Specificity and Structure-Function Studies of Human Drug-Metabolizing Enzymes

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AIMS: To determine the reason for drug and xenobiotic specificity of the CYP enzymes in humans. The CYP2C subfamily is highly homologous at the gene level, but differ markedly in their substrate specificity. CYP2C9 specifically metabolizes a number of anti-inflammatory drugs such as ibuprofen and diclofenac. In contrast, the closely related CYP2C19 specifically metabolizes other drugs such as the anticonvulsant mephenytoin. The aims of this study are to define the amino acids involved in substrate specificity and solve the structure of these CYP2Cs. Structure activity studies will help predict which drugs will be metabolized by these polymorphic enzymes.ACCOMPLISHMENTS:We used ibuprofen and diclofenac as specific substrates for CYP2C9 and mephenytoin as a prototype substrate for CYP2C19. Chimeras and site directed mutants were constructed in a cDNA expression system in bacteria. Substrate recognition sites (SRS) 3 and 4 (proposed by Gotoh for mammalian P450s) appeared to confer specificity for specific CYP2C9 substrates such as ibuprofen and diclofenac. Both SRS 3 and 4 appeared necessary for activity or proper expression. Site directed mutagenesis experiments showed that mutations at 286(N S and 289 I N) conferred high specific activity toward ibuprofen and diclofenac to 2C19. However, the converse is not true. SRS 3 and 4 of CYP2C19 alone do not confer mephenytoin hydroxylase to CYP2C9. Preliminary experiments suggest that I99H (SRS1) in combination with 220Ser Pro and 220Pro Thr in the FG loop and SRS 3 and 14 of 2C19 are required to convert CYP2C9 to a high turnover mephenytoin hydroxylase. Therefore multiple regions appear to be required for CYP2C19 specificity, but much fewer changes are required for CYP2C9 specificity. - Structure-activity, CYP, P-450, CYP2C19, CYP2C8, CYP2C9