Development Of Yeast Expression And In Vitro Metabolic Assay Systems For Human Cytochrome 3A4 Polymorphic Enzymes

The human cytochrome P450 (CYP450) superfamily members metabolize a vast array of clinically, physiologically, and toxicologically important compounds. CYP3A4 is the most abundant isoform of CYP in adult human liver and is involved in the metabolism of 45-60% of the drugs metabolized by CYPs. The expression of CYP3A4 varies 40-fold in individual human livers, and metabolism of CYP3A4 substrates varies at least 10-fold in vivo. This variation can affect drug efficacy and toxicity. One of the causes for these drug metabolism variations is genetic polymorphism. To date, several CYP3A4 alleles have been found and may be a source of inter-individual drug metabolism difference.The SNPs were introduced into the prototype 3A4 gene by PCR mutagenesis method. A yeast expression vector pYES2/CT was used to clone the expression constructs. Following sequencing, the recombinants were expressed in yeast by galactose induction. The S9 microsome fractions containing recombinant 3A4 enzymes were isolated by differential centrifugation and were used to test the enzymatic activities in real-time kinetic assays using CYP3A4 fluorogenic substrates, DBF (Dibenzylfluorescein) and BOMCC, and CYP3A4-specific substrate nifedipine. The kinetic datas were fit to nonlinear regression analysis for determination of Km and Vmax values. Eight drugs were used in the inhibition assay, to investigate the influence of SNP on drug metabolism.These results showed budding yeast S.cerevisiae was a suitable host to express human CYP3A4 enzymes. Fluorogenic and HPLC assays using isolated microsomes showed robust catalytic activity of the prototype CYP 3A4 enzymes, and the values of Vmax and Km were comparable to the published data. Moreover, R162Q, L293P, M445T and P467S allelic enzymes have similar metabolic rates of DBF, BOMCC and nifedipine. T185S, F189S and S222P enzymes exhibited a 75.8%, 91.2% and 71.3% decrease in the intrinsic clearance for DBF, and an 89.9%, 94.5% and 95.2% for BOMCC, respectively. Furthermore, the IC50 values for the inhibition of these enzymes by 8 chemical compounds showed remarkable enzyme-selective inhibitory effects. And the IC50 values of all tested compounds did not change at various times.The panel of CYP3A4 polymorphic enzymes will be tested against various drugs, kinds of fluorogenic substrates and CYP3A4-specific substrate to identify potentially adverse drug-CYP in order to generate predictive information applicable in personalized medicine.