Kinase Photoaffinity Labeling Reveals Low Selectivity Profile of the IRE1 Targeting Imidazopyrazine-Based KIRA6 Inhibitor

Inositol-requiring enzyme 1α (IRE1α) is one of three endoplasmic reticulum stress sensors. Upon activation of its kinase domain, IRE1α splices the mRNA substrate XBP1, which activates the unfolded protein response. IRE1α has emerged as a therapeutic target as its hyperactivation is implicated in various diseases. Kinase inhibiting RNase attenuator 6 (KIRA6) is an allosteric IRE1α inhibitor targeting the ATP binding pocket, resulting in effective blockage of the IRE1α-XBP1 pathway in mouse models of diabetes and pain. However, recent studies indicate that KIRA6 is not as selective as initially thought. Here, we developed a photoaffinity-based KIRA6 probe to reveal its selectivity. Surprisingly, the majority of off-targets that we identified were not protein kinases but mostly nucleotide-binding proteins. Furthermore, we found that the promiscuous off-target profile of KIRA6 is not cell-line-dependent. Overall, this study calls for caution when KIRA6 is used in IRE1α-targeted studies and illustrates the power of kinase photoaffinity probes.