The overall objective of the proposed research is to design specific inhibitors of individual forms of cytochrome P-450. These enzymes play a pivotal role in both the detoxification and bioactivation of drugs, environmental contaminants, and other chemical toxicants. The balance between detoxification and bioactivation is largely governed by the relative amounts and activities of the various forms of cytochrome P-450 involved in metabolizing a particular compound. P-450 form-specific inhibitors should be very valuable tools for in vivo applications and could be used either 1) diagnostically to assess the role of the various cytochromes P-450 in mediating or protecting against chemical toxicity or 2) therapeutically to redirect the metabolism of xenobiotics from potentially harmful to innocuous pathways. The principle to be used in the design of specific cytochrome P-450 inhibitors is mechanism-based inactivation. This involves the identification or synthesis of substrates that in the process of catalytic conversion are changed into intermediates or products that inactivate the target enzyme. The requirement for catalysis adds an extra degree of potential selectivity compared with reversible inhibitors, which rely solely on binding. In the last two years it has become evident that cytochromes P-450 differing in structure by as few as one amino acid residue may have very different substrate specificities. The hypothesis of the current proposal is that mechanism- based inactivators can be used to distinguish between cytochromes P-450 highly related in primary structure and that differences in selectivity can be rationalized in terms of enzyme and inactivator structure. The basic approach will be to examine in liver microsomes, reconstituted systems, and heterologous expression systems the inactivation by appropriate compounds of highly related cytochromes P-450. Three examples will be investigated involving pairs of cytochromes P-450, the individual members of which differ from each other in primary structure by one, eleven, or twenty-five amino acid residues, respectively. These are 1) two allelic variants of rat liver cytochrome P450IIB1, 2) rabbit liver P450IIB4 and P450IIB5, and 3) rabbit liver P450IIC4 and IIC5. Inactivators have already been identified that distinguish the two IIB1 variants from each other, and several progesterone and pregnenolone derivatives that inactivate P450IIC5 have been designed. Once inactivators have been identified that select between the individual members of these pairs of highly related cytochromes P-450, hybrid and mutant cDNAs will be made and expressed in order to pinpoint the structural basis for differences in inactivator specificity. These studies should provide the rational basis for the design of superior inhibitors of individual cytochrome P-450 forms.
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