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 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.


33.
13. Methods for generation of an HEK293TN XBP1 luciferase reporter cell line.
14. Methods to determine inhibition of an XBP1 luciferase reporter in HEK293TN cells and assess compound cytotoxicity to HEK293TN cells.
15. Methods to quantify XBP1s expression in NCI-H929 cells by immunofluorescence.
16. Methods to quantify XBP1s and DNAJB9 mRNA expression in NCI-H929 cells by qPCR.
18. Methods to assess total IRE1 and pIRE1 expression in H929 myeloma cells.

20.
As XBP1s showed a nuclear localisation the same minimum area criteria was used to measure the intensity in the 480_40X -HQ535_50M green channel. The mean well background intensity was then subtracted from the mean XBP1 nuclear intensity to determine the IC 50 relative to the 7-(1-(3,5-difluorobenzyl)-1H-pyrazol-4-yl)-3Himidazo[4,5-b]pyridine + tunicamycin and the tunicamycin only controls using Dotmatics Studies (Dotmatics, UK).

Quantification of XBP1s and DNAJB9 mRNA expression in NCI-H929 cells by qPCR
Unless otherwise stated all reagents were purchased from ThermoFisher Scientific.

X-ray crystallography of 33-IRE1
hIRE1 was expressed and purified as previously described 6 . hIRE1 was crystallized by hanging-drop vapor diffusion. A protein-ligand mixture was prepared comprising hIRE1 at 10 mg/mL and compound 33 at 1.4 mM. This was mixed with reservoir solution containing 20% polyethylene glycol 3350, 0.2 M sodium acetate and 0.1 bistris propane (pH 6.5) in a 1:1 ratio. Crystallization experiments were conducted at 18°C. Crystals were briefly soaked in reservoir buffer supplemented with 25% ethylene glycol before plunging into liquid nitrogen. X-ray diffraction data were collected at 100 K from a single cryo-cooled crystal using an in-house X-ray system (Rigaku 007-HF, Saturn 944+ CCD detector). Diffraction data were integrated and scaled using CCP4 software 50 . The structure of apo-hIRE1 was solved by molecular replacement using PHASER S1 with a single protomer from the apo-hIRE1 crystal structure (PDB code 4Z7G 6 ). Manual model building was performed in COOT 51 and refinement was performed in PHENIX S2 . The structure was validated using MolProbity S3 .