Abstract

A series of imidazo[1,2-b]pyridazin-8-amine kinase inhibitors was discovered to allosterically inhibit the endoribonuclease function of the dual kinase-endoribonuclease inositol-requiring enzyme 1 (IRE1, a key component of the unfolded protein response in mammalian cells and a potential drug target in multiple human diseases. Inhibitor optimization gave compounds with high kinome selectivity that prevented endoplasmic reticulum stress-induced IRE1oligomerization and phosphorylation, and inhibited endoribonuclease activity in human cells. X-ray crystallography showed the inhibitors to bind to a previously unreported and unusually disordered conformation of the IRE1 kinase domain that would be incompatible with back-to-back dimerization of the IRE1 protein and activation of the endoribonuclease function. These findings increase the repertoire of known IRE1 protein conformations and can guide the discovery of highly selective ligands for the IRE1 kinase site that allosterically inhibit the endoribonuclease.