Abstract
Oral anticoagulants such as warfarin, which function as vitamin K antagonists (VKAs), have long been central to anticoagulation therapy. Warfarin inhibits vitamin K epoxide reductase (VKOR), thereby restricting the availability of reduced vitamin K and impairing γ-carboxylation and activation of vitamin K-dependent clotting factors (VKDCFs). However, warfarin's clinical use is hindered by a narrow therapeutic index and marked interindividual variability, largely attributable to genetic variation in VKOR, yet it remains the only oral anticoagulant option for clinical conditions such as antiphospholipid antibody syndrome. γ-Glutamyl carboxylase (GGCX), the enzyme that directly modifies VKDCFs, represents an attractive but largely unexplored target for anticoagulation. Using a cell-based high-throughput screen of the Maybridge Hit-Finder compound library (comprising 14 400 drug-like molecules), we identified 21 compounds that inhibited γ-carboxylation of VKDCFs. Subsequent gene-deficient cell-based assays and in vitro enzymatic characterization revealed that compound HF13141-H5 selectively and potently inhibits GGCX activity, with nanomolar half-maximal inhibitory concentrations. Molecular docking with the recently resolved GGCX structure suggested that HF13141-H5 mimics the binding of reduced vitamin K, and site-directed mutagenesis confirmed the critical hydrogen bond with residue N290 within the substrate-binding pocket. Importantly, HF13141-H5 maintained full inhibitory potency against VKOR variants associated with warfarin resistance and demonstrated robust anticoagulant activity in a zebrafish laser-induced endothelial injury model. In summary, we describe the discovery and mechanistic characterization of HF13141-H5 as the first small-molecule GGCX inhibitor. By directly targeting GGCX, HF13141-H5 presents a promising strategy for developing next-generation anticoagulants that could overcome the limitations of warfarin, such as genetic variability and resistance.