The molecular basis for selective inhibition of unconventional mRNA splicing by an IRE1-binding small molecule

Abstract

IRE1 couples endoplasmic reticulum unfolded protein load to RNA cleavage events that culminate in the sequence-specific splicing of the Xbp1 mRNA and in the regulated degradation of diverse membrane-bound mRNAs. We report on the identification of a small molecule inhibitor that attains its selectivity by forming an unusually stable Schiff base with lysine 907 in the IRE1 endonuclease domain, explained by solvent inaccessibility of the imine bond in the enzyme-inhibitor complex. The inhibitor (abbreviated 4μ8C) blocks substrate access to the active site of IRE1 and selectively inactivates both Xbp1 splicing and IRE1-mediated mRNA degradation. Surprisingly, inhibition of IRE1 endonuclease activity does not sensitize cells to the consequences of acute endoplasmic reticulum stress, but rather interferes with the expansion of secretory capacity. Thus, the chemical reactivity and sterics of a unique residue in the endonuclease active site of IRE1 can be exploited by selective inhibitors to interfere with protein secretion in pathological settings.8-formyl-umbelliferone | unfolded protein response | high-throughput screening | reversible covalent inhibitor | aldehydes P erturbation of the protein folding environment in the endoplasmic reticulum (ER) leads to rectifying changes in gene expression and protein synthesis. These are mediated by an unfolded protein response (UPR), whose most conserved arm is initiated by an ER localized transmembrane protein, IRE1 (1, 2). The lumenal domain of IRE1 senses the perturbation in the ER and transmits the ER stress signal across the ER membrane to the effector cytosolic domain of the protein. This effector domain is endowed with two linked enzymatic activities: a protein kinase and an RNase, and both are activated by ER stress.The most conserved output of IRE1 signaling is the sitespecific cleavage of an mRNA, the product of the yeast HAC1 gene (3) and its metazoan orthologue Xbp1 (4, 5). Cleavage occurs at two distinct sites and is followed by ligation of the 5′ and 3′ fragments to generate an ER stress-dependent spliced mRNA encoding a potent transcription factor. The target genes of spliced XBP1 (and Hac1p) enhance the ability of the ER to cope with unfolded proteins (6) and also act more broadly to upregulate secretory capacity (7). In addition, mammalian IRE1 contributes to the promiscuous degradation of membrane-associated mRNAs in a process known as regulated IRE1-dependent degradation (or RIDD), but whose mechanistic basis and functional consequences are incompletely understood (8, 9).Fluctuating levels of ER stress accompany diverse physiological conditions. In metazoans, IRE1 signaling constitutes one arm of a three-pronged UPR. The other two arms are mediated by the translation initiation factor 2α (eIF2α) kinase PERK, which attenuates ER load by inhibiting protein synthesis in stressed cells, and by a parallel transcriptional pathway mediated by ATF6. Redundancy between the long-term transcriptional programs mediated by the three arms of the UPR has obscured the interp...

Targeting IRE1 with small molecules counteracts progression of atherosclerosis

“…S1 A-E). Both of these drugs were used at concentrations that do not compromise cell viability or induce any other toxicity (21,34,35,(37)(38)(39)(40). Consistent with earlier reports, these IRE1 inhibitors had no effect on the kinase function of IRE1 (Fig.…”

“…4A). Although limited pharmacodynamic data are available for the IRE1 inhibitors that we used, target engagement has been shown in cells and tissues (21,39). We based our in vivo drug dosage and delivery on earlier in vivo studies that successfully administered the IRE1 modulators without toxicity (21,24,31).…”

“…The action of both compounds is wellunderstood mechanistically. Both form a Schiff base with a specific lysine positioned in the active site of the IRE1 RNase domain, blocking its function, and both show no overt toxicity when administered systemically (21,32,33). Thus, we reasoned that these drugs might have therapeutic applicability in atherosclerosis.…”

“…Third, experimentally sustained Xbp1 mRNA splicing in the vessel wall promotes atherosclerosis, whereas its ablation ameliorates hypercholesterolemia in obese or apolipoprotein E-deficient (ApoE −/− ) mice (10,30). Two small molecules, STF-083010 and 4μ8C, which selectively inhibit IRE1's RNase function, have been used in cells and animals to produce favorable therapeutic outcomes in other disease settings: STF-083010 reduced growth of multiple myeloma (21,24,31,32), and 4μ8c suppressed inflammation in a murine arthritis model (33). The action of both compounds is wellunderstood mechanistically.…”

4μ8C Inhibits Insulin Secretion Independent of IRE1α RNase Activity

“…4μ8C (4-methyl umbelliferone 8-carbaldehyde) inhibits IRE1α RNase activity (Cross et al, 2012) by covalently modifying the K907 lysine residue in the RNase domain. It inhibits XBP1u mRNA splicing, but does not inhibit RNase L, which supports the idea that 4μ8C is a specific inhibitor of IRE1α RNase activity (Cross et al, 2012).…”