Covalent modification of Keap1 Cys489 by NU6300 activates Nrf2 signaling and suppresses NLRP3 inflammasome-mediated pyroptosis.

Nuclear factor erythroid 2-related factor 2 (Nrf2), a master regulator of oxidative stress and inflammasome, plays a critical role in modulating pyroptosis. In this study, we identified NU6300 as a novel small-molecule activator of Nrf2 that restores mitochondrial function, alleviates oxidative stress, and suppresses inflammasome activation and pyroptosis. Mechanistically, NU6300 covalently modified Kelch-like ECH-associated protein 1 (Keap1) at cysteine-489, disrupting the Keap1-Nrf2 interaction, thereby promoting Nrf2 nuclear translocation and transcription of antioxidant genes. Notably, NU6300 inhibits NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation and gasdermin D (GSDMD)-mediated pyroptosis through redox-dependent mechanisms, representing the first evidence that covalent modification of Keap1 at cysteine-489 by NU6300 bridges Nrf2 activation and inflammasome suppression. In vivo, NU6300 exhibits potent antioxidant and anti-inflammatory protection against acetaminophen (APAP)-induced acute liver injury in mice. Collectively, these findings demonstrate that NU6300 is a novel Nrf2 activator and offers a promising therapeutic strategy for pyroptosis-driven inflammatory diseases.