Discovery of novel IKKβ allosteric inhibitors as dual-antioxidant for cerebral ischemia-reperfusion injury therapy by upregulating HO-1 and SLC7A11.

Antioxidant therapy represents a promising intervention for ischemic stroke, yet no targeted drug has successfully entered clinical application. While IKKβ is a known target in inflammatory and oncological diseases, its potential as an antioxidant target in stroke remains underexplored, and no IKKβ inhibitor has been approved clinically. Starting from the lead compound EF24, an IKKβ inhibitor with undefined mechanism, we first optimized its scaffold to obtain the safer derivative JH, then developed more potent analogs. A Random Forest-based QSAR model (R2 = 0.9098) highlighted molecular charge distribution, topology, and lipophilicity as key activity determinants. The optimized compound JHB-17 showed ∼70-fold higher potency than EF24 and acted as a non-ATP competitive inhibitor, targeting a novel allosteric site on IKKβ. In an H₂O₂-induced SH-SY5Y cell injury model, JHB-17 demonstrated significant dual antioxidant cytoprotective effects, and IKKβ overexpression reversed the protective effect of JHB-17. H₂O₂ exposure induced IKKβ phosphorylation. Conversely, silencing IKKβ exerts antioxidant cytoprotective effects by upregulating heme oxygenase-1 (HO-1). JHB-17 markedly inhibited IKKβ phosphorylation, promoted Nrf2 nuclear translocation, subsequently upregulated HO-1, and concurrently elevated the ferroptosis suppressor SLC7A11 and glutathione. In a rat MCAO/R model, JHB-17 reduced cerebral infarct volume and improved neurobehavioral outcomes. JHB-17 exhibits favorable pharmacokinetic parameters, and the comet assay and acute toxicity test demonstrate its good safety profile. This study preliminarily reveals a novel allosteric site on IKKβ, identifies JHB-17 as a novel IKKβ allosteric inhibitor with dual antioxidant activity, proposes IKKβ as a novel antioxidant target for cerebral ischemia-reperfusion injury, and reveals a novel mechanism by which IKKβ inhibition upregulates SLC7A11 to suppress ferroptosis, providing insights for targeted ischemic stroke therapy.