Development of a Chemical Toolset for Studying the Paralog-Specific Function of IRE1

The dual kinase endoribonuclease IRE1 is a master regulator of cell fate decisions in cells experiencing endoplasmic reticulum (ER) stress. In mammalian cells, there are two paralogs of IRE1: IRE1α and IRE1β. While IRE1α has been extensively studied, much less is understood about IRE1β and its role in signaling. In addition, whether the regulation of IRE1β’s enzymatic activities varies compared to IRE1α is not known. Here, we show that the RNase domain of IRE1β is enzymatically active and capable of cleaving an XBP1 RNA mini-substrate in vitro. Using ATP-competitive inhibitors, we find that, like IRE1α, there is an allosteric relationship between the kinase and RNase domains of IRE1β. This allowed us to develop a novel toolset of both paralog specific and dual-IRE1α/β kinase inhibitors that attenuate RNase activity (KIRAs). Using sequence alignments of IRE1α and IRE1β, we propose a model for paralog-selective inhibition through interactions with nonconserved residues that differentiate the ATP-binding pockets of IRE1α and IRE1β.