Discovery of toxoflavin, a potent IRE1 alpha inhibitor acting through structure-dependent oxidative inhibition

Inositol-requiring enzyme 1 alpha (IRE1 alpha) is the most conserved endoplasmic reticulum (ER) stress sensor with two catalytic domains, kinase and RNase, in its cytosolic portion. IRE1 alpha inhibitors have been used to improve existing clinical treatments against various cancers. In this study we identified toxoflavin (TXF) as a new-type potent small molecule IRE1 alpha inhibitor. We used luciferase reporter systems to screen compounds that inhibited the IRE1 alpha-XBP1s signaling pathway. As a result, TXF was found to be the most potent IRE1 alpha RNase inhibitor with an IC50 value of 0.226 mu M. Its inhibitory potencies on IRE1 alpha kinase and RNase were confirmed in a series of cellular and in vitro biochemical assays. Kinetic analysis showed that TXF caused time- and reducing reagent-dependent irreversible inhibition on IRE1 alpha, implying that ROS might participate in the inhibition process. ROS scavengers decreased the inhibition of IRE1 alpha by TXF, confirming that ROS mediated the inhibition process. Mass spectrometry analysis revealed that the thiol groups of four conserved cysteine residues (CYS-605, CYS-630, CYS-715 and CYS-951) in IRE1 alpha were oxidized to sulfonic groups by ROS. In molecular docking experiments we affirmed the binding of TXF with IRE1 alpha, and predicted its binding site, suggesting that the structure of TXF itself participates in the inhibition of IRE1 alpha. Interestingly, CYS-951 was just near the docked site. In addition, the RNase IC50 and ROS production in vitro induced by TXF and its derivatives were negative correlated (r = -0.872). In conclusion, this study discovers a new type of IRE1 alpha inhibitor that targets a predicted new alternative site located in the junction between RNase domain and kinase domain, and oxidizes conserved cysteine residues of IRE1 alpha active sites to inhibit IRE1 alpha. TXF could be used as a small molecule tool to study IRE1 alpha's role in ER stress.