<i>In Silico</i> and <i>In Vitro</i> Studies of Potential Novel Vitamin K Epoxide Reductase (VKOR) Inhibitors Suggest an Updated Structure–Activity Relationship

Vitamin K epoxide reductase (VKOR) is an enzyme involved in the activation of several clotting factors, making its inhibition a well-known mechanism of anticoagulant action. Though the VKOR inhibitor warfarin remains essential and popular today, its adverse effects still make it a high-risk drug. This study intends to identify novel compounds with predicted VKOR inhibition and pharmacokinetic stability and explain the mechanistic basis of their action. A combination of pharmacophore modeling, druglikeness prediction, and molecular docking was used to find hits from existing compound libraries. The top hits showed <i>in vitro</i> activity at the micromolar level in a cell-based VKOR inhibition assay using human embryonic kidney 293 cells, led by A114 (IC₅₀ = 5.51 μM) and A116 (IC₅₀ = 5.53 μM). The docked protein-ligand complexes of the <i>in vitro</i> hits were also subjected to molecular dynamics to study atomic-level interactions. The combined data suggest that VKOR inhibitors need a bicyclic nucleus to elicit hydrophobic forces with the luminal residues 55 to 63 and TM3 residues 120 to 128, and extensively hydrogen bond to N80 and Y139 or C135. Shorter side chains should interact primarily with F83, while longer side chains must interact with residues G84 to F87. An extra side chain, like warfarin's alkyl ketone, may not be essential. This proposed structure-activity relationship can serve as a useful guide for designing improved and novel VKOR inhibitors in the future.