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

Inositol-requiring enzyme 1 alpha (IRE1 alpha) is one of three endoplasmic reticulum stress sensors. Upon activation of its kinase domain, IRE1 alpha splices the mRNA substrate XBP1, which activates the unfolded protein response. IRE1 alpha has emerged as a therapeutic target as its hyperactivation is implicated in various diseases. Kinase inhibiting RNase attenuator 6 (KIRA6) is an allosteric IRE1 alpha inhibitor targeting the ATP binding pocket, resulting in effective blockage of the IRE1 alpha-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 IRE1 alpha-targeted studies and illustrates the power of kinase photoaffinity probes.