Development of a Rapid in vivo Assay to Evaluate the Efficacy of IRE1-specific Inhibitors of the Unfolded Protein Response using Medaka Fish.

Three types of transmembrane protein, IRE1alpha/IRE1beta, PERK, and ATF6alpha/ATF6beta, are expressed ubiquitously in vertebrates as transducers of the unfolded protein response (UPR), which maintains the homeostasis of the endoplasmic reticulum. IRE1 is highly conserved from yeast to mammals, and transmits a signal by a unique mechanism, namely splicing of mRNA encoding XBP1, the transcription factor downstream of IRE1 in metazoans. IRE1 contains a ribonuclease domain in its cytoplasmic region which initiates splicing reaction by direct cleavage of XBP1 mRNA at the two stem loop structures. As the UPR is considered to be involved in the development and progression of various diseases, as well as in the survival and growth of tumor cells, UPR inhibitors have been sought. To date, IRE1 inhibitors have been screened using cell-based reporter assays and fluorescent-based in vitro cleavage assays. Here, we used medaka fish to develop an in vivo assay for IRE1alpha inhibitors. IRE1alpha, IRE1beta, ATF6alpha and ATF6beta are ubiquitously expressed in medaka. We found that IRE1alpha/ATF6alpha-double knockout is lethal, similarly to IRE1alpha/IRE1beta- and ATF6alpha/ATF6beta-double knockout. Therefore, IRE1 inhibitors are expected to confer lethality to ATF6alpha-knockout medaka but not to wild-type medaka. One compound named K114 was obtained from 1,280 compounds using this phenotypic screening. K114 inhibited ER stress-induced splicing of XBP1 mRNA as well as reporter luciferase expression in HCT116 cells derived from human colorectal carcinoma, and inhibited ribonuclease activity of human IRE1alpha in vitro. Thus, this phenotypic assay can be used as a quick test for the efficacy of IRE1alpha inhibitors in vivo.Key words: endoplasmic reticulum, inhibitor screening, mRNA splicing, phenotypic assay, unfolded protein response.