The Build Of Cyp2d6 * 14b Yeast Expression System, The Fluorescent Drug Screening Platform For The Establishment And Of Cyp2d6 * 10 Gene Type

Cytochrome 450 (CYP,P450)are major phase I drug metabolism enzymesHuman CYP2D6 is responsible for the metabolism of large number of prescription drugs.The activity of CYP2D6 can be influenced by many factors,such genetic,age,diet.The disparity of the metabolism ability lead to the heterogeneity of treatment effects (HTE) when taking the same dose. CYP2D6 is polymorphic,more than 70 alleles and 100 SNPs have been reported.CYP2D6*10 is an important subtype in Asian,40.8-49.5% people were classified with this subtype.CYP2D6*10 is main cause of the IM phenotype in Asian people and has an impact on metabolic enzymes of drugs.CYP2D6*14 is an unique subtype in Asian,1%-2% people were classified with this subtype.CYP2D6*14A may be an important cause of the PM phenotype in the Asian,and in-vitro researches about CYP2D6*14B only involves activity or kinetics of the enzyme,but the role and effect of CYP2D6*14B in drug-drug interaction remains unknown.The research cloned and expressed the cDNA of CYP2D6*14B in yeast Saccharomyces cerevisiae,then these recombinant enzymes were validated by a CYP2D6-specific fluorogenic substrate AMMC through enzyme kinetics experiment.Eight drugs were used in the inhibition asssy.The results can provide valuable data and information for studying the genetic polymorphic effects on drug metabolism and predicting in vivo drug-drug interaction.CYP450s need POR to catalyze the reaction,which is in low expression level in yeast Saccharomyces cerevisiae.We intergreted POR with yeast cells,then co-expressed with CYP2D6*14B.CYP2D6*14B allele was obtained by site-directed mutagenesis PCR.The cDNA of CYP2D6*14B were constructed into pYES2/CT vector.Following sequencing,the recombinant was co-expressed with POR in yeast by galactose induction,western blotting testified a 55KD protein.A large quantities of recombinant enzyme were isolated by differential centrifugation and were used to measure the enzymatic activities using CYP2D6 flurogenic substrate AMMC.Compared with CYP2D6*1,the Km of CYP2D6*14B is similar,but the Vmax decrease to 31.7%,Clint decrease to 30.9%.Inhibition assay were carried out using fluorogenic detection method.The results showed the substrate of CYP2D6 and specific inhibitor quinidine had inhibition.Drugs belongs to the same kind had similar inhibition trend;the degree of inhibition on genotype varied and was irrelevant to enzyme activity.Moreover,we investigated gene polymorphisms of CYP2D6*10 in 74 sample by PCR-RFLP. The frequencies of CYP2D6*10 allele in Chinese are 55.1%, which was consistent with other reports.