P4502C9--ACTIVE SITE STRUCTURE AND GENETIC POLYMORPHISM

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Cytochrome P4502C9 plays a central role in the etiology of warfarin drug interactions which have an inhibitory basis. It is the sole monoxygenase responsible for the metabolic clearance of (s)-warfarin, the enantiomer of the drug in which essentially all of the anticoagulant effect resides. Therefore, inhibition of P4502C9 by drugs which are co-administered with the anticoagulant is likely to be a primary cause of many of the drug interactions frequently encountered in the clinic. The identification of numerous (allelic) variants of P4502C9 is a notable complication because some of these variants appear to be catalytically deficient in their ability to metabolize (S)-warfarin and other substrates such as phenytoin and tolbutamide. Expression of these mutant alleles may underlie the slow metabolizer phenotype identified for some P4502C9 substrates. Therefore, we propose to evaluate the metabolic consequences of expression of structural variants of P4502C9 in the human population, and to define the structural features, both P4502C9 and of interacting drugs, which govern the formation of enzyme-substrate and enzyme- inhibitor complexes. This will be accomplished by; 1) determining common structural and conformational elements for both substrates and inhibitors using molecular mechanics, 2) using photoaffinity probes to covalently modify, and fast atom bombardment/mass spectrometry to identify, critical amino acids present in the active site of P4502C9, 3) expressing site-directed mutants of the enzyme and comparing their metabolic capabilities with that of the wild-type enzyme, 4) undertaking a pharmacogenetic study with 200 subjects, using the metabolism of sub-therapeutic doses of (s)-warfarin as a reporter of individual P4502C9 phenotype, 5) genotyping these same individuals using an allele-specific polymerase chain-reaction assay, 6) investigating the extent of co-segregation of (S)-warfarin and phenytoin in pre-selected heterozygotes and homozygotes, in order to assess the substrate-dependency of the phenomenon. The long-term aim of project 2 is to gain a detailed understanding of the active-site topography of proteins encoded by the CYP2C9 gene. The above studies will provide complementary data on the structural features which govern the interactions of substrates and inhibitors with the enzyme and its structural variants. At all stages of this proposal the accrued information will be integrated into our homology model in order to refine it sufficiently to permit, ultimately, a prospective assessment of the extent to which a co-administered agent might provoke inhibitory drug interactions with P4502C9 substrates.