The cytochrome P-450 dependent monooxygenase system plays a pivotal role in both the detoxification and bioactivation of drugs, environmental contaminants, and other potential chemical toxicants. The balance between detoxification and activation is largely dependent on the relative amounts and activities of different isozymes of cytochrome P-450. The long-term objective of the research proposed in this application is to design irreversible inhibitors of specific isozymes of cytochrome P-450. Such isozyme-specific inhibitors could be used in vivo either 1) diagnostically to assess the role of the various cytochromes in mediating or protecting against chemical toxicity or 2) therapeutically to redirect the metabolism of xenobiotics from potentially harmful to innocuous pathways. This proposal will focus on the mechanism, isozyme specificity, and structural requirements of the suicide inactivation of rat liver cytochromes P-450 by dihalomethyl-compounds. The best example of such a compound is the antibiotic chloramphenicol. In contrast to the vast majority of irreversible inhibitors of cytochrome P-450, chloramphenicol acts by virtue of the modification of the protein rather than the heme moiety. Therefore, chloramphenicol and its analogs should prove valuable tools for studying and modulating the various functions of cytochrome P-450. Emphasis will first be placed on elucidation of the mechanism by which rat liver cytochromes P-450 are inactivated upon covalent modification of specific amino acid residues in the proteins by metabolites of chloramphenicol or of its analog 1-p-nitrophenyl-2-dichloroacetamidoethane. Subsequent studies will focus on determining the specificity of dihalomethyl-compounds as inhibitors of different isozymes of rat liver cytochrome P-450 and on elucidating which structural features of the molecules are responsible for their effectiveness and specificity as suicide substrates of cytochromes P-450. Finally, attempts will be made to synthesize specific inhibitors of particular isozymes. These studies should provide the rational basis for the design of isozyme-specific inhibitors for modulating monooxygenase function in humans.
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