Structure-Guided Discovery of Non-Peptide Macrocyclic KEAP1−NRF2 Inhibitors Targeting Cuproptosis in Depression

Macrocyclic molecules are promising for targeting intractable protein−protein interactions (PPIs). The small-molecule KEAP1−NRF2 PPI inhibitors have shown potential for treating major depressive disorder (MDD). Herein, we report a crystallographic structure-guided strategy for the design of non-peptide macrocyclic KEAP1−NRF2 PPI inhibitors. The best macrocyclic compound T7 exhibits rapid binding and slow dissociation with the KEAP1 protein, significant in vitro anti-oxidative stress and anti-inflammatory activities, and capacity for brain penetration. In a chronic unpredictable mild stress (CUMS) mouse model, T7 treatment improves the depressive behavior and serum neurotransmitter levels by activating the NRF2 pathway. Furthermore, we found that the activation suppresses CUMS-induced cuproptotic pathologies including mitochondrial morphological disruption/reduced density, synaptic vesicle depletion, and dysregulated expression of cuproptosis mediators in hippocampal and cortical regions. Collectively, our study presents a structure-based strategy to develop macrocyclic KEAP1−NRF2 PPI inhibitors, which hold promise for treating MDD by engaging NRF2-mediated cuproptosis suppression in stress-related neuropathology.