Discovery of an allosteric 14-3-3 inhibitor for suppressing NRF2-driven cancer phenotypic screening and chemoproteomic-based target deconvolution.

The NRF2 transcription factor is constitutively active in various cancers, functioning as an oncogenic driver for tumor progression and chemo/radiotherapy resistance. Despite the well-documented role of NRF2 overactivation in cancer, no targeted therapy is currently available. In this study, using a combination of phenotypic screening, chemoproteomics, and biochemical and cellular assays, we identified WS3 as a potent allosteric inhibitor of 14-3-3 that selectively inhibits NRF2 activity in tumor cells. Mechanistically, WS3 binds allosterically to the 14-3-3 dimer, inducing a conformational change and disrupting the 14-3-3-pGSK3β interaction, thereby releasing pGSK3β for dephosphorylation. This activation of GSK3β subsequently enhances the ubiquitination and degradation of NRF2 by the CUL1-β-TrCP E3 ligase. WS3 effectively elicits oxidative stress and potentiates chemotherapeutics and ferroptosis in NRF2-driven cancers. Our findings uncover a previously unrecognized role of 14-3-3 in the hyperactivation of NRF2 and present a first-in-class sub-micromolar 14-3-3 allosteric inhibitor as an effective therapeutic strategy to suppress NRF2 overactivation, especially in Keap1 defective cancers.