Pharmacokinetic Tuning Redirects β‐Lapachone From NQO1 Bioactivation to Vimentin‐Targeted Oxidation in Cancer

The "single molecule, multi-target" paradigm is increasingly recognized among bioactive small molecules, yet the role of pharmacokinetics as a determinant of mechanism selectivity remains unexplored. Here, we demonstrate that dosing schedule governs the dominant cell death mechanism of β-lapachone (β-lap), an ortho-naphthoquinone antitumor natural product, switching it from NQO1-bioactivation to targeted oxidative modification of vimentin (VIM), a type III intermediate filament protein overexpressed in aggressive cancers. Using a photoaffinity probe and mass spectrometry-based proteomics, we identified VIM as a direct binding target driving the dominant cell death mechanism under sustained low-concentration exposure. Vimentin directly binds β-lap with a dissociation constant at micromolar levels, and VIM essentiality for β-lap cytotoxicity was established via knockdown and overexpression in cancer cell lines. Mechanistically, β-lap selectively oxidizes the C328 residue of VIM, triggering disulfide crosslinking of VIM filaments, proteasomal overload, and ferroptotic cell death, a cascade independent of NQO1. A sustained-release formulation of β-lap achieves tumor suppression comparable to gemcitabine at its maximum tolerated dose in MIA Paca-2 xenograft models. Taken together, these findings establish pharmacokinetic tuning as a programmable mechanistic switch, repurposing β-lap as a broader-spectrum, safer VIM-targeted therapy for NQO1-deficient malignancies and offering a generalizable strategy for natural product therapeutic expansion.