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-ARE activators, exert
an extensive therapeutic effect on inflammation, cancer, neurodegeneration,
and microbial infection. Among these analogues, DDO-7263 is the most potent Nrf2 activator and used as the core structure
for bioactive probes to explore the precise mechanism. In this work,
we obtained compound 7, a mimic of DDO-7263, and biotin-labeled and fluorescein-based probes, which exhibited
homologous biological activities to DDO-7263, including
activating Nrf2 and its downstream target genes, anti-oxidative stress,
and anti-inflammatory effects. Affinity chromatography and mass analysis
techniques revealed Rpn6 as the potential target protein regulating
the Nrf2 signaling pathway. In vitro affinity experiments further
confirmed that DDO-7263 upregulated Nrf2 through binding
to Rpn6 to block the assembly of 26S proteasome 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.