P450-GloTM bioluminescent CYP assay
Three concentrations of PTV (1, 10, 100 µM) were conducted to detect the luciferin PPXE activity against recombinant CYP3A4 and CYP3A5. A significant decrease in the luminescene activity in both recombinant CYP3A4 and CYP3A5 was observed in 10 µM and 100 µM of PTV (Fig 1). It seems that PTV is a candidate drug for metabolism against CYP3A4 and CYP3A5 enzymes.
Metabolite profiling and Identification
After incubating PTV with HLMs in the presence of a NADPH regenerating system, a metabolite (M2) of PTV was profiled, characterized, and identified by LC-MS/MS. A representative chromatograms are provided in Fig 2 (a) and (b). The LC-MS/MS analysis of the unchanged PTV and its metabolite produced informative and prominent product ions. The MS/MS spectrum of PTV, with a protonated molecular ion [M+H]+ at m/z …show more content…
The kinetic plots indicated that the Km values for HLMs CYP3A5 expressers and CYP3A5 non-expressers were 23.40 ± 1.74 and 28.63 ± 2.38 µM, respectively. The Vmax for HLMs CYP3A5 expressers and CYP3A5 non-expressers were 6.10 ± 1.04 pmol/mg protein/min and 6.93 ± 1.26 pmol/mg protein/min, respectively. The intrinsic clearance (Clint) was 0.26 ± 0.03 µl/mg protein/min for CYP3A5 expressers and 0.24 ± 0.04 µl/mg protein/min for CYP3A5 non-expressers. The results are demonstrated in Table …show more content…
Ketoconazole (1 µM), a potent CYP3A4/3A5 inhibitor, completely inhibited the formation of major metabolite. CYP3cide (0.5 µM) selectively inhibited the metabolic activity of CYP3A4
Three concentrations of PTV (1, 10, 100 µM) were conducted to detect the luciferin PPXE activity against recombinant CYP3A4 and CYP3A5. A significant decrease in the luminescene activity in both recombinant CYP3A4 and CYP3A5 was observed in 10 µM and 100 µM of PTV (Fig 1). It seems that PTV is a candidate drug for metabolism against CYP3A4 and CYP3A5 enzymes.
Metabolite profiling and Identification
After incubating PTV with HLMs in the presence of a NADPH regenerating system, a metabolite (M2) of PTV was profiled, characterized, and identified by LC-MS/MS. A representative chromatograms are provided in Fig 2 (a) and (b). The LC-MS/MS analysis of the unchanged PTV and its metabolite produced informative and prominent product ions. The MS/MS spectrum of PTV, with a protonated molecular ion [M+H]+ at m/z …show more content…
The kinetic plots indicated that the Km values for HLMs CYP3A5 expressers and CYP3A5 non-expressers were 23.40 ± 1.74 and 28.63 ± 2.38 µM, respectively. The Vmax for HLMs CYP3A5 expressers and CYP3A5 non-expressers were 6.10 ± 1.04 pmol/mg protein/min and 6.93 ± 1.26 pmol/mg protein/min, respectively. The intrinsic clearance (Clint) was 0.26 ± 0.03 µl/mg protein/min for CYP3A5 expressers and 0.24 ± 0.04 µl/mg protein/min for CYP3A5 non-expressers. The results are demonstrated in Table …show more content…
Ketoconazole (1 µM), a potent CYP3A4/3A5 inhibitor, completely inhibited the formation of major metabolite. CYP3cide (0.5 µM) selectively inhibited the metabolic activity of CYP3A4