Binding to an Unusual Inactive Kinase Conformation by Highly Selective Inhibitors of Inositol-Requiring Enzyme 1α Kinase-Endoribonuclease

A series of imidazo[1,2-b]pyridazin-8-amine kinase inhibitors were discovered to allosterically inhibit the endoribonuclease function of the dual kinase-endoribonuclease inositol-requiring enzyme 1 alpha (IRE1 alpha), 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 alpha 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 IREl1 alpha kinase domain that would be incompatible with back-to-back dimerization of the IRE1 alpha protein and activation of the endoribonuclease function. These findings increase the repertoire of known IRE1 alpha protein conformations and can guide the discovery of highly selective ligands for the IRE1 alpha kinase site that allosterically inhibit the endoribonuclease.