Two major goals of drug metabolism studies are to identify and characterize pathways of biotransformation that lead to toxic and active metabolites, and to utilize this information to therapeutic advantage. The research described in this proposal uses minor changes to the chemical structures of various drugs to alter their metabolism, and thereby modify their toxicity or therapeutic activity. Thus, the application is an extension of our previous work to elucidate molecular mechanisms of reactive metabolite formation and disposition, to characterize mechanisms of oxidative N-dealkylation, and to synthesize suicide inhibitors of aromatase as antitumor agents and to characterize their reactions with aromatase.
The aims of the present project are: (l) To use dimethylated and regioisomeric analogs of the widely used analgesic and antipyretic, acetaminophen, to determine the roles of oxidative stress and covalent binding to proteins in hepatotoxicity caused by the drug. A combination of radiochemical and immunochemical techniques coupled with methods for protein resolution and purification (l- and 2-D gel electrophoresis, preparative isoelectric focusing, gravity-flow chromatography and HPLC) will be used to isolate modified proteins for characterization by microsequencing and mass spectral analysis of peptide fragments. Enzyme activity assays and DNA fragmentation will be used to assess the roles of mitochondria and nuclei in acute lethal cell injury caused by acetaminophen. (2) To characterize the metabolism of two natural product furans, menthofuran and teucrin A, that have been ingested as herbal medicines by humans and have caused hepatotoxicity. An antibody to menthofuran protein adducts will be used to isolate and characterize damaged liver proteins. (3) To determine the effects of deuterium substitution on N-demethylations by human CYP1A1 and CYP1A2 of the widely used drug, caffeine. 15N- and 13C-NMR relaxation methods will be used to determine the effects of the orientation of caffeine relative to the heme iron of each P450 isoform which regioselectively oxidize caffeine to different metabolites. (4) To investigate the mechanism of inactivation of CYP19A1 (aromatase) by three different suicide inhibitors of this important enzyme, one of which was synthesized during the last grant period and is in clinical trials for breast cancer. Radiolabeled analogs will be synthesized and reacted with aromatase, either purified from human placenta or as cDNA expressed protein, to determine what apoprotein and/or heme residues are modified. The overall project is expected to advance our knowledge of mechanisms of drug induced cell death and mechanisms of metabolite formation, and may lead to drugs with enhanced therapeutic benefit.
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