Target Fishing Reveals a Novel Mechanism of 1,2,4-Oxadiazole Derivatives Targeting Rpn6, a Subunit of 26S Proteasome

1,2,4-Oxadiazole derivatives, a class of Nrf2-AREactivators, exert an extensive therapeutic effect on inflammation, cancer, neurodegeneration, and microbial infection. Among theseanalogues,DDO-7263is the most potent Nrf2 activator and used as the core structure for bioactive probes to explore the precisemechanism. In this work, we obtained compound7, a mimic ofDDO-7263, and biotin-labeled and fluorescein-based probes, which exhibited homologous biological activities toDDO-7263,including activating Nrf2 and its downstream target genes, anti-oxidative stress, and anti-inflammatory effects. Affinity chromatog-raphy and mass analysis techniques revealed Rpn6 as the potential target protein regulating the Nrf2 signaling pathway. In vitro affinity experiments further confirmed thatDDO-7263upregulated Nrf2 through binding to Rpn6 to block the assembly of 26Sproteasome and the subsequent degradation of ubiquitinated Nrf2. These results indicated that Rpn6 is a promising candidate target to activate the Nrf2 pathway for protecting cells and tissues from oxidative, electrophilic, and exogenous microbial stimulation