Inositol-requiring enzyme 1α (IRE1α) is the most conserved endoplasmic reticulum (ER) stress sensor with two catalytic domains, kinase and RNase, in its cytosolic portion. IRE1α 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 IRE1α inhibitor. We used luciferase reporter systems to screen compounds that inhibited the IRE1α-XBP1s signaling pathway. As a result, TXF was found to be the most potent IRE1α RNase inhibitor with an IC₅₀ value of 0.226 μM. Its inhibitory potencies on IRE1α 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 IRE1α, implying that ROS might participate in the inhibition process. ROS scavengers decreased the inhibition of IRE1α 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 IRE1α were oxidized to sulfonic groups by ROS. In molecular docking experiments we affirmed the binding of TXF with IRE1α, and predicted its binding site, suggesting that the structure of TXF itself participates in the inhibition of IRE1α. 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 IRE1α inhibitor that targets a predicted new alternative site located in the junction between RNase domain and kinase domain, and oxidizes conserved cysteine residues of IRE1α active sites to inhibit IRE1α. TXF could be used as a small molecule tool to study IRE1α’s role in ER stress.

肌醇需求酶 1α (IRE1α) 是最保守的内质网 (ER) 应激传感器，在其胞质部分具有两个催化结构域：激酶和 RNase。IRE1α 抑制剂已被用于改善现有的针对各种癌症的临床治疗。在这项研究中，我们确定了毒黄素 (TXF) 是一种新型有效的小分子 IRE1α 抑制剂。我们使用荧光素酶报告系统来筛选抑制 IRE1α-XBP1s 信号通路的化合物。结果发现，TXF 是最有效的 IRE1α RNase 抑制剂，IC ₅₀值为 0.226 μM。一系列细胞和体外生化测定证实了其对 IRE1α 激酶和 RNase 的抑制效力。动力学分析表明，TXF 对 IRE1α 造成时间依赖性和还原剂依赖性的不可逆抑制，这表明 ROS 可能参与了抑制过程。ROS 清除剂减少了 TXF 对 IRE1α 的抑制，证实了 ROS 介导了抑制过程。质谱分析表明，IRE1α 中四个保守半胱氨酸残基（CYS-605、CYS-630、CYS-715 和 CYS-951）的硫醇基团被 ROS 氧化为磺酸基团。在分子对接实验中我们证实了TXF与IRE1α的结合，并预测了其结合位点，提示TXF本身的结构参与了IRE1α的抑制。有趣的是，CYS-951就在停靠地点附近。此外，TXF及其衍生物体外诱导的RNase IC _{50与ROS产生呈负相关（} r  = -0.872）。总之，本研究发现了一种新型的 IRE1α 抑制剂，其靶向位于 RNase 结构域和激酶结构域之间连接处的预测新替代位点，并氧化 IRE1α 活性位点的保守半胱氨酸残基来抑制 IRE1α。TXF 可作为小分子工具来研究 IRE1α 在 ER 应激中的作用。