Enzyme Kinetics Analysis Of Human Polymorphic CYP2C19 And Its Primary Study In The Application Of Drug Screening

Human cytochrome P450 2C19 plays an important role in the metabolism of drugs and xenobiotics. Pharmacogenetic studies have shown that genetic polymorphisms in this gene are important determinants of inter-individual and inter-ethnic variation in drug metabolism and toxicity. To develop a broadly applicable assay system for studying human CYP2C19 polymorphic enzymes, we cloned and expressed the cDNA of CYP2C19 wild-type(WT) and 10 viarants in budding yeast Saccharomyces cerevisiaee, which was already integreted with CYP450 Oxidoreductase(POR). Using these recombinant enzymes in real-time biochemical assays and high throughput drug screening, the kinetic constants as well as inhibition constants for the test compounds were measured. The results we got can provid valuable information for studying the effects of polymorphic genes on drug metabolism and evluating new drugs in the early phase of drug discovery.The wild-type cDNA of CYP 2C19 was obtained by RT-PCR from human liver tissue and cloned into pYES2/CT vector for galactose-inducible expression in yeast. The cloned cDNA was subsequently used as a template to introduce SNP by site-directed mutagenesis ,the viarants are named I331V, W120R, L17P, E92D, R132Q, R144H, R150H, P227L, R410C, R433W. All the viarants were cloned into the same vector. Transformed yeasts produced large quantities of microsome-bound 2C19 enzymes as determined by Western analysis, a 55 kDa protein was observed.The P450 content was assayed by reduced CO difference spectrum.There was a 450-nm peak observed in 2C19-WT, I331V and R150H , and their content respectively were 22pmol/mg, 16 pmol/mg, 15 pmol/mg; Other SNPs only had a very strong 420-nm peak ,it was a denatured form of CYP2C19. The isolated microsomes were used to measure the kinetic constants of 2C19 enzymes in real-time assays using a fluorogenic substrate CEC. The results showed that all enzymes possess robust activity with the exception of R132Q and R433W, the Km value of 9 catalytically active enzymes showed little difference(no more than two-fold),but the Vmax value were markedly different: I331V and R410C were very close to 2C19-WT, the other viarants had a much lower Vmax .The inhibition of recombinat CYP2C19-WT enzyme by known inhibitor drug was tested by serial titration of drugs in the fluorogenic assays. The results showed that: CYP2C19 was strongly inhibited by its known inhibitor tranylcypromine, IC50 was about 7.3μM, but CYP2C9'known inhibitor Sulfaphenazole can't inhibit CYP2C19.Used fluorogenic high throughput technique to detect the inhibition of 20 drugs on 9 catalytically active enzymes,the results showed : Except 8 drugs such as statins and Thiozol nediones(TZDs), drugs matabolized by CYP2C19 and its known inhibitors all had specific inhibition on CYP2C19 ; Drugs had similar structure or therapy had different inhibition on CYP2C19; The inhibitory degree of one drug on different genotype varied a lot.For the first time,we successfully constructed the Saccharomyces cerevisiae expressing system for 11 CYP2C19 polymorphic genes; The catalytic activity of yeast microsome protein were robust and stable; Established an in vitro detection system for biochemical analysis and drug high throughput screening of CYP2C19 polymorphic enzymes,which were efficient, sensitive and convenient, This work can guide the further study of CYP2C19 related drug-drug interaction , as well as can aid the future personalized prescription, reducing adverse reaction and enhancing drug efficiency in vivo.