Discovery of toxoflavin, a potent IRE1α inhibitor acting through structure-dependent oxidative inhibition

Kai-long Jiang1,2,3,4,5, Chang-mei Liu3,6, Li-tong Nie3, Hai-ni Jiang2,7, Lei Xu2,3, Kun-zhi Zhang3,8, Li-xia Fan3, An-hui Gao3, Lu-ling Lin1, Xiang-yu Wang5, Ming-jia Tan3, Qi-qing Zhang1, Yu-bo Zhou2,3,4, Jia Li2,3,4,7,9
1 Institute of Biomedical Engineering, The Second Clinical Medical College, Jinan University (Shenzhen People’s Hospital), Shenzhen 518020, China
2 Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan 528400, China
3 State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China
4 University of Chinese Academy of Sciences, Beijing 100049, China
5 The First Affiliated Hospital, Jinan University, Guangzhou 510632, China
6 School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, China
7 School of Pharmacy, Zunyi Medical University, Zunyi 563006, China
8 Zhejiang Center for Medical Device Evaluation, Zhejiang Medical Products Administration, Hangzhou 311121, China
9 Shanghai Tech University, Shanghai 201210, China
Correspondence to: Qi-qing Zhang:, Yu-bo Zhou:, Jia Li:,
DOI: 10.1038/s41401-022-00949-9
Received: 5 January 2022
Accepted: 24 June 2022
Advance online: 15 July 2022


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.
Keywords: endoplasmic reticulum stress; toxoflavin; reactive oxygen species; IRE1α; XBP1

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