DAPSONE ACTIVATION OF C4P2C9: A MOLECULAR MODELING STUDY

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。细胞色素P450酶参与多种外源和内源化合物的代谢，并能激活苯并[a]芘等致癌物质。许多药物-药物相互作用是由于其中一种药物对第二种药物代谢所涉及的P450亚型活性的影响而引起的。某些异构体表现出某些底物代谢的非典型动力学。我们和其他人已经提出，活性位点上两个底物的同时结合(双位点模型)是造成大多数非典型动力学图谱的原因。氨苯砜和结构相关的底物，已被证明激活了氟比洛芬、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。在实验上，我们进行了动力学和核磁共振研究，证明了氟比洛芬和氨苯砜同时结合在CYP2C9的活性部位，尽管分辨率很低。在这里，我们建议建立一个分子模型，可以用来预测是否有可能同时结合，因为动力学和核磁共振方法不能作为高通量的筛选方法。我们拟通过对接方法和分子动力学(MD)模拟来研究氟比洛芬和氨苯砜与CYP2C9活性中心的结合结构以及氨苯砜对CYP2C9的激活作用。具体地说，我们将i)单独使用氟比波芬、萘普生和吡罗昔康并在氨苯砜停靠在活性部位的情况下对CYP2C9进行扩展的MD模拟，ii)通过对从分子建模方法中选择的氨苯酮类似物进行动力学筛选并与核磁共振数据相关联来测试和验证计算模型，iii)比较野生型和F114L突变体在活性部位的底物取向，以及iv)比较野生型和F476L突变体，也就活性部位的底物方向进行比较。通过这些研究，我们将证实氨苯砜激活CYP2C9的双位点模型的可能性，并将结果与核磁共振和动力学数据相关联。该项目的成功完成将利用计算技术深入了解氨苯磺隆对氟比洛芬、萘普生和吡罗昔康代谢的激活机制。这种方法也可能是一种有用的工具，用来确定双位模型是否能解释所有种类的非典型动力学。这些计算研究的结果将提供一种方法，可以在昂贵、耗时的体内研究之前使用计算工具预测有害和/或有益的药物-药物相互作用。它还将在毒品和毒品助手设计领域产生重大影响。最后，尽管该方法专用于细胞色素P450 C9，但也适用于其他P450异构体和底物。

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.