ACCOMPLISHMENTS: We have identified new genetic polymorphisms in the CYP family in humans which are responsible for introducing variability in the way humans metabolize of specific drugs and environmental chemicals and thus altered susceptibility of humans to these toxicity and disease states. CYP2C9 metabolizes numerous clinically important drugs including phenytoin, tolbutamide, warfarin, glipizide and numerous nonsteroidal antiinflammatory drugs (NSAIDs). By resequencing CYP2C9 we from the three different racial groups with ethnically diverse backgrounds we discovered 12 new alleles of CYP2C9. The recombinant human drug metabolizing enzymes were expressed in an E. coli expression system, and the catalytic activity toward tolbutamide compared with that of wild-type 2C9*1. The newly discovered CYP2C9 alleles include the amino acid changes L19I (2C9*7), R150H (2C9*8), H251R (2C9*9), E272G (2C9*10), R335W (2C9*11) and P489S (2C9*12). The CYP2C9*11 allele is markedly impaired (90% ). Last year, we discovered a homozygous null mutation of CY2C9 which decreased metabolism of the phenytoin by 85% in a clinical case study of an African-American patient hospitalized from severe clinical toxicity to the anticonvulsant phenytoin. This year we identified a patient homozygous for the same null mutation of CYP2C9 who had markedly impaired ability to metabolize the commonly used clinical anticoagulant warfarin. CYP2C19 metabolizes the common antiulcer drug omeprazole, the anticonvulsant mephenytoin, the anxiolytic valium, barbiturates, activates certain antimalarials, and sulfoxidizes the pesticide phorate. Nine new polymorphisms were discovered by resequencing CYP2C19. One P227L (2C19*10) showed a 90% decrease in catalytic activity toward a prototype substrate in vitro while a second allele, R144H (2C19*9), showed modest impairment. Almost all of the new defective alleles of CYP2C9 and CYP2C19 were prevalent in African-American or African populations, suggesting that these populations have not received sufficient study in the past. Two new important drug metabolizing genes are being resequenced. We have discovered 5 alleles of CYP3A5 which contain amino acid changes and these have been expressed in cDNA expression systems. Metabolism of testosterone is dramatically decreased by one of the alleles. We will look at metabolism of at least one clinical drug and one pesticide. We have discovered at several mutations in CY2B6. Future studies are addressing whether some of these are linked, and the degree to which they affect metabolism of testosterone and several environmental compounds. This includes the organophoshate insectide pesticide chlorpyrifos and the widely used carbamate pesticide carbaryl. Mechanistic structure-activity studies have addressed why the two similar CYP2C enzymes CYP2C9 and CYP2C19 (93% identical) have distinct substrate specificities. We have identified multiple key residues 99, 220, 221, and three residues in helix I (286, 289, 295) which appear to confer the ability to metabolize mephenytoin.
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