Cytochromes P450 (P450s) are heme-containing monooxygenases that play a key role in the metabolic activation of chemical carcinogens and in the metabolism of drugs. The members of the P450 1A subfamily, P450 1A1 and 1A2, are both considered important in carcinogenesis. Human P450 1A1, which is present in lungs, is thought to be linked to lung cancer because this enzyme is able to metabolize environmental pollutants, such as polycyclic aromatic hydrocarbons (PAHs), to their carcinogenic derivatives. P450 1A2, a typical hepatic P450, oxidizes a number of drugs and plays a major role in the metabolic activation of heterocyclic amines to genotoxic products. Therefore, the elucidation of the catalytic mechanism of these enzymes and the structural basis for activity would give us some insight into P450-linked carcinogenesis and have important implications for the rational design of anticancer drugs. Since P450s require a redox partner, cytochrome P450 reductase (CPR), during catalysis, we intend to focus on the investigation of P450-CPR interactions. The studies proposed here focus both on the investigation of enzyme-substrate/inhibitor interactions and the interactions of P450s with a larger ligand, namely P450 reductase (CPR), a P450 redox partner. The central hypothesis is that both substrate dynamics in the active site and P450-CPR interactions determine enzyme specificity. These interactions will be affected by the identity of P450 residues at the CPR binding site, defining regio- and stereospecificity of a given P450. This will be studied by a combination of theoretical techniques including homology modeling and molecular dynamics, and experimental approaches, including NMR, site-directed mutagenesis and functional analyses. The model P450s will be human P450 1A1 and 1A2, currently under investigation in our laboratory. These enzymes constitute an ideal system for these studies due to their high sequence similarity (72%), but different substrate specificities. The projected outcomes will help us to explain the metabolism of xenobiotics, in particular carcinogen activation. The findings will be widely disseminated and available for drug discovery purposes both in academia and industry. The resulting computational methodology will significantly reduce time, save materials, and limit animal and/or human research in the area of drug metabolism and design. These studies will also provide training in advanced modeling, biophysical and biochemical techniques to postdoctoral fellows and graduate students. Due to the interdisciplinary nature of this project, they will be exposed to various facets of biological research and will learn a comprehensive approach to biological problems. Moreover, the work on this project will foster cooperation and collaboration among students and postdocs involved in different aspects of the studies. They will be also trained in manuscript preparation and submission procedures. This type of experience will help students and postdocs to succeed in their future research careers. Furthermore, this project will also provide opportunities for summer research by undergraduate students, as part of WV-BRIN and WV-INBRE programs. ? ? ? ? ? ?
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