An integrated framework identified potent inhibitors targeting IRE1α

Inositol-requiring enzyme 1 (IRE1α) is one of the key sensors and signaling effectors of the unfolded protein response (UPR), which is essential for preserving endoplasmic reticulum (ER) homeostasis. Dysregulation of IRE1α signaling can lead to several illnesses. Initially, we employed the multidocking approach utilizing AutoDock Vina, AutoDock Wizard, and iGEMDOCK to predict the accurate binding affinities of the flavonoid library against IRE1α and rank them based on their affinities. Subsequently, post-docking approaches were used to refine the hit selection. Finally, the top-ranked flavonoids were selected based on their consistent high binding affinity and exhibited strong binding within the ATP-binding pocket of the kinase active site. In vitro kinase assays revealed that both amentoflavone and glycitein significantly inhibited IRE1α kinase activity, with IC₅₀ values of 16.4 μM and 23.68 μM, respectively. Cell-based studies demonstrated that these flavonoids have anti-inflammatory properties and significantly promote robust activation of XBP1 splicing and IRE1α expression under normal conditions. In contrast, flavonoid pretreatment significantly attenuated LPS-induced IRE1α-XBP1 signaling and reduced inflammatory responses. Overall, our results indicate that both glycitein and amentoflavone exhibit promising modulatory effects on IRE1α. To the best of our knowledge, this is the first study to report the modulatory potential of flavonoids amentoflavone and glycitein to possess both IRE1α kinase inhibitory and RNase-activating properties. This work provides a valuable basis for the development of flavonoid-based IRE1α inhibitors to treat ER stress and associated inflammatory diseases.