Characterization of drugs that inactivate VKOR for KO reduction. (A) Inhibition of VKD carboxylation by warfarin. FIXgla-PC/HEK293 cells were incubated with increasing concentrations of warfarin in cell culture medium containing 5 µM KO (KO_HEK293) or vitamin K (K_HEK293) for cell-based activity and cell viability assays, as described in the legend to Figure 1C. DGKO cells were used for a similar assay, with vitamin K serving as the substrate (K_DGKO). Inhibition of VKD carboxylation by nitazoxanide (B) and lansoprazole (C). FIXgla-PC/HEK293 and DGKO cells were incubated with increasing concentrations of the test drug in cell culture medium containing 5 µM KO (KO_HEK293) and vitamin K (K_DGKO), respectively, for cell-based activity and cell viability assays. (D) Inhibition of KO reduction by warfarin, nitazoxanide, and lansoprazole in HEK293 cells. GGCX-knockout HEK293 cells were incubated with 10 µM KO in the presence or absence of 10 µM test drug for 5 hours. DMSO-treated cells were used as the control. KO reduction activity was evaluated by the production of vitamin K (red) in the chromatogram. (E) Inhibition of vitamin K reduction by warfarin, nitazoxanide, and lansoprazole in HEK293 cells. DGKO cells were incubated with 10 µM vitamin K in the presence or absence of 10 µM of the test drug for 5 hours. DMSO-treated cells were used as the control. Vitamin K reduction activity was evaluated by the production of KO (red) in the chromatogram.