In HEK 293T knockout-cell assays supplied with vitamin K1 quinone, phenindione inhibited FIX activity with IC50 values of 2.9 nM for endogenous VKORC1 and 258.0 nM for VKORC1L1. These values depend on the cellular model, substrate and downstream coagulation readout. [2]
Phenindione recognition by human VKOR
Biochemical assay · Crystallized human VKOR (residues 3–155) fused at its termini to split superfolder GFP. [1]
The ligand-bound structure identifies hydrogen bonds from Asn80 and Tyr139 to the phenindione indandione ring. The nearly planar ligand does not reach the isoprenyl-chain tunnel.
Literature-Reported Treatment Conditions
- Method
- Ligand-bound protein purification followed by lipidic cubic phase crystallography.
- Reported limits
- No cellular treatment dose applies to this structural experiment. Ligand concentrations vary across purification steps; they are not potency estimates. Compound vendor/lot and purity are not established by the reviewed locations.
Methods and source locations
Compound reported in the paper: Phenindione; Compound label in the paper: Phen
Binding observations concern an engineered human protein construct; they do not establish efficacy or dose in animals.
Figure 2B–C; HsVKOR stabilization and VKA-recognition results; Materials and Methods: constructs, ligand-bound purification, crystallization and structure determination
Phenindione binding pose in human VKOR: X-ray crystallography; deposited HsVKOR–phenindione complex 6WV6. — Figure 2B–C and caption; VKA-recognition results; data availability (6WV6)
Differential cellular inhibition of endogenous VKORC1 and VKORC1L1
Cell-based study · F9-transfected HEK 293T single-knockout cells: VKORC1L1 KO to assess endogenous VKORC1; VKORC1 KO to assess endogenous VKORC1L1; 12 µM vitamin K1 quinone. [2]
Phenindione suppressed FIX activity with greater apparent potency in the VKORC1 assay. Table 1 reports an IC50 ratio (VKORC1L1/VKORC1) of 89.0. These are cellular vitamin K reductase readouts with K1 substrate.
Literature-Reported Treatment Conditions
- Treatment concentration
- Phenindione concentration series; 12 µM vitamin K1 quinone substrate.
- Exposure duration
- Compounds added at medium replacement 4 hours after transfection; medium collected 72 hours after transfection.
- Method
- Secreted FIX activity measured by an aPTT-based coagulation assay and normalized to cells secreting FIX without oral anticoagulant.
- Vehicle
- Phenindione stock in ethanol; vitamin K1 quinone stock in dimethyl sulfoxide.
- Co-treatment
- Vitamin K1 quinone supplied as assay substrate; no second anticoagulant in the phenindione series.
- Reported limits
- Exact phenindione concentration series and raw curves were not retrieved because the supplement link returned challenge HTML. Final solvent percentages and independent biological replicate count are not specified in the reviewed main-text locations; Table 1 says triplicates.
Methods and source locations
Compound reported in the paper: Phenindione
Model-specific cellular potency with K1 quinone; not a purified-enzyme epoxide-reduction IC50 or a treatment-dose recommendation.
Table 1 phenindione row and footnote; Materials and methods: Determination of vitamin K reductase and VKOR activity and IC50 values; Figure 1B and caption
Inhibition of vitamin K1-supported FIX activity in VKORC1L1 KO cells: aPTT-based FIX activity; variable-slope nonlinear dose-response fit; measurements in triplicate. — Table 1 phenindione row, headers and footnote; activity/IC50 methods
Inhibition of vitamin K1-supported FIX activity in VKORC1 KO cells: Same cellular assay design, with endogenous VKORC1L1 isolated by VKORC1 knockout. — Table 1 phenindione row, headers and footnote; activity/IC50 methods
