Constructing Stable Expression Cytochrome P4502C19Cell Lines And Its Preliminary Study In The Screening Of Traditional Chinese Medicine In Vitro

Aims: CYP2C19is an important metabolic enzyme in cytochromeP450family and play an important role in the metabofism of drugs. In thisstudy, we used recombinant DNA technology, cloning to build theCYP2C19wild-type vector and other two mutant gene carriers, Then,expressed in human hepatoma cells (HepG2), establishment of stabletransfection cell lines stably expressing the target gene, also establishmentof a research CYP2C19activity in high-throughput fluorescence detectiontechnology platform. The majoy job is enzyme kinetics analysis and druginhibition of high-throughput screening, determination of enzyme kineticconstants and drug inhibition constants of cytochrome CYP2C19SNPs ondrugs in vitro. Provide an effective experimental platform for innovativedrugs or new drug lead compounds for preclinical metabolic nature of thescreening and predictive as well as for the clinical potential druginteractions.Methods: The full-length CYP2C19wild-type cDNA fragment wasamplified by PCR from the human liver cDNA library and mutagenesisbuild*2,*3(mutant) cDNA in vitro, then those cDNA were inserted intoeukaryotic expression vector pcDNA3.l(-). After identification ofrestriction digestion and PCR，the recombinant plasmid was transfectedinto HepG2cells by lipofectamine. After screening culture by G4l8，astably-transfected cell line was established，and the transcription and expression of the CYP2C19gene SNPs were identified by RT-PCR,Western blot assay. CEC (3-cynao-7-ethoxycoumarin) as aCYP2C19-specific fluorescent substrate, which used for enzymatic reactionand detection of CYP2C19SNPs activity, using the nonlinear regressionmethod to calculate the enzymatic kinetic parameters(Km value, Vmax ofthe value and intrinsicclear rate CLint value). Then, the specific fluorescentsubstrate CEC (concentration selected near the Km value) and thetraditional Chinese medicine monomer (concentration gradient from128to1uM geometric dilution), as well as other experimental conditions simulatethe in vivo environment combinationed to inhibit the enzyme reaction invitro, and analysis the inhibitory effect of Chinese medicine monomer tocytochrome CYP2C19SNPs, also calculate the half inhibitoryconcentration. In this study, we screening a total of over100kinds ofChinese medicine monomer, and selected22kinds of drugs whichinhibition obvious in-depth study.Results: RT-PCR showed the target gene of CYP2C19SNPsamplification good and all the target gene expression in the HepG2celllines, Western-blotting was also imprinted target gene of CYP2C19in thissystem, successful expression of target gene protein. The specificfluorescent substrate CEC with the cells incubated for enzyme kineticsobtained the kinetic parameters,for CYP2C19*1, Km value is17.3±1.093，Vmax value is86.8±4.518，CLint value is4.945±0.132；CYP2C19*2Kmvalue is19.62±1.384， Vmax value is17.24±2.364， CLint value is0.847±0.086； CYP2C19*3Km value is15.21±2.271， Vmax value is9.93±0.479，CLint value is0.671±0.059. CEC concentration near the Kmvalue, cells directly incubated with CYP2C19known specific inhibitor oftranylcypromine, the results indicate that the tranylcypromine inhibitiedCYP2C19*1, the IC50value approximately7.73uM. The inhibition screening experiments of used fluorescent high-throughput detectiontechnology analysis allozyme polymorphism and about100kinds oftraditional Chinese medicine monomer show that:2C19involved in themetabolism of substrates and known inhibitors all were its inhibitor, themajority of Chinese medicine monomer weak or no inhibitory effect onCYP2C19inhibition; Chemical structure or similar drugs with similartherapeutic effects on inhibition of CYP2C19different；The same druginhibitied different genotypes are not the same result，evern a largerdifference.Conclusions:1. This study successfully constructed stably expressingCYP2C19SNPs cell lines in vitro. The target gene protein have goodactivity and expresss tability.2. We Verified the pharmacokinetic activity ofthe CYP2C19SNPs in vitro and investigated the inhibition of a number oftraditional Chinese medicine monomer in the preliminary study.3.Establishment a efficient, stable, and easy fluorescent high-throughput drugscreening platform, it bulited a basement applied before the metabolicnature of innovative drugs or new drug lead compounds for clinicalscreening and predictive for late-stage study,providing experimental basisfor the clinical potential drug interactions...