Discovery of a novel covalent allosteric site CYS434 in Keap1 and its ligand for the treatment of acute lung injury.

Keap1 is a central negative regulator of the Nrf2 antioxidant pathway, yet its covalent allosteric modulation remains incompletely understood. Here, we provide evidence that Cys434 may represent a previously unrecognized covalent allosteric site in Keap1. Using site-directed mutagenesis, high-resolution protein mass spectrometry, molecular dynamics simulations, MST binding assays, and covalent docking, we show that dimethyl fumarate (DMF) can form a covalent linkage with Cys434, suggesting a potential structural target of DMF and an alternative mode of Keap1 regulation. Leveraging this candidate site, a deep-learning-based screening of 350,000 small molecules yielded six predicted covalent ligands. Among them, fumaramide (Fum) was prioritized and further shown to covalently modify Keap1 at Cys434, as supported by mutagenesis, high-resolution MS, and MST assays. Functionally, Fum activates the Nrf2 pathway and promotes Nrf2 nuclear translocation in vitro. In a murine acute lung injury model, Fum alleviates pulmonary damage, suppresses inflammatory cytokines, and enhances Nrf2 nuclear accumulation in lung tissue. Collectively, these findings suggest that Cys434 may act as a covalent regulatory site within Keap1 and that DMF and related electrophiles could engage noncanonical cysteine residues. This work provides mechanistic insight and highlights a potential molecular scaffold for the development of next-generation Keap1 allosteric modulators.