Figure 3

Identification of CYP450 isoenzymes that transform BBR into its metabolites. Interaction between CYP450 and BBR were analyzed with docking score generated from SYBYL 7.3 software analysis. CYP450 isoenzymes with docking scores over 5 were labelled with the score value (a). The 3-D structrual docking patterns between BBR and CYP2D6 (upper), or CYP1A2 (middle), or CYP3A4 (lower) were generated with SYBYL 7.3. BBR is rendered in sticks (red, oxygen atoms; white, carbon atoms; blue, nitrogen atoms). The heme prosthetic group is rendered in sticks in red. The amino acid residues constituting the active site cavity are in cyan, most of which are shown in lines. Amino acid residues rendered in sticks may help BBR binding to CYP450. Specific hydrogen bonds and a water molecule are showed as yellow broken lines and a red sphere, respectively (b). For the transformation test in rCYP450 isoenzymes reaction system, BBR (20 μM) was incubated with each of the rCYP450 isoenzymes (50 pmol/ml of rCYP450) for 0.5 hr, followed by a detection of the metabolites. The experiment was repeated twice (c). In the chemical inhibition assay, the α-naphthoflavone (ANF) was for CYP1A2 inhibition, quinidine (QND) for CYP2D6, ketoconazole (KET) for CYP3A, sulfaphenazole (SUP) for CYP2C9 and troglitazone (TGL) for CYP2C19. BBR was incubated with HLMs in the presence or absence of CYP450 specific inhibitors. The final concentration of BBR and the inhibitor in the reaction was 15 μM and 5 μM, respectively. The enzyme catalyzing activity of sample free of inhibitors (control) was defined as 100%. Presented is mean and sd of the percent of control (d).