This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor 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 simultaneousbinding 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 theactive 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 harmfuland/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.
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