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West Virginia IDeA Network of Biomedical
Research Excellence |
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Jarrett Aguilar
West Liberty State CollegeDapsone
Activation of CYP2C9: A Molecular Modeling Study
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| Abstract: 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 develope here, though
specific for CYP2C9, will be applicable to other P450 isoforms and
substrates. |
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