Cytochrome P450's (CYPs) are a major source of metabolic drug interactions. The allosteric behavior ofCYPs, including the major drug metabolizing isoform CYP3A4, continues to confound in vitro - In vivocorrelations and the prediction of drug interactions based on in vitro data. The molecular mechanisms thatconfer allosteric behavior are incompletely understood, and improved mechanistic models are likely toimprove predictive drug metabolism. Moreover, although allosteric behavior in vitro is well known and widelyobserved, examples of in vivo allosterism are limited. Among the multiple mechanisms that contribute,occupancy by small molecule drugs at a peripheral effector site of CYP3A4, and conformational changesinduced by the electron transfer partner Cytochrome bs (Cyt bs), are likely to converge on a set of commoneffects, wherein the active site is more efficiently desolvated or 'well-packed' through protein-proteininteractions or multiple drug binding. In turn this, hypothetically, leads to more efficient coupling of NADPHconsumption and O2 reduction, concomitant with drug oxidation, with decreased 'uncoupling' to form reducedoxygen species. This proposal aims to: 1) increase our understanding of allosteric CYP mechanisms withmodel probe drugs, and 2) to complete a comparative, and mechanistic, analysis of CYP-Cyt b5 interactions.The fluorescent probes Nile Red and TNS provide a comparison of the active site hydration and stericconstraints of CYP3A4 when the peripheral allosteric site is occupied or empty. Mass spectrometry andcomputational models provide methods to map binding interactions in Cyt b5-CYP complexes and theresulting conformational changes linked to effector functions of Cyt bs. Each of these approaches will becombined with functional studies to characterize reaction intermediates that determine the efficiency of theflux through the catalytic cycle. A major goal of these studies is to develop their translational utility inconceptually analogous studies with P-glycoprotein in Project 4 of this Program. In addition, the importanceof in vivo allosteric effects remains speculative, and the final aim, aim 3, of this proposal will utilize a novel invivo, human, clinical study to explore heterotropic effects between the antiepileptic drug carbamazapine andthe antianxiety drug midazolam. This study not only seeks to establish definitive proof-of-principle for in vivoallosteric effects in human CYPs, but also to provide a platform experimental design to be exploited in otherin vivo studies.
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