Expression, Characterization And Inhibitor Studies Of Human CYP2C19.1 And CYP2C19.2

The lentivirus-mediated, pcDNA3.1 (+)-mediated and baculovirus expression system were evaluated. The Bac-to-BacTM system was utilized to express CYP2C19 recombinant protein finally. The heterogeneous expressed CYP2C19 protein was proved to be active by using the classic substrate and inhibitors, and can be used to screen substrates and inhibitors.Aims This project was to utilize an effective expression system to express CYP2C19.1 wild type and CYP2C19.2 variants recombinant protein, to study the enzyme kinetics and the ability of inhibitor screening of the recombinant protein, and to help the research of phase I drug metabolism and DDIs studies.Methods The ability of three expression systems to heterogeneous express CYP2C19 was examined in vitro. The Bac-to-BacTM baculovirus expression system was used to coexpress recombinant CYP2C19 with CYPOR and CYPb5. The recombinant CYP2C19 with His-tag was detected by Western blot using anti-His antibody. The expression conditions were optimized to obtain the enzyme of highest activity, including the multiplicity of infection (MOI) of total viruses and MOI ratio among the 3 viruses. The enzyme activity was determined using omeprazole as the probe substrate which was determinated by HPLC. IC50 and Ki of ticlopidine and fluvoxamine were determined to validate the recombinant protein for inhibitor screening. Then the recombinant enzyme was used to screening the inhibition activities to CYP2C19 of 70 phytochemicals.Results Baculovirus expression system was proved to be the most effective expression system. Successful recombinant viruses containing genes of interest were verified by each construction step. After Sf9 cells were infected with each recombinant CYP2C19 baculovirus, the result from western blot suggested that the protein of interest were expressed on the level of translation. Activities of recombinant CYP2C19.1 wild type and CYP2C19.2 variants were determined by using omeprazole as the probe substrates. Their enzyme kinetics parameters were Km= (4.99±0.22)μmol·L-1, Vmax=(0.2539±0.0024)μmol·min-1·mg-1 protein, CLint= 0.0509 L·min-1·mg-1 protein for CYP2C19.1; Km= (5.822±0.27)μmol·L-1, Vmax= (0.5481±0.0058)μmol·min-1·mg-1 protein, CLint= 0.0941 L-min-1·mg-1 protein for CYP2C19.2, respectively. IC5o of ticlopidine to CYP2C19.1 and CYP2C19.2 was 2.28μmol·L-1 and 2.47μmol·L-1, Kt was (0.64±0.025)μmol·L-1 and 2.038μmol·L-1, respectively. IC50 of fluvoxamine to CYP2C19.1 and CYP2C19.2 was 0.19μmol·L-1 and 0.19μmol·L-1, Ki was (0.29±0.090)μmol·L-1 and 0.041μmol·L-1, respectively. The inhibitor screening analysis revealed that resveratrol, emodin, isoalantolactone, curcumol, DL-menthol, podophyllotoxin, psoralen, schisandrin A, schisandrin B and chamaejasmine B showed inhibition to CYP2C19.Conclusions The recombinant proteins of human CYP2C19 could be used as in vitro model for studying phase I drug metabolism and DDIs, especially for screening the substrates and inhibitors of human CYP2C19.