Vitamin K3 (Menadione) Targets TBK1 to Enhance the Efficacy of Pirfenidone in a Mouse Model of Bleomycin-Induced Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive and irreversible interstitial lung disease. Current antifibrotic drugs, exemplified by pirfenidone (PFD), only slow disease progression with limited efficacy and adverse effects, underscoring the need for novel therapeutic strategies. By integrating single-cell transcriptomics with the Drug2Cell algorithm, we performed drug prioritization at single-cell resolution and identified menadione (vitamin K3, VK3), a synthetic naphthoquinone and the minimal core of naturally occurring vitamin K, as a candidate preferentially active in alveolar epithelial cells. In bleomycin (BLM)-stimulated A549 cells and a mouse model of pulmonary fibrosis, therapeutic co-administration of low-dose VK3 and PFD reduced vimentin expression by 44.8% and collagen deposition by 50.3%, and decreased fibroblast abundance by 36.7%, thereby attenuating histopathological damage and restoring BLM-disrupted alveolar epithelial homeostasis. Experiments with VK3 alone further demonstrated that its antifibrotic activity is independent of conversion to vitamin K2. Mechanistically, thermal proteome profiling identified TANK-binding kinase 1 (TBK1) as the principal candidate target; molecular docking and molecular dynamics simulations predicted a stable binding mode, independently confirmed by biolayer interferometry and differential scanning fluorimetry, demonstrating direct binding of VK3 to TBK1 with nanomolar affinity. TBK1 knockdown suppressed BLM-induced fibrotic responses, establishing TBK1 as a positive regulator of pulmonary fibrosis. In vitro signaling analysis revealed that VK3 plus PFD inhibits phosphorylation of TBK1 and STAT3, whereas PFD alone reduces phosphorylation of JAK1 and STAT3, suggesting that both agents converge on STAT3 through distinct upstream nodes. Although VK3 exhibits dose-dependent cytotoxicity precluding direct clinical use as monotherapy, its validated high-affinity target TBK1 and privileged naphthoquinone scaffold lay a rational foundation for lead optimization toward safer anti-IPF agents, and its ability to enhance PFD efficacy supports further development as an adjunct to existing antifibrotic therapy.