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

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 IC 50 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 IC 50 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.

Unlocking the potential of bacterial endophytes from medicinal plants for drug discovery

“…Toxoflavin was shown to be the key virulence factor of phytopathogenic bacteria, for example, Burkholderia glumae , acting as an electron carrier that sequesters electrons from the respiratory chain of the plant and transfers them to oxygen, thereby generating toxic hydrogen peroxide (Iqbal et al., 2021). Recently published study suggested that toxoflavin can be useful as a molecular tool to study enzymes involved in sensing endoplasmic reticulum stress (Jiang et al., 2023).…”

Toxoflavin analog D43 exerts antiproliferative effects on breast cancer by inducing ROS-mediated apoptosis and DNA damage

“…In addition, toxoflavin can target the junction between the RNase and kinase domains and inhibit IRE1α by oxidizing the conserved cysteine residues in the active site of IRE1α, inhibiting its activity through oxidation of conserved cysteine residues within its active site. This property enables its application as a small molecule tool to investigate the involvement of IRE1α in ER stress 26 .…”