Abstract
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 1α (IRE1α), 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 IRE1α 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 IRE1α kinase domain that would be incompatible with back-to-back dimerization of the IRE1α protein and activation of the endoribonuclease function. These findings increase the repertoire of known IRE1α protein conformations and can guide the discovery of highly selective ligands for the IRE1α kinase site that allosterically inhibit the endoribonuclease.
Highlights
The dual kinase-endoribonuclease inositol-requiring enzyme 1α (IRE1α; ERN1) is a central and conserved component of the unfolded protein response (UPR) activated by eukaryotic cells in reaction to endoplasmic reticulum (ER) stress, caused by an excess of misfolded proteins in the ER lumen.[1,2] Activation of the UPR reduces protein translation and increases the protein-folding capacity
Suggesting a different allosteric effect on the RNase compared to typical type I IRE1α kinase inhibitors.[21]
We hypothesized that the orientation of 2 within the IRE1α adenosine 5′-triphosphate (ATP) site would be similar to that determined for 1 (PDB 4Z7H).[6]
Summary
The dual kinase-endoribonuclease inositol-requiring enzyme 1α (IRE1α; ERN1) is a central and conserved component of the unfolded protein response (UPR) activated by eukaryotic cells in reaction to endoplasmic reticulum (ER) stress, caused by an excess of misfolded proteins in the ER lumen.[1,2] Activation of the UPR reduces protein translation and increases the protein-folding capacity. We show how 2 and more potent analogues bind to a previously undescribed inactive conformation of monomeric IRE1α involving major rearrangements of key secondary structure in the kinase domain, including disordering of the αC-helix and disruption of the hydrophobic spine of the protein, which together confer high IRE1α kinase binding selectivity and inhibition of both kinase and RNase functions We confirm that these allosteric modulators disrupt all downstream signaling through IRE1α in human cells, validating targeting of this unusual binding mode as a new way to generate selective and cell active inhibitors of IRE1α. Fluorine substituents were introduced into the 2-(N-phenyl)amino-1H-benzo[d]imidazole group starting from the appropriate fluorinated 4-bromobenzene-1,2-diamines via condensation with isothiocyanatobenzene, and the route was telescoped to generate compounds 30−32
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