Expressed In Saccharomyces Cerevisiae And Enzyme Kinetics Analysis Of Human Polymorphic CYP2D6 And Its Primary Application In Drug Screen

Human cytochrome P450 2D6 is an important member of CYPs family with polymorphism,its genetic polymorphisms result in increased,decreased or even dead enzyme,the population carried different genotypes were divided into poor metabolizer,intermedium metabolizer and extensive metabolizer.CYP2D6 plays an important role in the metabolism of drugs and xenobiotics.In the average person, CYP2D6 accounts for～5%of total P450,however,this enzyme is involved in the oxidation of～30%of all drugs oxidized by P450s.In our research,we cloned the cDNA of CYP2D6 wild type and five single nucleotide polymorphisms(SNPs) including A237S,E418Q,R343G,V11M and T107I,then all the cDNA were cloned into galactose-inducible vector pYES2/CT.The recombinants were expressed in Saccharomyces cerevisiae and CYP proteins were identified by Western blotting.The enzyme kinetics were deteced using Fluorometry Assay and HPLC towards substrates AMMC and bufuralol respectively and To analyze the impact of single nucleotide polymorphisms(SNPs)on enzyme catalytic activities.23 drugs were screened with high throughput Fluorometry Assay.We successfully established stable human CYPs heterogenous expression system and drug screen system.The wild-type cDNA of CYP 2D6 was obtained by PCR from commercial plasmid(1.5Kbp)and cloned into pYES2/CT vector for galactose-inducible expression in yeast.The cDNA of wild-type was subsequently used as a template to introduce SNP by site-directed mutagenesis.The recombinants pYES2/CT-CYP2D6 were transformed into Saccharomyces cerevisiae,the proteins were expressed and identified by Western blotting.When the CYP2D6 protein was expressed,microsomes were obtained through mechanical lysis and differential centrifugation,the protein concentration was determined by Bradford assay.The isolated microsomes were used to measure the kinetic parameters of 2D6 enzymes in real-time assays using a fluorogenic substrate AMMC and high performance liquid chromatography using a probe substrate bufuralol.The Assay was carried under the condition,which the products formed in the linearity with time and protein concentration.Apparent K_m and V_maxparameters from the kinetic studies were determined by nonlinear regression fitting using Prism^?Version 4.03(GraphPad Software,Inc.;San Diego,CA).Intrinsic clearance(CL_int)is determined by V_max/K_m. Kinetics studies using fluorogenic substrate AMMC revealed that the A237S,R343G and E418Q substitutions had no apparent effect on K_m retative to 2D6_WT,the V_max values changed to different extent,of three SNPs,A237S was similar to WT,E418Q decreased 50%and R343G was 25%of WT.The V11M mutation resulted in a 50% reduction in K_m and a 2-fold higher V_max.Interestingly,the T107I mutation caused a 4-fold higher K_m but no apparent effect on V_maxvalue.Kinetics studies using probe substrate bufuralol revealed that except for T107I,all the other SNPs decreased 50% in K_m value,the T107I had no apparent change in K_m.A237S and E418Q showed a 50%reduction in V_max,R343G and T107I substitutions decreased 85%and 70%in V_maxrespectively.However,The V11M exhibited a 2-fold higher V_max.Inhibition of CYP2D6_WT using AMMC and bufuralol both showed that quinidine had strong inhibitory effects on these two substrates,the IC₅₀values were 0.09±0.01μM and 0.11±0.02μM respectively.In this study,23 frequently prescribed drugs and some inhibitors were chosen for in vitro fluorometry assay as a high throughput method.Withing these drugs, antidepressants fluoxetine,Sertraline,chlomipramine and amitriptyline and antipsychotic drugs chlorpromazine and thioridazine showed strong inhibition on CYP2D6 WT and SNPs.The inhibitory degree of one drug on different genotype varied ratherish.Antidiabetic and drugs for decrease cholesterol had no inhibition on CYP2D6.The 23 drugs showed similar effect on metabolism of bufuralol by CYP2D6 WT.We have successfully cloned the cDNA of CYP2D6 wild type and its five SNPs and expressed them in Saccharomyces cerevisiae.The enzyme kinetics were analyzed by Fluorometry Assay and HPLC respectively,the impact of single nucleotide polymorphisms on enzyme activities were analyzed according to enzyme kinetic parameters we obtained.We also have established drug screen system.