2. Academic Validation
  3. Discovery of toxoflavin, a potent IRE1α inhibitor acting through structure-dependent oxidative inhibition

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

  • Acta Pharmacol Sin. 2022 Jul 15. doi: 10.1038/s41401-022-00949-9.
Kai-Long Jiang 1 2 3 4 5,  Chang-Mei Liu 3 6,  Li-Tong Nie 3,  Hai-Ni Jiang 2 7,  Lei Xu 2 3,  Kun-Zhi Zhang 3 8,  Li-Xia Fan 3,  An-Hui Gao 3,  Lu-Ling Lin 1,  Xiang-Yu Wang 5,  Ming-Jia Tan 3,  Qi-Qing Zhang 9,  Yu-Bo Zhou 10 11 12,  Jia Li 13 14 15 16 17
Affiliations

Affiliations

  • 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 Institute of Biomedical Engineering, The Second Clinical Medical College, Jinan University (Shenzhen People's Hospital), Shenzhen, 518020, China. zhang.qiqing@szhospital.com.
  • 10 Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan, 528400, China. ybzhou@simm.ac.cn.
  • 11 State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China. ybzhou@simm.ac.cn.
  • 12 University of Chinese Academy of Sciences, Beijing, 100049, China. ybzhou@simm.ac.cn.
  • 13 Zhongshan Institute for Drug Discovery, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Zhongshan, 528400, China. jli@simm.ac.cn.
  • 14 State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China. jli@simm.ac.cn.
  • 15 University of Chinese Academy of Sciences, Beijing, 100049, China. jli@simm.ac.cn.
  • 16 School of Pharmacy, Zunyi Medical University, Zunyi, 563006, China. jli@simm.ac.cn.
  • 17 Shanghai Tech University, Shanghai, 201210, China. jli@simm.ac.cn.
PMID: 35840659 DOI: 10.1038/s41401-022-00949-9
Abstract

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

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

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