S-lactoyl modification of KEAP1 by a reactive glycolytic metabolite activates NRF2 signaling

KEAP1, a cytoplasmic repressor of the oxidative stress responsive transcription factor NRF2, senses the presence of electrophilic agents by modification of its sensor cysteine residues. In addition to xenobiotics, several reactive metabolites have been shown to covalently modify key cysteines on KEAP1, although the full repertoire of these molecules and their respective modifications remains undefined. Here, we report the discovery of sAKZ692, a small molecule identified by high throughput screening that stimulates NRF2 transcriptional activity in cells by inhibiting the glycolytic enzyme pyruvate kinase. sAKZ692 treatment promotes the buildup of glyceraldehyde 3-phosphate, a metabolite which leads to S-lactate modification of cysteine sensor residues of KEAP1, resulting in NRF2 dependent transcription. This work identifies a novel posttranslational modification of cysteine derived from a reactive central carbon metabolite and helps further define the complex relationship between metabolism and the oxidative stress sensing machinery of the cell. To identify the transcriptional program activated by the small molecule NRF2 activator sAKZ692, we evaluated the transcriptional signature of compound treatment (20 µM, 24 hours) relative to DMSO in the IMR32 cell line.