γ-glutamyl carboxylase inhibition by Anisindione

The maturation of key coagulation factors requires γ-carboxylation catalyzed by γ-glutamyl carboxylase (GGCX), in which vitamin K hydroquinone (VKH₂) is oxidized to vitamin K epoxide (VKO) and recycled by vitamin K epoxide reductase (VKORC1). Clinically, vitamin K antagonists (VKAs) inhibit VKORC1 but are thought not to target GGCX. Here, we demonstrate that a member of VKA, anisindione and its analogs can dock within the VK-binding pocket of GGCX. Importantly, both in vitro and cell-based γ-carboxylation assays showed that anisindione inhibits GGCX activity. Furthermore, our cryo-electron microscopy structure of the GGCX-BGP (bone Gla protein or osteocalcin)-anisindione complex reveals that anisindione directly occupies the VK-binding pocket of GGCX, consistent with competitive inhibition with VK. These results establish anisindione as a structural prototype for direct GGCX inhibition and provide a structural framework for developing anticoagulants beyond VKORC1.