Long-Range Inhibitor-Induced Conformational Regulation of Human IRE1αEndoribonuclease Activity

Abstract

Activation of the inositol-requiring enzyme-1 alpha (IRE1a) protein caused by endoplasmic reticulum stress results in the homodimerization of the N-terminal endoplasmic reticulum luminal domains, autophosphorylation of the cytoplasmic kinase domains, and conformational changes to the cytoplasmic endoribonuclease (RNase) domains, which render them functional and can lead to the splicing of X-box binding protein 1 (XBP 1) mRNA. Herein, we report the first crystal structures of the cytoplasmic portion of a human phosphorylated IRE1a dimer in complex with (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)-2,7-diazaspiro(4.5)decane-7-carboxamide, a novel, IRE1a-selective kinase inhibitor, and staurosporine, a broad spectrum kinase inhibitor. (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)-2,7-diazaspiro(4.5)decane-7-carboxamide inhibits both the kinase and RNase activities of IRE1a. The inhibitor interacts with the catalytic residues Lys599 and Glu612 and displaces the kinase activation loop to the DFG-out conformation. Inactivation of IRE1a RNase activity appears to be caused by a conformational change, whereby the aC helix is displaced, resulting in the rearrangement of the kinase domain-dimer interface and a rotation of the RNase domains away from each other. In contrast, staurosporine binds at the ATP-binding site of IRE1a, resulting in a dimer consistent with RNase active yeast Ire1 dimers. Activation of IRE1a RNase activity appears to be promoted by a network of hydrogen bond interactions between highly conserved residues across the RNase dimer interface that place key catalytic residues poised for reaction. These data implicate that the intermolecular interactions between conserved residues in the RNase domain are required for activity, and that the disruption of these interactions can be achieved pharmacologically by small molecule kinase domain inhibitors.

Hydroxycarboxylic Acid Receptor 2 Is a Zika Virus Restriction Factor That Can Be Induced by Zika Virus Infection Through the IRE1-XBP1 Pathway

“…To verify this hypothesis, two kinds of IRE1 inhibitors (GSK2850163 and 4µ8c) were used in the context of ZIKV infection (MOI 3) to detect their effects on the expression of HCAR2 by qRT-PCR assay. As shown in the Figure 3B, GSK2850163 (an inhibitor for IRE1 kinase activity) (Concha et al, 2015) and 4µ8c (an inhibitor for IRE1 endoribonulease activity) (Cross et al, 2012) dramatically inhibited the splicing of XBP1 in both mock and infected cells. Moreover, the mRNA level of HCAR2 was not increased by ZIKV infection with GSK2850163 or 4µ8c treatment ( Figure 3C).…”

Phosphorylation of IRE1 at S729 regulates RIDD in B cells and antibody production after immunization

“…S2 C). Compared with staurosporine (a broad-spectrum kinase inhibitor that binds to IRE1; Concha et al, 2015 ), imatinib (a tyrosine kinase inhibitor that activates IRE1 in cardiomyocytes; Kerkelä et al, 2006 ), or sunitinib (a receptor tyrosine kinase inhibitor that binds to IRE1; Korennykh et al, 2009 ; Ali et al, 2011 ), KIRA6 was effective in suppressing S729 phosphorylation of IRE1 ( Figs. 2 F and S2 D).…”

IRE1α is critical for kaempferol induced neuroblastoma differentiation

“…4D). The interactions of kaempferol with Glu 643, Cys 645 and His 692 residues of human IRE1α had been shown for kinase activity inhibition by staurosporine, a pan kinase inhibitor (Concha et al, 2015). APY29 has been known to interact with amino acid residues of kinase domain to activate the endoribonuclease activity of IRE1α (Korennykh et al, 2009).…”