title: Potent and selective inhibitors of the inositol-requiring enzyme 1 endoribonuclease.
abstract: Inositol-requiring enzyme 1 (IRE1) is the most highly conserved signaling node of the unfolded protein response (UPR) and represents a potential therapeutic target for a number of diseases associated with endoplasmic reticulum stress. IRE1 activates the XBP-1 transcription factor by site-specific cleavage of two hairpin loops within its mRNA to facilitate its nonconventional splicing and alternative translation. We screened for inhibitors using a construct containing the unique cytosolic kinase and endoribonuclease domains of human IRE1α (hIRE1α-cyto) and a mini-XBP-1 stem-loop RNA as the substrate. One class compounds was salicylaldehyde analogs from the hydrolyzed product of salicylaldimines in the library. Salicylaldehyde analogs were active in inhibiting the site-specific cleavage of several mini-XBP-1 stem-loop RNAs in a dose-dependent manner. Salicyaldehyde analogs were also active in inhibiting yeast Ire1 but had little activity inhibiting RNase L or the unrelated RNases A and T1. Kinetic analysis revealed that one potent salicylaldehyde analog, 3-ethoxy-5,6-dibromosalicylaldehyde, is a non-competitive inhibitor with respect to the XBP-1 RNA substrate. Surface plasmon resonance studies confirmed this compound bound to IRE1 in a specific, reversible and dose-dependent manner. Salicylaldehydes inhibited XBP-1 splicing induced pharmacologically in human cells. These compounds also blocked transcriptional up-regulation of known XBP-1 targets as well as mRNAs targeted for degradation by IRE1. Finally, the salicylaldehyde analog 3-methoxy-6-bromosalicylaldehyde strongly inhibited XBP-1 splicing in an in vivo model of acute endoplasmic reticulum stress. To our knowledge, salicylaldehyde analogs are the first reported specific IRE1 endoribonuclease inhibitors.
authors.affiliation: MannKind Corporation, Valencia, California 91355, USA.
authors.forename: Kori
authors.initials: K
authors.lastname: Volkmann
authors.forename: Julie L
authors.initials: JL
authors.lastname: Lucas
authors.forename: Danka
authors.initials: D
authors.lastname: Vuga
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Wang
authors.forename: Duane
authors.initials: D
authors.lastname: Brumm
authors.forename: Caryn
authors.initials: C
authors.lastname: Stiles
authors.forename: David
authors.initials: D
authors.lastname: Kriebel
authors.forename: Ani
authors.initials: A
authors.lastname: Der-Sarkissian
authors.forename: Kris
authors.initials: K
authors.lastname: Krishnan
authors.forename: Colleen
authors.initials: C
authors.lastname: Schweitzer
authors.forename: Zheng
authors.initials: Z
authors.lastname: Liu
authors.forename: Uriel M
authors.initials: UM
authors.lastname: Malyankar
authors.forename: David
authors.initials: D
authors.lastname: Chiovitti
authors.forename: Marella
authors.initials: M
authors.lastname: Canny
authors.forename: Dan
authors.initials: D
authors.lastname: Durocher
authors.forename: Frank
authors.initials: F
authors.lastname: Sicheri
authors.forename: John B
authors.initials: JB
authors.lastname: Patterson
mesh_terms: D000447:Aldehydes / Q000737:chemistry*; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004722:Endoribonucleases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology*; D005260:Female; D006801:Humans; D020128:Inhibitory Concentration 50; D008565:Membrane Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding; D017510:Protein Folding / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D000071701:Regulatory Factor X Transcription Factors; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D000071717:X-Box Binding Protein 1
chemical_list: D000447:Aldehydes; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D000071701:Regulatory Factor X Transcription Factors; D014157:Transcription Factors; D000071717:X-Box Binding Protein 1; C000605543:XBP1 protein, human; C000605544:Xbp1 protein, mouse; C013243:salicylaldehyde; C403982:ERN2 protein, human; D017346:Protein Serine-Threonine Kinases; D004722:Endoribonucleases