构体显示某些底物代谢的非典型动力学。我们和其他人已经提出，同时结合的两个底物的活性位点（两个网站模型）是负责最不典型的动力学配置文件。已证明氨苯砜和结构相关底物可激活氟比洛芬、萘普生和吡罗昔康的CYP 2C 9代谢。动力学数据表明底物和活化剂都存在于活性位点。在实验上，我们进行了动力学和NMR研究，并证明了氟比洛芬和氨苯砜的CYP 2C 9的活性位点的同时结合，虽然在低分辨率。在这里，我们建议开发一个分子模型，可以用来预测是否同时结合是可能的动力学和NMR方法不能被用作高通量筛选方法。我们建议利用对接方法和分子动力学（MD）模拟来探索氟比洛芬和氨苯砜与CYP 2C 9活性位点结合的结构以及氨苯砜对CYP 2C 9的激活作用。具体而言，我们将i）单独使用氟吡泊芬、萘普生和吡罗昔康以及在活性位点中对接的氨苯砜存在下，对CYP 2C 9进行扩展的MD模拟，ii）通过从分子建模方法中对选定的氨苯砜类似物进行动力学筛选并与NMR数据相关联来测试和验证计算模型，iii）比较野生型CYP 2C 9与F114 L突变体在活性位点中底物的取向，和iv）比较野生型CYP 2C 9与F476 L突变体，也比较活性位点中底物的取向。从这些研究中，我们将确认氨苯砜激活CYP 2C 9的双位点模型的可能性，并将结果与NMR和动力学数据相关联。该项目的成功完成将提供深入了解氨苯砜激活CYP 2C 9代谢氟比洛芬，萘普生和吡罗昔康的机制，使用计算技术。这种方法也可能是一个有用的工具，在确定是否一个双网站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，通过该方法，在昂贵的、时间密集的体内研究之前，可以用计算工具预测有害和/或有益的药物-药物相互作用。它也将在药物和药物辅助者设计领域产生重大影响。最后，本文开发的方法虽然对CYP 2C 9具有特异性，但将适用于其他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.