Cell-based screening of the NCC library for drugs that off-target the vitamin K cycle. (A) Vitamin K redox cycle. When VKD proteins are carboxylated by GGCX, by using vitamin K hydroquinone (KH₂), carbon dioxide (CO₂), and oxygen (O₂) as cofactors, KH₂ is oxidized to KO, which must be reduced to KH₂ by a 2-step reduction. (B) Functional screenings of the 727 drugs in the NCC library. FIXgla-PC/HEK293 cells were incubated with the drugs identified in the NCC library (final concentration, 10 µM) in cell culture medium containing 5 µM KO. After a 48-hour incubation, reporter protein carboxylation was evaluated by ELISA. Carboxylation activity of DMSO-treated cells was normalized to 100%. The effect of lansoprazole (C) and tegaserod maleate (D) on VKD carboxylation and cell viability. FIXgla-PC/HEK293 cells were incubated with increasing concentrations of the test drug in cell culture medium containing 5 µM KO. Twenty-four hours later, cell culture medium was used for the cell-based activity assay and the drug-treated cells were used for the cell viability assay.