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Small molecule strategies to harness the unfolded protein response: where do we go from here?

Open AccessPublished:September 04, 2020DOI:https://doi.org/10.1074/jbc.REV120.010218
      The unfolded protein response (UPR) plays a central role in regulating endoplasmic reticulum (ER) and global cellular physiology in response to pathologic ER stress. The UPR is comprised of three signaling pathways activated downstream of the ER membrane proteins IRE1, ATF6, and PERK. Once activated, these proteins initiate transcriptional and translational signaling that functions to alleviate ER stress, adapt cellular physiology, and dictate cell fate. Imbalances in UPR signaling are implicated in the pathogenesis of numerous, etiologically-diverse diseases, including many neurodegenerative diseases, protein misfolding diseases, diabetes, ischemic disorders, and cancer. This has led to significant interest in establishing pharmacologic strategies to selectively modulate IRE1, ATF6, or PERK signaling to both ameliorate pathologic imbalances in UPR signaling implicated in these different diseases and define the importance of the UPR in diverse cellular and organismal contexts. Recently, there has been significant progress in the identification and characterization of UPR modulating compounds, providing new opportunities to probe the pathologic and potentially therapeutic implications of UPR signaling in human disease. Here, we describe currently available UPR modulating compounds, specifically highlighting the strategies used for their discovery and specific advantages and disadvantages in their application for probing UPR function. Furthermore, we discuss lessons learned from the application of these compounds in cellular and in vivo models to identify favorable compound properties that can help drive the further translational development of selective UPR modulators for human disease.
      The endoplasmic reticulum (ER) is associated with critical biological functions, including protein secretion, lipid synthesis, and calcium regulation (
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      The role of the endoplasmic reticulum protein calreticulin in mediating TGF-β-stimulated extracellular matrix production in fibrotic disease.
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      Calcium and apoptosis: ER-mitochondria Ca2+ transfer in the control of apoptosis.
      ). Therefore, proper regulation of ER function in response to a constantly changing physiologic environment is crucial for organismal survival. Consistent with this, defects in ER biology are linked to nearly all types of human disease, including cancer, diabetes, infectious disease, amyloid diseases, ischemic disorders, and neurodegenerative diseases (
      • Wang S.
      • Kaufman R.J.
      The impact of the unfolded protein response on human disease.
      ,
      • Cnop M.
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      Endoplasmic reticulum stress, obesity and diabetes.
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      Regulating secretory proteostasis through the unfolded protein response: from function to therapy.
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      ER stress and the unfolded protein response in neurodegeneration.
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      Endoplasmic reticulum stress and type 2 diabetes.
      ,
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      • Hedou E.
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      • Roussel B.D.
      Proteostasis during cerebral ischemia.
      ). Considering the importance of the ER in disease pathogenesis, pharmacologic intervention to ameliorate pathologic imbalances in ER function has emerged as a promising therapeutic approach for a wide variety of disorders.
      One of the most attractive strategies to alter ER function in the context of disease is through targeting the unfolded protein response (UPR)—the predominant stress-responsive signaling pathway responsible for regulating ER and cellular physiology following an ER insult (i.e. ER stress) (
      • Zhang K.
      • Kaufman R.J.
      Signaling the unfolded protein response from the endoplasmic reticulum.
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      • Bernales S.
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      Intracellular signaling by the unfolded protein response.
      ,
      • Schroder M.
      • Kaufman R.J.
      ER stress and the unfolded protein response.
      ,
      • Hetz C.
      • Zhang K.
      • Kaufman R.J.
      Mechanisms, regulation and functions of the unfolded protein response.
      ,
      • Karagöz G.E.
      • Acosta-Alvear D.
      • Walter P.
      The unfolded protein response: detecting and responding to fluctuations in the protein-folding capacity of the endoplasmic reticulum.
      ,
      • Preissler S.
      • Ron D.
      Early events in the endoplasmic reticulum unfolded protein response.
      ). The UPR comprises three signaling pathways activated downstream of the ER stress–sensing transmembrane proteins inositol-requiring enzyme 1 (IRE1), protein kinase R–like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF6) (Fig. 1) (
      • Schroder M.
      • Kaufman R.J.
      ER stress and the unfolded protein response.
      ,
      • Hetz C.
      • Zhang K.
      • Kaufman R.J.
      Mechanisms, regulation and functions of the unfolded protein response.
      ,
      • Karagöz G.E.
      • Acosta-Alvear D.
      • Walter P.
      The unfolded protein response: detecting and responding to fluctuations in the protein-folding capacity of the endoplasmic reticulum.
      ,
      • Preissler S.
      • Ron D.
      Early events in the endoplasmic reticulum unfolded protein response.
      ,
      • Hollien J.
      Evolution of the unfolded protein response.
      ). These three signaling pathways are activated in response to diverse types of ER stress, including the accumulation of non-native proteins within the ER lumen and lipid disequilibrium within the ER membrane. Activation of these UPR pathways elicits transcriptional and translational remodeling of ER and global cellular physiology that functions to alleviate the ER stress and promote cellular adaption following an acute insult (Fig. 1). Through this activity, the UPR functions as a protective signaling pathway that is involved in regulating diverse aspects of cellular physiology, including maintenance of secretory proteostasis, proliferation, redox regulation, differentiation, and metabolism (
      • Zhang K.
      • Kaufman R.J.
      Signaling the unfolded protein response from the endoplasmic reticulum.
      ,
      • Bernales S.
      • Papa F.R.
      • Walter P.
      Intracellular signaling by the unfolded protein response.
      ). However, in response to chronic or severe ER insults that cannot be alleviated through protective remodeling, prolonged UPR activation leads to pro-apoptotic signaling (
      • Hetz C.
      • Papa F.R.
      The unfolded protein response and cell fate control.
      ,
      • Hetz C.
      • Zhang K.
      • Kaufman R.J.
      Mechanisms, regulation and functions of the unfolded protein response.
      ). Thus, the UPR serves a critical role in dictating both protective and apoptotic signaling in response to pathologic ER insults.
      Figure thumbnail gr1
      Figure 1The three ER stress–sensing proteins that activate UPR signaling. Activation of IRE1, PERK, and ATF6 promotes integrated signaling that translationally and transcriptionally remodels ER and cellular proteostasis.
      Due to the importance of UPR signaling for regulating ER function, it is not surprising that alterations in UPR signaling contribute to human disease pathogenesis. For example, hypomorphic or “loss-of-function” mutations in the EIF2AK3 gene, which encodes the PERK protein, are associated with multiple diseases, including Wolcott–Rallison syndrome, progressive supranuclear palsy, and late-stage Alzheimer's disease (
      • Bell M.C.
      • Meier S.E.
      • Ingram A.L.
      • Abisambra J.F.
      PERK-opathies: an endoplasmic reticulum stress mechanism underlying neurodegeneration.
      ,
      • Delepine M.
      • Nicolino M.
      • Barrett T.
      • Golamaully M.
      • Lathrop G.M.
      • Julier C.
      EIF2AK3, encoding translation initiation factor 2-α kinase 3, is mutated in patients with Wolcott-Rallison syndrome.
      ,
      • Yuan S.H.
      • Hiramatsu N.
      • Liu Q.
      • Sun X.V.
      • Lenh D.
      • Chan P.
      • Chiang K.
      • Koo E.H.
      • Kao A.W.
      • Litvan I.
      • Lin J.H.
      Tauopathy-associated PERK alleles are functional hypomorphs that increase neuronal vulnerability to ER stress.
      ,
      • Höglinger G.U.
      • Melhem N.M.
      • Dickson D.W.
      • Sleiman P.M.
      • Wang L.S.
      • Klei L.
      • Rademakers R.
      • de Silva R.
      • Litvan I.
      • Riley D.E.
      • van Swieten J.C.
      • Heutink P.
      • Wszolek Z.K.
      • Uitti R.J.
      • Vandrovcova J.
      • et al.
      Identification of common variants influencing risk of the tauopathy progressive supranuclear palsy.
      ). Similarly, environmental or aging-related deficiencies in UPR signaling contribute to diverse types of disease, including cardiovascular disorders and neurodegenerative diseases (
      • Hetz C.
      • Papa F.R.
      The unfolded protein response and cell fate control.
      ,
      • Hetz C.
      • Saxena S.
      ER stress and the unfolded protein response in neurodegeneration.
      ). In contrast, overactivity of UPR signaling is also associated with disease pathogenesis. For example, overactive PERK signaling is implicated in many different neurodegenerative diseases (
      • Hetz C.
      • Saxena S.
      ER stress and the unfolded protein response in neurodegeneration.
      ,
      • Hughes D.
      • Mallucci G.R.
      The unfolded protein response in neurodegenerative disorders—therapeutic modulation of the PERK pathway.
      ,
      • Remondelli P.
      • Renna M.
      The endoplasmic reticulum unfolded protein response in neurodegenerative disorders and its potential therapeutic significance.
      ). Similarly, chronic IRE1 activity is associated with atherosclerosis in mouse models (
      • Tufanli O.
      • Telkoparan Akillilar P.
      • Acosta-Alvear D.
      • Kocaturk B.
      • Onat U.I.
      • Hamid S.M.
      • Çimen I.
      • Walter P.
      • Weber C.
      • Erbay E.
      Targeting IRE1 with small molecules counteracts progression of atherosclerosis.
      ). Thus, either too much or too little signaling through UPR signaling pathways can promote pathogenesis in the context of human disease. This effect may be best demonstrated in the hereditary vision disorder achromatopsia, where mutations in the ATF6 gene that either increase or decrease ATF6 activity are both causatively implicated in the impaired retinal development central to disease pathogenesis (
      • Ansar M.
      • Santos-Cortez R.L.
      • Saqib M.A.
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      • Lee K.
      • Ashraf N.M.
      • Ullah E.
      • Wang X.
      • Sajid S.
      • Khan F.S.
      • Amin-Ud-Din M.
      University of Washington Center for Mendelian Genomics
      Mutation of ATF6 causes autosomal recessive achromatopsia.
      ,
      • Chiang W.-C.
      • Chan P.
      • Wissinger B.
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      • Krawczyński M.R.
      • Kaufman R.J.
      • Tsang S.H.
      • Héon E.
      • Kohl S.
      • Lin J.H.
      Achromatopsia mutations target sequential steps of ATF6 activation.
      ).
      The importance of altered UPR signaling in the pathogenesis of etiologically-diverse diseases makes these pathways attractive targets for therapeutic intervention (
      • Plate L.
      • Wiseman R.L.
      Regulating secretory proteostasis through the unfolded protein response: from function to therapy.
      ,
      • Glembotski C.C.
      • Rosarda J.D.
      • Wiseman R.L.
      Proteostasis and beyond: ATF6 in ischemic disease.
      ,
      • Hetz C.
      • Axten J.M.
      • Patterson J.B.
      Pharmacological targeting of the unfolded protein response for disease intervention.
      ). This has led to significant interest in establishing compounds that either activate or inhibit select UPR signaling pathways to provide new opportunities to define the therapeutic potential for targeting the UPR in human disease. Here, we discuss currently available compounds that target individual UPR pathways, specifically highlighting how they were discovered, their described mechanism of action, and their applicability for studying the importance of UPR signaling in cellular and in vivo models. In addition, we summarize lessons learned from these available UPR-modulating compounds to identify specific properties that confer increased translational potential for application in human disease to help guide the future development of next-generation compounds.

      The IRE1 arm of the UPR

      The IRE1 signaling pathway is the most highly conserved arm of the UPR, found in all organisms from yeast to humans (Fig. 1) (
      • Hollien J.
      Evolution of the unfolded protein response.
      ,
      • Nikawa J.
      • Yamashita S.
      IRE1 encodes a putative protein kinase containing a membrane-spanning domain and is required for inositol phototrophy in Saccharomyces cerevisiae.
      ). Notably, it was the first UPR pathway to be identified and is likely the most well-studied. IRE1 is a type I ER membrane protein comprising three domains: an ER luminal domain, a cytosolic kinase domain, and a cytosolic RNase domain (Fig. 2, A and B) (
      • Tirasophon W.
      • Welihinda A.A.
      • Kaufman R.J.
      A stress response pathway from the endoplasmic reticulum to the nucleus requires a novel bifunctional protein kinase/endoribonuclease (Ire1p) in mammalian cells.
      ,
      • Cox J.S.
      • Shamu C.E.
      • Walter P.
      Transcriptional induction of genes encoding endoplasmic reticulum resident proteins requires a transmembrane protein kinase.
      ). Mammals encode two different IRE1 isoforms: IRE1α and IRE1β. IRE1α is the predominant isoform associated with UPR signaling in most cell types, whereas IRE1β appears to primarily function through tissue-specific mechanisms and/or the regulation of IRE1α activity (
      • Bertolotti A.
      • Wang X.
      • Novoa I.
      • Jungreis R.
      • Schlessinger K.
      • Cho J.H.
      • West A.B.
      • Ron D.
      Increased sensitivity to dextran sodium sulfate colitis in IRE1β-deficient mice.
      ,
      • Grey M.J.
      • Cloots E.
      • Simpson M.S.
      • LeDuc N.
      • Serebrenik Y.V.
      • De Luca H.
      • De Sutter D.
      • Luong P.
      • Thiagarajah J.R.
      • Paton A.W.
      • Paton J.C.
      • Seeliger M.A.
      • Eyckerman S.
      • Janssens S.
      • Lencer W.I.
      IRE1β negatively regulates IRE1α signaling in response to endoplasmic reticulum stress.
      ,
      • Tsuru A.
      • Fujimoto N.
      • Takahashi S.
      • Saito M.
      • Nakamura D.
      • Iwano M.
      • Iwawaki T.
      • Kadokura H.
      • Ron D.
      • Kohno K.
      Negative feedback by IRE1β optimizes mucin production in goblet cells.
      ). For the purposes of this review, we primarily focus on the IRE1α isoform (herein referred to as IRE1 unless otherwise noted).
      Figure thumbnail gr2
      Figure 2Pharmacologic targeting of the IRE1 UPR signaling pathway. A, simplified mechanism of ER stress–dependent activation and downstream signaling of the IRE1 UPR signaling pathway. B, domain architecture of IRE1, including the luminal domain, transmembrane domain (TM), and the cytosolic kinase and RNase domains. Enzymatic activities of key domains are as shown in A, where the cytosolic kinase domain of IRE1 participates in trans-autophosphorylation, and, upon activation, the RNase domain functions through both XBP1 mRNA splicing and RIDD. C, image showing the binding of ADP to the nucleotide-binding pocket of human IRE1 (
      • Ali M.M.
      • Bagratuni T.
      • Davenport E.L.
      • Nowak P.R.
      • Silva-Santisteban M.C.
      • Hardcastle A.
      • McAndrews C.
      • Rowlands M.G.
      • Morgan G.J.
      • Aherne W.
      • Collins I.
      • Davies F.E.
      • Pearl L.H.
      Structure of the Ire1 autophosphorylation complex and implications for the unfolded protein response.
      ). D, structures of the IRE1 RNase active site inhibitors 4µ8c and STF-083010. E, structures of the Type II IRE1 kinase inhibitors Compound 3 and KIRA6 that inhibit both IRE1 kinase activity and RNase activity. F, structures of Type I IRE1 kinase inhibitors that inhibit IRE1 kinase activity while allosterically activating the IRE1 RNase. G, structures of the new IRE1/XBP1s-activating compounds IXA1, IXA4, and IXA6 identified through an HTS that prioritized transcriptional profiling.
      IRE1 is activated in response to diverse cellular insults, including ER stress and lipid disequilibrium (
      • Shamu C.E.
      • Walter P.
      Oligomerization and phosphorylation of the Ire1p kinase during intracellular signaling from the endoplasmic reticulum to the nucleus.
      ,
      • Volmer R.
      • van der Ploeg K.
      • Ron D.
      Membrane lipid saturation activates endoplasmic reticulum unfolded protein response transducers through their transmembrane domains.
      ). Despite being the most well-studied arm of the UPR, the molecular mechanism of IRE1 activation remains somewhat controversial, with multiple proposed models, all of which involve ER stress sensing by the IRE1 luminal domain. One prominent model suggests that IRE1 detects ER stress through dynamic interactions between the ER HSP70 chaperone binding immunoglobulin protein (BiP) and the IRE1 luminal domain through a process regulated by BiP co-chaperones, such as ER DNA J domain–containing protein 4 (ERdj4) (
      • Preissler S.
      • Ron D.
      Early events in the endoplasmic reticulum unfolded protein response.
      ,
      • Bertolotti A.
      • Zhang Y.
      • Hendershot L.M.
      • Harding H.P.
      • Ron D.
      Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response.
      ,
      • Amin-Wetzel N.
      • Saunders R.A.
      • Kamphuis M.J.
      • Rato C.
      • Preissler S.
      • Harding H.P.
      • Ron D.
      A J-protein co-chaperone recruits BiP to monomerize IRE1 and repress the unfolded protein response.
      ,
      • Amin-Wetzel N.
      • Neidhardt L.
      • Yan Y.
      • Mayer M.P.
      • Ron D.
      Unstructured regions in IRE1α specify BiP-mediated destabilisation of the luminal domain dimer and repression of the UPR.
      ). In this model, BiP dissociates from the IRE1 luminal domain in response to the accumulation of misfolded proteins with the ER lumen, thus activating signaling through this pathway (Fig. 2A). However, another model proposes that IRE1 directly binds non-native protein conformations in a putative peptide-binding groove found in the IRE1 luminal domain, suggesting that IRE1 directly senses the accumulation of non-native proteins during ER stress (Fig. 2A) (
      • Karagöz G.E.
      • Acosta-Alvear D.
      • Nguyen H.T.
      • Lee C.P.
      • Chu F.
      • Walter P.
      An unfolded protein-induced conformational switch activates mammalian IRE1.
      ,
      • Credle J.J.
      • Finer-Moore J.S.
      • Papa F.R.
      • Stroud R.M.
      • Walter P.
      On the mechanism of sensing unfolded protein in the endoplasmic reticulum.
      ,
      • Gardner B.M.
      • Walter P.
      Unfolded proteins are Ire1-activating ligands that directly induce the unfolded protein response.
      ). Whereas the precise mechanism of IRE1 activation is still being scrutinized, the downstream events of IRE1 signaling are well-established to involve IRE1 oligomerization, autophosphorylation, and RNase activation (Fig. 2A) (
      • Shamu C.E.
      • Walter P.
      Oligomerization and phosphorylation of the Ire1p kinase during intracellular signaling from the endoplasmic reticulum to the nucleus.
      ). IRE1 exists predominantly as a monomer in the ER membrane and in response to ER stress undergoes dimerization/oligomerization and subsequent trans-autophosphorylation (Fig. 2A) (
      • Karagöz G.E.
      • Acosta-Alvear D.
      • Walter P.
      The unfolded protein response: detecting and responding to fluctuations in the protein-folding capacity of the endoplasmic reticulum.
      ,
      • Preissler S.
      • Ron D.
      Early events in the endoplasmic reticulum unfolded protein response.
      ,
      • Shamu C.E.
      • Walter P.
      Oligomerization and phosphorylation of the Ire1p kinase during intracellular signaling from the endoplasmic reticulum to the nucleus.
      ). Whereas both dimer and higher-order IRE1 oligomers have been described in vitro, it appears that clustering of IRE1 into higher-order assemblies parallels maximal activation as measured by downstream signaling outputs (
      • Korennykh A.V.
      • Egea P.F.
      • Korostelev A.A.
      • Finer-Moore J.
      • Zhang C.
      • Shokat K.M.
      • Stroud R.M.
      • Walter P.
      The unfolded protein response signals through high-order assembly of Ire1.
      ,
      • Li H.
      • Korennykh A.V.
      • Behrman S.L.
      • Walter P.
      Mammalian endoplasmic reticulum stress sensor IRE1 signals by dynamic clustering.
      ). IRE1 autophosphorylation occurs at several sites on a conserved “activation loop” within its cytosolic kinase domain (Fig. 2C) (
      • Ali M.M.
      • Bagratuni T.
      • Davenport E.L.
      • Nowak P.R.
      • Silva-Santisteban M.C.
      • Hardcastle A.
      • McAndrews C.
      • Rowlands M.G.
      • Morgan G.J.
      • Aherne W.
      • Collins I.
      • Davies F.E.
      • Pearl L.H.
      Structure of the Ire1 autophosphorylation complex and implications for the unfolded protein response.
      ). Extensive structural and biochemical studies on yeast and human IRE1 have characterized the displacement of this loop upon ATP binding, due to direct interactions of bound ADP with a conserved DFG motif in the kinase active site (Fig. 2C) (
      • Ali M.M.
      • Bagratuni T.
      • Davenport E.L.
      • Nowak P.R.
      • Silva-Santisteban M.C.
      • Hardcastle A.
      • McAndrews C.
      • Rowlands M.G.
      • Morgan G.J.
      • Aherne W.
      • Collins I.
      • Davies F.E.
      • Pearl L.H.
      Structure of the Ire1 autophosphorylation complex and implications for the unfolded protein response.
      ). These events cause coordinated conformational changes through the IRE1 cytosolic domain, which allosterically activate the IRE1 RNase (
      • Ali M.M.
      • Bagratuni T.
      • Davenport E.L.
      • Nowak P.R.
      • Silva-Santisteban M.C.
      • Hardcastle A.
      • McAndrews C.
      • Rowlands M.G.
      • Morgan G.J.
      • Aherne W.
      • Collins I.
      • Davies F.E.
      • Pearl L.H.
      Structure of the Ire1 autophosphorylation complex and implications for the unfolded protein response.
      ). This RNase domain cleaves the mRNA encoding X-box binding protein 1 (XBP1), which is then re-ligated by the RTCB RNA ligase, resulting in a frameshift in this transcript (Fig. 2A) (
      • Sidrauski C.
      • Walter P.
      The transmembrane kinase Ire1p is a site-specific endonuclease that initiates mRNA splicing in the unfolded protein response.
      ,
      • Yoshida H.
      • Matsui T.
      • Yamamoto A.
      • Okada T.
      • Mori K.
      XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor.
      ,
      • Jurkin J.
      • Henkel T.
      • Nielsen A.F.
      • Minnich M.
      • Popow J.
      • Kaufmann T.
      • Heindl K.
      • Hoffmann T.
      • Busslinger M.
      • Martinez J.
      The mammalian tRNA ligase complex mediates splicing of XBP1 mRNA and controls antibody secretion in plasma cells.
      ,
      • Lu Y.
      • Liang F.X.
      • Wang X.
      A synthetic biology approach identifies the mammalian UPR RNA ligase RtcB.
      ,
      • Kosmaczewski S.G.
      • Edwards T.J.
      • Han S.M.
      • Eckwahl M.J.
      • Meyer B.I.
      • Peach S.
      • Hesselberth J.R.
      • Wolin S.L.
      • Hammarlund M.
      The RtcB RNA ligase is an essential component of the metazoan unfolded protein response.
      ). Spliced XBP1 mRNA (XBP1s) encodes the active transcription factor spliced XBP1 (XBP1s), which up-regulates transcriptional targets that promote ER proteostasis, including genes involved in ER-associated degradation (ERAD), ER chaperones and folding enzymes, and N-linked glycosylation (Fig. 2A) (
      • Yoshida H.
      • Matsui T.
      • Yamamoto A.
      • Okada T.
      • Mori K.
      XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor.
      ,
      • Shoulders M.D.
      • Ryno L.M.
      • Genereux J.C.
      • Moresco J.J.
      • Tu P.G.
      • Wu C.
      • Yates 3rd, J.R.
      • Su A.I.
      • Kelly J.W.
      • Wiseman R.L.
      Stress-independent activation of XBP1s and/or ATF6 reveals three functionally diverse ER proteostasis environments.
      ). In addition, XBP1s has also been proposed to up-regulate transcripts involved in other biological pathways, including lipid biosynthesis (Fig. 2A) (
      • Shoulders M.D.
      • Ryno L.M.
      • Genereux J.C.
      • Moresco J.J.
      • Tu P.G.
      • Wu C.
      • Yates 3rd, J.R.
      • Su A.I.
      • Kelly J.W.
      • Wiseman R.L.
      Stress-independent activation of XBP1s and/or ATF6 reveals three functionally diverse ER proteostasis environments.
      ,
      • Sriburi R.
      • Jackowski S.
      • Mori K.
      • Brewer J.W.
      XBP1: a link between the unfolded protein response, lipid biosynthesis, and biogenesis of the endoplasmic reticulum.
      ). In general, XBP1s transcriptional signaling is a protective mechanism to promote ER proteostasis and cellular adaptation in response to acute stress.
      Aside from inducing transcriptional changes via activation of XBP1s, IRE1 has other functionalities that play a role in stress-responsive signaling following ER stress. One of these additional functions is termed regulated IRE1-dependent decay (RIDD), in which the activated IRE1 RNase degrades a variety of ER-associated mRNAs (Fig. 2A) (
      • Hollien J.
      • Weissman J.S.
      Decay of endoplasmic reticulum-localized mRNAs during the unfolded protein response.
      ,
      • Hollien J.
      • Lin J.H.
      • Li H.
      • Stevens N.
      • Walter P.
      • Weissman J.S.
      Regulated Ire1-dependent decay of messenger RNAs in mammalian cells.
      ). Whereas the specificity for these RIDD substrates is not yet understood, many putative RIDD targets have been identified, including scavenger receptor class A member 3 (SCARA3) and biogenesis of lysosomal organelles complex 1 (BLOS1) (
      • Hollien J.
      • Lin J.H.
      • Li H.
      • Stevens N.
      • Walter P.
      • Weissman J.S.
      Regulated Ire1-dependent decay of messenger RNAs in mammalian cells.
      ,
      • Bae D.
      • Moore K.A.
      • Mella J.M.
      • Hayashi S.Y.
      • Hollien J.
      Degradation of Blos1 mRNA by IRE1 repositions lysosomes and protects cells from stress.
      ). Unlike protective XBP1s transcriptional signaling, IRE1 RIDD activity has been implicated in both protective and pro-apoptotic signaling. For example, RIDD activity reduces incoming protein folding load within the ER and suppresses apoptotic signaling through degradation of mRNA encoding death receptor 5 (DR5) (
      • Lu M.
      • Lawrence D.A.
      • Marsters S.
      • Acosta-Alvear D.
      • Kimmig P.
      • Mendez A.S.
      • Paton A.W.
      • Paton J.C.
      • Walter P.
      • Ashkenazi A.
      Opposing unfolded-protein-response signals converge on death receptor 5 to control apoptosis.
      ). Similarly, IRE1-dependent degradation of BLOS1 mRNA through RIDD promotes repositioning of late endosomes for degradation of protein aggregates (
      • Bae D.
      • Moore K.A.
      • Mella J.M.
      • Hayashi S.Y.
      • Hollien J.
      Degradation of Blos1 mRNA by IRE1 repositions lysosomes and protects cells from stress.
      ). In contrast, RIDD has also been suggested to promote apoptotic signaling through the degradation of mRNA encoding protective UPR-regulated chaperones (e.g. BiP) and the degradation of anti-apoptotic pre-miRNAs (
      • Han D.
      • Lerner A.G.
      • Vande Walle L.
      • Upton J.P.
      • Xu W.
      • Hagen A.
      • Backes B.J.
      • Oakes S.A.
      • Papa F.R.
      IRE1α kinase activation modes control alternate endoribonuclease outputs to determine divergent cell fates.
      ,
      • Upton J.P.
      • Wang L.
      • Han D.
      • Wang E.S.
      • Huskey N.E.
      • Lim L.
      • Truitt M.
      • McManus M.T.
      • Ruggero D.
      • Goga A.
      • Papa F.R.
      • Oakes S.A.
      IRE1α cleaves select microRNAs during ER stress to derepress translation of proapoptotic Caspase-2.
      ). Thus, RIDD activity appears to be involved in the transitioning of IRE1 signaling from adaptive to pro-apoptotic in response to severe or prolonged ER stress. Interestingly, IRE1-dependent degradation of BLOS1 and other RIDD targets has been suggested to involve signaling through the PERK arm of the UPR, although PERK activation on its own is not sufficient to promote RIDD, highlighting the importance of integration between UPR signaling pathways in regulating cellular responses to ER stress (
      • Moore K.
      • Hollien J.
      Ire1-mediated decay in mammalian cells relies on mRNA sequence, structure, and translational status.
      ).
      IRE1 also promotes signaling independent of its RNase activity. Active, phosphorylated IRE1 can bind tumor necrosis factor receptor–associated factor 2 (TRAF2) to promote apoptotic signaling downstream of the ASK-JNK signaling axis and inflammatory signaling downstream of nuclear factor κB (NFκB) (Fig. 2A) (
      • Urano F.
      • Wang X.
      • Bertolotti A.
      • Zhang Y.
      • Chung P.
      • Harding H.P.
      • Ron D.
      Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1.
      ,
      • Tam A.B.
      • Mercado E.L.
      • Hoffmann A.
      • Niwa M.
      ER stress activates NF-κB by integrating functions of basal IKK activity, IRE1 and PERK.
      ). This IRE1-TRAF2 signaling promotes cell death and inflammation in response to severe or chronic ER insults associated with pathologic conditions, including fatty liver disease and neurodegeneration (
      • Lee H.
      • Noh J.Y.
      • Oh Y.
      • Kim Y.
      • Chang J.W.
      • Chung C.W.
      • Lee S.T.
      • Kim M.
      • Ryu H.
      • Jung Y.K.
      IRE1 plays an essential role in ER stress-mediated aggregation of mutant huntingtin via the inhibition of autophagy flux.
      ,
      • Hu P.
      • Han Z.
      • Couvillon A.D.
      • Kaufman R.J.
      • Exton J.H.
      Autocrine tumor necrosis factor α links endoplasmic reticulum stress to the membrane death receptor pathway through IRE1α-mediated NF-κB activation and down-regulation of TRAF2 expression.
      ,
      • Wang J.
      • He W.
      • Tsai P.J.
      • Chen P.H.
      • Ye M.
      • Guo J.
      • Su Z.
      Mutual interaction between endoplasmic reticulum and mitochondria in nonalcoholic fatty liver disease.
      ). This ability of IRE1 to promote cell death independent of its RNase activity is an important consideration when developing pharmacologic approaches to modulate IRE1 activity in the context of human disease.
      The potential to influence diverse aspects of IRE1 signaling using pharmacologic approaches represents a unique opportunity to alter pathologic imbalances in UPR signaling implicated in diverse diseases. Below, we discuss the different types of compounds available to activate or inhibit IRE1 signaling and their application to probe IRE1 function in cellular and in vivo models of disease.

      Preventing IRE1 signaling with RNase inhibitors

      Whereas IRE1 activity is often protective during acute ER stress, chronic activity of this pathway has been associated with numerous disease etiologies and can support the persistence of certain cancers (
      • Wang M.
      • Kaufman R.J.
      The impact of the endoplasmic reticulum protein-folding environment on cancer development.
      ,
      • Chevet E.
      • Hetz C.
      • Samali A.
      Endoplasmic reticulum stress-activated cell reprogramming in oncogenesis.
      ). Thus, multiple strategies have been employed to develop potent inhibitors of IRE1 signaling. One such strategy focuses on developing compounds that block IRE1 RNase activity to prevent XBP1 splicing. To this end, high-throughput screening using in vitro FRET assays for XBP1 cleavage has been commonly utilized to identify these types of IRE1 RNase inhibitors. This approach has been used to identify classes of salicylaldehyde analogs (e.g. MK0186893) and umbelliferones (e.g. 4µ8c), as promising IRE1 inhibitors that selectively prevent IRE1 RNase activity but do not elicit cellular toxicity (Fig. 2D) (
      • Cross B.C.
      • Bond P.J.
      • Sadowski P.G.
      • Jha B.K.
      • Zak J.
      • Goodman J.M.
      • Silverman R.H.
      • Neubert T.A.
      • Baxendale I.R.
      • Ron D.
      • Harding H.P.
      The molecular basis for selective inhibition of unconventional mRNA splicing by an IRE1-binding small molecule.
      ,
      • Volkmann K.
      • Lucas J.L.
      • Vuga D.
      • Wang X.
      • Brumm D.
      • Stiles C.
      • Kriebel D.
      • Der-Sarkissian A.
      • Krishnan K.
      • Schweitzer C.
      • Liu Z.
      • Malyankar U.M.
      • Chiovitti D.
      • Canny M.
      • Durocher D.
      • et al.
      Potent and selective inhibitors of the inositol-requiring enzyme 1 endoribonuclease.
      ). Another IRE1 RNase inhibitor, STF-083010, was identified using a phenotypic cell-based high-throughput screen monitoring the activity of an XBP1-splicing luciferase reporter in human fibrosarcoma cells subjected to ER stress, demonstrating that cell-based strategies can also be applied to identify this class of inhibitor (Fig. 2D) (
      • Papandreou I.
      • Denko N.C.
      • Olson M.
      • Van Melckebeke H.
      • Lust S.
      • Tam A.
      • Solow-Cordero D.E.
      • Bouley D.M.
      • Offner F.
      • Niwa M.
      • Koong A.C.
      Identification of an Ire1α endonuclease specific inhibitor with cytotoxic activity against human multiple myeloma.
      ). All of these IRE1 RNase inhibitors exhibit selectivity for IRE1 in cell-based models and have been shown to covalently modify residues in the IRE1 RNase domain via Schiff base formation (
      • Cross B.C.
      • Bond P.J.
      • Sadowski P.G.
      • Jha B.K.
      • Zak J.
      • Goodman J.M.
      • Silverman R.H.
      • Neubert T.A.
      • Baxendale I.R.
      • Ron D.
      • Harding H.P.
      The molecular basis for selective inhibition of unconventional mRNA splicing by an IRE1-binding small molecule.
      ). Importantly, 4µ8c and STF-083010, but not MK0186893, directly bind the IRE1 RNase domain without modification of the kinase domain, indicating that these compounds are able to block IRE1 signaling activated by the RNase domain (e.g. RIDD and XBP1 splicing), without interfering with IRE1 phosphorylation status (
      • Cross B.C.
      • Bond P.J.
      • Sadowski P.G.
      • Jha B.K.
      • Zak J.
      • Goodman J.M.
      • Silverman R.H.
      • Neubert T.A.
      • Baxendale I.R.
      • Ron D.
      • Harding H.P.
      The molecular basis for selective inhibition of unconventional mRNA splicing by an IRE1-binding small molecule.
      ). This specific activity provides a useful strategy to separate the enzymatic activities involved in IRE1 signaling and to probe the direct role of the RNase domain in the various IRE1 signaling functionalities.
      Importantly, use of IRE1 RNase inhibitors has shown promise as a potential therapeutic strategy to counteract disease pathogenesis associated with overactive IRE1 signaling. For example, both STF-083010 and 4µ8c prevent the systemic up-regulation of inflammatory factors IL-1β and IL-18 downstream of IRE1 in response to hyperlipidemia in mice, preventing the chronic “metainflammation” known to play a role in the development of metabolic disorders (
      • Tufanli O.
      • Telkoparan Akillilar P.
      • Acosta-Alvear D.
      • Kocaturk B.
      • Onat U.I.
      • Hamid S.M.
      • Çimen I.
      • Walter P.
      • Weber C.
      • Erbay E.
      Targeting IRE1 with small molecules counteracts progression of atherosclerosis.
      ). Consistent with this, these compounds lower immune responses and atherosclerotic plaque development in mouse models of atherosclerosis (
      • Tufanli O.
      • Telkoparan Akillilar P.
      • Acosta-Alvear D.
      • Kocaturk B.
      • Onat U.I.
      • Hamid S.M.
      • Çimen I.
      • Walter P.
      • Weber C.
      • Erbay E.
      Targeting IRE1 with small molecules counteracts progression of atherosclerosis.
      ). Additionally, 4µ8c and STF-083010 slow cancer cell proliferation in models of multiple myeloma, demonstrating the potential of inhibiting prosurvival UPR functionality in carcinogenic cells as a mode of chemotherapy (
      • Cross B.C.
      • Bond P.J.
      • Sadowski P.G.
      • Jha B.K.
      • Zak J.
      • Goodman J.M.
      • Silverman R.H.
      • Neubert T.A.
      • Baxendale I.R.
      • Ron D.
      • Harding H.P.
      The molecular basis for selective inhibition of unconventional mRNA splicing by an IRE1-binding small molecule.
      ,
      • Papandreou I.
      • Denko N.C.
      • Olson M.
      • Van Melckebeke H.
      • Lust S.
      • Tam A.
      • Solow-Cordero D.E.
      • Bouley D.M.
      • Offner F.
      • Niwa M.
      • Koong A.C.
      Identification of an Ire1α endonuclease specific inhibitor with cytotoxic activity against human multiple myeloma.
      ). Importantly, whereas both 4µ8c and STF-083010 are widely used and exhibit limited toxicity, 4µ8c has some reported off-target effects. For example, treatment of pancreatic β-cells with 4µ8c resulted in reduced insulin secretion independent of IRE1 RNase inhibition (
      • Sato H.
      • Shiba Y.
      • Tsuchiya Y.
      • Saito M.
      • Kohno K.
      4mu8C inhibits insulin secretion independent of IRE1α RNase activity.
      ). In addition, 4µ8c appears to have antioxidant properties, demonstrated by decreases in angiotensin II–induced reactive oxygen species production (
      • Chan S.M.H.
      • Lowe M.P.
      • Bernard A.
      • Miller A.A.
      • Herbert T.P.
      The inositol-requiring enzyme 1 (IRE1α) RNAse inhibitor, 4micro8C, is also a potent cellular antioxidant.
      ). Therefore, whereas STF-083010, 4µ8c, and related analogs have distinguished themselves as useful tools to inhibit IRE1 RNase signaling, special consideration must be given when using 4µ8c for investigating the specific consequences of IRE1 inhibition in cellular and in vivo models.

      Inhibiting IRE1 autophosphorylation with kinase inhibitors

      Similar in vitro approaches to those described above have also been used to establish compounds that inhibit IRE1 kinase activity. Generally, kinase inhibitors are classified as Type I or Type II based on their ability to stabilize kinase active sites in opposing conformations (
      • Liu Y.
      • Gray N.S.
      Rational design of inhibitors that bind to inactive kinase conformations.
      ). In the context of IRE1, Type II inhibitors have been shown to stabilize the IRE1 activation loop in a conformation that blocks both IRE1 autophosphorylation and allosteric activation of the IRE1 RNase (
      • Wang L.
      • Perera B.G.
      • Hari S.B.
      • Bhhatarai B.
      • Backes B.J.
      • Seeliger M.A.
      • Schurer S.C.
      • Oakes S.A.
      • Papa F.R.
      • Maly D.J.
      Divergent allosteric control of the IRE1α endoribonuclease using kinase inhibitors.
      ). Thus, Type II kinase inhibitors are predicted to inhibit both IRE1 RNase-dependent activities (e.g. XBP1 splicing and RIDD) and RNase-independent functions dependent on IRE1 phosphorylation (e.g. TRAF2 binding). A class of pyrazolopyrimidine-based Type II kinase inhibitors was identified by FRET-based screening, leading to the development of Compound 3, which prevented XBP1 cleavage to a similar extent as the RNase inhibitor, STF-083010 (Fig. 2E) (
      • Wang L.
      • Perera B.G.
      • Hari S.B.
      • Bhhatarai B.
      • Backes B.J.
      • Seeliger M.A.
      • Schurer S.C.
      • Oakes S.A.
      • Papa F.R.
      • Maly D.J.
      Divergent allosteric control of the IRE1α endoribonuclease using kinase inhibitors.
      ). Whereas Compound 3 was further validated to inhibit IRE1 in INS-1 cells, its selectivity for IRE1 remains to be defined in cell-based models (
      • Wang L.
      • Perera B.G.
      • Hari S.B.
      • Bhhatarai B.
      • Backes B.J.
      • Seeliger M.A.
      • Schurer S.C.
      • Oakes S.A.
      • Papa F.R.
      • Maly D.J.
      Divergent allosteric control of the IRE1α endoribonuclease using kinase inhibitors.
      ). A second generation of IRE1 Type II kinase inhibitors, called kinase-inhibiting RNase attenuators (KIRAs), included KIRA6, which had the same mechanistic properties as Compound 3 (Fig. 2E) (
      • Ghosh R.
      • Wang L.
      • Wang E.S.
      • Perera B.G.
      • Igbaria A.
      • Morita S.
      • Prado K.
      • Thamsen M.
      • Caswell D.
      • Macias H.
      • Weiberth K.F.
      • Gliedt M.J.
      • Alavi M.V.
      • Hari S.B.
      • Mitra A.K.
      • et al.
      Allosteric inhibition of the IRE1α RNase preserves cell viability and function during endoplasmic reticulum stress.
      ). However, KIRA6 afforded increased potency for inhibition of cytosolic IRE1 activity in vitro (
      • Ghosh R.
      • Wang L.
      • Wang E.S.
      • Perera B.G.
      • Igbaria A.
      • Morita S.
      • Prado K.
      • Thamsen M.
      • Caswell D.
      • Macias H.
      • Weiberth K.F.
      • Gliedt M.J.
      • Alavi M.V.
      • Hari S.B.
      • Mitra A.K.
      • et al.
      Allosteric inhibition of the IRE1α RNase preserves cell viability and function during endoplasmic reticulum stress.
      ). Further characterization in INS-1 cells demonstrated that KIRA6 prevented IRE1 phosphorylation and XBP1 splicing induced by the ER stressor, tunicamycin, in a dose-dependent manner (
      • Ghosh R.
      • Wang L.
      • Wang E.S.
      • Perera B.G.
      • Igbaria A.
      • Morita S.
      • Prado K.
      • Thamsen M.
      • Caswell D.
      • Macias H.
      • Weiberth K.F.
      • Gliedt M.J.
      • Alavi M.V.
      • Hari S.B.
      • Mitra A.K.
      • et al.
      Allosteric inhibition of the IRE1α RNase preserves cell viability and function during endoplasmic reticulum stress.
      ). Studies in other cellular and in vitro models, however, describe KIRA6-induced cell death at nanomolar concentrations and poor specificity against a panel of diverse kinases, thus demonstrating the need for validating the selectivity of these types of UPR modulators in multiple contexts (
      • Mahameed M.
      • Wilhelm T.
      • Darawshi O.
      • Obiedat A.
      • Tommy W.S.
      • Chintha C.
      • Schubert T.
      • Samali A.
      • Chevet E.
      • Eriksson L.A.
      • Huber M.
      • Tirosh B.
      The unfolded protein response modulators GSK2606414 and KIRA6 are potent KIT inhibitors.
      ,
      • Harnoss J.M.
      • Le Thomas A.
      • Shemorry A.
      • Marsters S.A.
      • Lawrence D.A.
      • Lu M.
      • Chen Y.A.
      • Qing J.
      • Totpal K.
      • Kan D.
      • Segal E.
      • Merchant M.
      • Reichelt M.
      • Ackerly Wallweber H.
      • Wang W.
      • et al.
      Disruption of IRE1α through its kinase domain attenuates multiple myeloma.
      ). Despite these challenges, KIRA6 and the related compound KIRA8 have been shown to reduce pancreatic damage associated with Type I diabetes, protect the retina against ER stress–induced apoptosis, and decrease Zika virus infection, demonstrating the potential for these types of kinase inhibitors to mitigate pathologic events associated with IRE1 signaling in disease models (
      • Ghosh R.
      • Wang L.
      • Wang E.S.
      • Perera B.G.
      • Igbaria A.
      • Morita S.
      • Prado K.
      • Thamsen M.
      • Caswell D.
      • Macias H.
      • Weiberth K.F.
      • Gliedt M.J.
      • Alavi M.V.
      • Hari S.B.
      • Mitra A.K.
      • et al.
      Allosteric inhibition of the IRE1α RNase preserves cell viability and function during endoplasmic reticulum stress.
      ,
      • Chu H.
      • Yuen T.T.T.
      • Chik K.K.H.
      • Yuan S.
      • Shuai H.
      • Zou Z.
      • Wang Y.
      • Zhu Z.
      • Yang D.
      • Poon V.K.M.
      • Chan C.C.S.
      • Zhou J.
      • Yin F.
      • Kok K.H.
      • Yuen K.Y.
      • et al.
      Targeting the inositol-requiring enzyme-1 pathway efficiently reverts Zika virus-induced neurogenesis and spermatogenesis marker perturbations.
      ,
      • Morita S.
      • Villalta S.A.
      • Feldman H.C.
      • Register A.C.
      • Rosenthal W.
      • Hoffmann-Petersen I.T.
      • Mehdizadeh M.
      • Ghosh R.
      • Wang L.
      • Colon-Negron K.
      • Meza-Acevedo R.
      • Backes B.J.
      • Maly D.J.
      • Bluestone J.A.
      • Papa F.R.
      Targeting ABL-IRE1α signaling spares ER-stressed pancreatic β cells to reverse autoimmune diabetes.
      ). However, further studies are still needed to validate the dependence of these observed benefits on the specific inhibition of IRE1 activity as opposed to off-target effects.

      Allosteric activation of IRE1 RNase activity using kinase inhibitors

      Type I kinase inhibitors have opposing effects to Type II inhibitors on the conformation of the activation loop within the IRE1 kinase active site (
      • Papa F.R.
      • Zhang C.
      • Shokat K.
      • Walter P.
      Bypassing a kinase activity with an ATP-competitive drug.
      ,
      • Lin J.H.
      • Li H.
      • Yasumura D.
      • Cohen H.R.
      • Zhang C.
      • Panning B.
      • Shokat K.M.
      • Lavail M.M.
      • Walter P.
      IRE1 signaling affects cell fate during the unfolded protein response.
      ). Thus, Type I kinase inhibitors are predicted to allosterically activate the IRE1 RNase domain, while inhibiting IRE1 autophosphorylation. This class of kinase inhibitors unsurprisingly includes ATP mimics, such as the clinically approved kinase inhibitor sunitinib and the aminopyrazole APY29 (Fig. 2F) (
      • Korennykh A.V.
      • Egea P.F.
      • Korostelev A.A.
      • Finer-Moore J.
      • Zhang C.
      • Shokat K.M.
      • Stroud R.M.
      • Walter P.
      The unfolded protein response signals through high-order assembly of Ire1.
      ). Co-crystal structures of yeast Ire1 bound to APY29 demonstrated this mechanism of allosteric RNase activation by Type I kinase inhibitors (
      • Korennykh A.V.
      • Egea P.F.
      • Korostelev A.A.
      • Finer-Moore J.
      • Zhang C.
      • Shokat K.M.
      • Stroud R.M.
      • Walter P.
      The unfolded protein response signals through high-order assembly of Ire1.
      ). Subsequent studies with APY29 in INS-1 cells showed divergent effects of APY29 and the Type II IRE1 kinase inhibitor, Compound 3, on XBP1 splicing and multiple other downstream consequences of IRE1 signaling, further highlighting the distinct activities of these two classes of kinase inhibitors on IRE1 RNase activity (
      • Wang L.
      • Perera B.G.
      • Hari S.B.
      • Bhhatarai B.
      • Backes B.J.
      • Seeliger M.A.
      • Schurer S.C.
      • Oakes S.A.
      • Papa F.R.
      • Maly D.J.
      Divergent allosteric control of the IRE1α endoribonuclease using kinase inhibitors.
      ). Importantly, cell-based studies indicated pleiotropic toxicity from APY29 treatment at low micromolar concentrations, making it difficult to apply this compound to diverse cellular contexts (
      • Ghosh R.
      • Wang L.
      • Wang E.S.
      • Perera B.G.
      • Igbaria A.
      • Morita S.
      • Prado K.
      • Thamsen M.
      • Caswell D.
      • Macias H.
      • Weiberth K.F.
      • Gliedt M.J.
      • Alavi M.V.
      • Hari S.B.
      • Mitra A.K.
      • et al.
      Allosteric inhibition of the IRE1α RNase preserves cell viability and function during endoplasmic reticulum stress.
      ).
      Targeted compound-engineering efforts have additionally resulted in the development of other IRE1 kinase inhibitors, such as IPA (Fig. 2F)(
      • Mendez A.S.
      • Alfaro J.
      • Morales-Soto M.A.
      • Dar A.C.
      • McCullagh E.
      • Gotthardt K.
      • Li H.
      • Acosta-Alvear D.
      • Sidrauski C.
      • Korennykh A.V.
      • Bernales S.
      • Shokat K.M.
      • Walter P.
      Endoplasmic reticulum stress-independent activation of unfolded protein response kinases by a small molecule ATP-mimic.
      ). This compound binds the IRE1 kinase active site and stabilizes its active conformation to increase IRE1 oligomerization and subsequent RNase activation (
      • Mendez A.S.
      • Alfaro J.
      • Morales-Soto M.A.
      • Dar A.C.
      • McCullagh E.
      • Gotthardt K.
      • Li H.
      • Acosta-Alvear D.
      • Sidrauski C.
      • Korennykh A.V.
      • Bernales S.
      • Shokat K.M.
      • Walter P.
      Endoplasmic reticulum stress-independent activation of unfolded protein response kinases by a small molecule ATP-mimic.
      ). However, like APY29, IPA demonstrated cellular toxicity at nanomolar concentrations in cells, limiting the potential for this compound to probe IRE1 activation in cellular and organismal models (
      • Mendez A.S.
      • Alfaro J.
      • Morales-Soto M.A.
      • Dar A.C.
      • McCullagh E.
      • Gotthardt K.
      • Li H.
      • Acosta-Alvear D.
      • Sidrauski C.
      • Korennykh A.V.
      • Bernales S.
      • Shokat K.M.
      • Walter P.
      Endoplasmic reticulum stress-independent activation of unfolded protein response kinases by a small molecule ATP-mimic.
      ). This toxicity is likely associated with off-target binding of IPA to other kinases. Consistent with this, the IPA scaffold also bound to the PERK kinase active site to activate PERK signaling at low concentrations (<2 μm) and inhibit PERK signaling at higher concentrations (>2 μm) in HEK293T cells (
      • Mendez A.S.
      • Alfaro J.
      • Morales-Soto M.A.
      • Dar A.C.
      • McCullagh E.
      • Gotthardt K.
      • Li H.
      • Acosta-Alvear D.
      • Sidrauski C.
      • Korennykh A.V.
      • Bernales S.
      • Shokat K.M.
      • Walter P.
      Endoplasmic reticulum stress-independent activation of unfolded protein response kinases by a small molecule ATP-mimic.
      ).
      Type I and Type II IRE1 kinase inhibitors have proven very useful for deconvoluting the molecular mechanism of IRE1 activation and allow a unique opportunity to inhibit or activate IRE1 RNase signaling through kinase inhibition without directly targeting the RNase domain. However, due to the off-target activity of these compounds (likely associated with binding to other kinases), toxicity, and/or poor bioavailability, these compounds have proven less useful for determining the functional implications of IRE1 signaling in the context of complex cellular and in vivo systems, as compared with other IRE1-modulating compounds. Regardless, these compounds can be used in certain cellular contexts, as well as in tandem with compounds that directly modulate IRE1 RNase activity, to validate the role of IRE1 signaling in a variety of biological and pathological settings.

      Phenotypic screening for IRE1-activating compounds with a novel mechanism

      In addition to targeted screening for IRE1 activators, phenotypic screening provides a useful approach to identify compounds that activate specific aspects of IRE1 signaling. Recently, a cell-based phenotypic high-throughput screen (HTS) was applied to identify compounds that selectively activate the protective IRE1/XBP1s signaling arm of the UPR. This screening strategy prioritized transcriptional selectivity to identify compounds that show preferential activation of IRE1/XBP1s over other arms of the UPR or other stress-responsive signaling pathways. This HTS used an IRE1-dependent XBP1 splicing luciferase reporter to screen a >650,000-compound library to identify IRE1-activating compounds (
      • Grandjean J.M.D.
      • Madhavan A.
      • Cech L.
      • Seguinot B.O.
      • Paxman R.J.
      • Smith E.
      • Scampavia L.
      • Powers E.T.
      • Cooley C.B.
      • Plate L.
      • Spicer T.P.
      • Kelly J.W.
      • Wiseman R.L.
      Pharmacologic IRE1/XBP1s activation confers targeted ER proteostasis reprogramming.
      ). Hits were then counterscreened against a luciferase reporter for the ATF6 UPR signaling pathway to identify compounds that preferentially activate IRE1/XBP1s signaling over other arms of the UPR. Through this screen, three compounds were identified, IXA1, IXA4, and IXA6, which were shown to selectively activate IRE1-dependent XBP1s signaling without significantly activating RIDD or TRAF2-dependent signaling (Fig. 2G). Importantly, these compounds were non-toxic in HEK293TREX cells (IC50 > 3 μm) (
      • Grandjean J.M.D.
      • Madhavan A.
      • Cech L.
      • Seguinot B.O.
      • Paxman R.J.
      • Smith E.
      • Scampavia L.
      • Powers E.T.
      • Cooley C.B.
      • Plate L.
      • Spicer T.P.
      • Kelly J.W.
      • Wiseman R.L.
      Pharmacologic IRE1/XBP1s activation confers targeted ER proteostasis reprogramming.
      ). The selectivity of these compounds for the IRE1/XBP1s pathway was further defined by RNA-seq transcriptional profiling, confirming that these compounds do not activate other arms of the UPR or other stress-responsive signaling pathways (
      • Grandjean J.M.D.
      • Madhavan A.
      • Cech L.
      • Seguinot B.O.
      • Paxman R.J.
      • Smith E.
      • Scampavia L.
      • Powers E.T.
      • Cooley C.B.
      • Plate L.
      • Spicer T.P.
      • Kelly J.W.
      • Wiseman R.L.
      Pharmacologic IRE1/XBP1s activation confers targeted ER proteostasis reprogramming.
      ). Importantly, these compounds increase IRE1/XBP1s signaling without inhibiting kinase activity, indicating that these compounds activate IRE1 through a mechanism distinct from the above-mentioned Type I kinase inhibitors (
      • Grandjean J.M.D.
      • Madhavan A.
      • Cech L.
      • Seguinot B.O.
      • Paxman R.J.
      • Smith E.
      • Scampavia L.
      • Powers E.T.
      • Cooley C.B.
      • Plate L.
      • Spicer T.P.
      • Kelly J.W.
      • Wiseman R.L.
      Pharmacologic IRE1/XBP1s activation confers targeted ER proteostasis reprogramming.
      ). Although the mechanism of action for these compounds as well as in vivo efficacy remains to be established, these compounds have already proven useful for demonstrating the potential for pharmacologic IRE1 activation to reduce the production and toxicity of amyloid precursor protein proteolytic fragments in cell culture models (
      • Grandjean J.M.D.
      • Madhavan A.
      • Cech L.
      • Seguinot B.O.
      • Paxman R.J.
      • Smith E.
      • Scampavia L.
      • Powers E.T.
      • Cooley C.B.
      • Plate L.
      • Spicer T.P.
      • Kelly J.W.
      • Wiseman R.L.
      Pharmacologic IRE1/XBP1s activation confers targeted ER proteostasis reprogramming.
      ).

      The PERK arm of the UPR

      The PERK UPR signaling pathway has proven to be an extremely attractive target for pharmacologic intervention. PERK is composed of an N-terminal luminal domain and a cytosolic effector kinase domain (Fig. 3, A and B) (
      • Shi Y.
      • Vattem K.M.
      • Sood R.
      • An J.
      • Liang J.
      • Stramm L.
      • Wek R.C.
      Identification and characterization of pancreatic eukaryotic initiation factor 2 α-subunit kinase, PEK, involved in translational control.
      ,
      • Harding H.P.
      • Zhang Y.
      • Ron D.
      Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase.
      ). Like IRE1, PERK can be activated by both ER stress and lipid disequilibrium (
      • Volmer R.
      • van der Ploeg K.
      • Ron D.
      Membrane lipid saturation activates endoplasmic reticulum unfolded protein response transducers through their transmembrane domains.
      ,
      • Harding H.P.
      • Zhang Y.
      • Ron D.
      Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase.
      ). Interestingly, the mechanism of PERK activation is similar, although not identical, to that observed for IRE1. In response to ER stress, BiP dissociates from the PERK luminal domain, promoting oligomerization and autophosphorylation of the cytosolic PERK kinase domain (Fig. 3A) (
      • Bertolotti A.
      • Zhang Y.
      • Hendershot L.M.
      • Harding H.P.
      • Ron D.
      Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response.
      ,
      • Carrara M.
      • Prischi F.
      • Nowak P.R.
      • Kopp M.C.
      • Ali M.M.
      Noncanonical binding of BiP ATPase domain to Ire1 and Perk is dissociated by unfolded protein CH1 to initiate ER stress signaling.
      ,
      • Ma K.
      • Vattem K.M.
      • Wek R.C.
      Dimerization and release of molecular chaperone inhibition facilitate activation of eukaryotic initiation factor-2 kinase in response to endoplasmic reticulum stress.
      ). However, unlike IRE1, BiP co-chaperones such as ERdj4 do not appear to be involved in the regulation of PERK signaling, highlighting subtle differences between the activation of these different ER stress sensors (
      • Amin-Wetzel N.
      • Saunders R.A.
      • Kamphuis M.J.
      • Rato C.
      • Preissler S.
      • Harding H.P.
      • Ron D.
      A J-protein co-chaperone recruits BiP to monomerize IRE1 and repress the unfolded protein response.
      ). Once activated, PERK primarily functions through phosphorylation of the α-subunit of eukaryotic initiation factor 2 (eIF2α) at residue Ser-51 (
      • Scheuner D.
      • Song B.
      • McEwen E.
      • Liu C.
      • Laybutt R.
      • Gillespie P.
      • Saunders T.
      • Bonner-Weir S.
      • Kaufman R.J.
      Translational control is required for the unfolded protein response and in vivo glucose homeostasis.
      ), although other PERK kinase targets, such as nuclear factor erythroid 2–related factor 2 (NRF2), have also been reported (
      • Cullinan S.B.
      • Zhang D.
      • Hannink M.
      • Arvisais E.
      • Kaufman R.J.
      • Diehl J.A.
      Nrf2 is a direct PERK substrate and effector of PERK-dependent cell survival.
      ). Phosphorylation of eIF2α disrupts protein translation by increasing the affinity of the eIF2 complex for the eIF2B GTP exchange factor. This prevents eIF2B activity, which is required for translation initiation (
      • Costa-Mattioli M.
      • Walter P.
      The integrated stress response: From mechanism to disease.
      ,
      • Pakos-Zebrucka K.
      • Koryga I.
      • Mnich K.
      • Ljujic M.
      • Samali A.
      • Gorman A.M.
      The integrated stress response.
      ). As a result, ribosomal protein synthesis is globally reduced. This reduction protects the ER by reducing the load of newly synthesized, unfolded proteins entering into the ER lumen, allowing ER proteostasis factors (e.g. chaperones and folding enzymes) to engage existing misfolded ER proteins and facilitate their refolding or clearance through mechanisms including ERAD or autophagy (Fig. 3A) (
      • Shi Y.
      • Vattem K.M.
      • Sood R.
      • An J.
      • Liang J.
      • Stramm L.
      • Wek R.C.
      Identification and characterization of pancreatic eukaryotic initiation factor 2 α-subunit kinase, PEK, involved in translational control.
      ,
      • Harding H.P.
      • Zhang Y.
      • Ron D.
      Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase.
      ,
      • Gupta S.
      • McGrath B.
      • Cavener D.R.
      PERK (EIF2AK3) regulates proinsulin trafficking and quality control in the secretory pathway.
      ,
      • Kondratyev M.
      • Avezov E.
      • Shenkman M.
      • Groisman B.
      • Lederkremer G.Z.
      PERK-dependent compartmentalization of ERAD and unfolded protein response machineries during ER stress.
      ). Apart from reducing folding load, PERK-dependent translational attenuation has also been shown to regulate other aspects of cellular physiology, including cell-cycle progression, mitochondrial protein import, and mitochondrial morphology, through mechanisms such as the increased degradation of short-lived proteins (
      • Brewer J.W.
      • Diehl J.A.
      PERK mediates cell-cycle exit during the mammalian unfolded protein response.
      ,
      • Lebeau J.
      • Saunders J.M.
      • Moraes V.W.R.
      • Madhavan A.
      • Madrazo N.
      • Anthony M.C.
      • Wiseman R.L.
      The PERK arm of the unfolded protein response regulates mitochondrial morphology during acute endoplasmic reticulum stress.
      ,
      • Rainbolt T.K.
      • Atanassova N.
      • Genereux J.C.
      • Wiseman R.L.
      Stress-regulated translational attenuation adapts mitochondrial protein import through Tim17A degradation.
      ,
      • Rainbolt T.K.
      • Saunders J.M.
      • Wiseman R.L.
      Stress-responsive regulation of mitochondria through the ER unfolded protein response.
      ).
      Figure thumbnail gr3
      Figure 3Pharmacologic PERK-modulating compounds. A, mechanism of activation and signaling for the PERK signaling arm of the UPR. B, domain architecture of PERK, including the luminal domain, transmembrane domain (TM), and cytosolic protein kinase domain. The protein kinase domain functions as shown in A through both PERK autophosphorylation and the phosphorylating eIF2α, the latter a key step of the PERK signaling cascade. C, structures of the PERK kinase inhibitors GSK2606414, AMG52, and AMG44. WRS, Wolcott–Rallison syndrome associated mutations are indicated. D, structures of the inhibitors of PERK signaling ISRIB and trazodone that block PERK signaling downstream of eIF2α phosphorylation. E, structures of the putative eIF2α phosphatase inhibitors salubrinal, guanabenz, and sephin 1. F, structures of PERK activators CCT020312, compound A, and MK-28 identified through phenotypic and computational screening approaches.
      Whereas general protein synthesis is decreased by PERK-dependent eIF2α phosphorylation, a specific group of proteins is selectively translated under these conditions. These include the stress-responsive transcription factor activating transcription factor 4 (ATF4) (Fig. 3A) (
      • Vattem K.M.
      • Wek R.C.
      Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells.
      ,
      • Lu P.D.
      • Harding H.P.
      • Ron D.
      Translation reinitiation at alternative open reading frames regulates gene expression in an integrated stress response.
      ,
      • Jousse C.
      • Bruhat A.
      • Carraro V.
      • Urano F.
      • Ferrara M.
      • Ron D.
      • Fafournoux P.
      Inhibition of CHOP translation by a peptide encoded by an open reading frame localized in the chop 5'UTR.
      ). The mRNA encoding this protein escapes translational inhibition afforded by eIF2α phosphorylation through upstream ORFs found in its 5′-UTR (
      • Young S.K.
      • Wek R.C.
      Upstream open reading frames differentially regulate gene-specific translation in the integrated stress response.
      ). ATF4 induces the expression of stress-responsive genes involved in diverse biological functions, including cellular redox, mitochondrial proteostasis, tRNA charging, and nutrient transport (Fig. 3A) (
      • Han J.
      • Back S.H.
      • Hur J.
      • Lin Y.H.
      • Gildersleeve R.
      • Shan J.
      • Yuan C.L.
      • Krokowski D.
      • Wang S.
      • Hatzoglou M.
      • Kilberg M.S.
      • Sartor M.A.
      • Kaufman R.J.
      ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death.
      ,
      • Harding H.P.
      • Zhang Y.
      • Zeng H.
      • Novoa I.
      • Lu P.D.
      • Calfon M.
      • Sadri N.
      • Yun C.
      • Popko B.
      • Paules R.
      • Stojdl D.F.
      • Bell J.C.
      • Hettmann T.
      • Leiden J.M.
      • Ron D.
      An integrated stress response regulates amino acid metabolism and resistance to oxidative stress.
      ). Many of these genes are also transcriptionally regulated via upstream ORFs and thus are translated during PERK activation (
      • Young S.K.
      • Wek R.C.
      Upstream open reading frames differentially regulate gene-specific translation in the integrated stress response.
      ). ATF4 also induces the expression of the eIF2α phosphatase regulator subunit growth arrest and DNA damage–inducible protein 34 (GADD34), which is involved in dephosphorylating eIF2α and restoring translation in a negative feedback loop that suppresses PERK signaling (
      • Novoa I.
      • Zeng H.
      • Harding H.P.
      • Ron D.
      Feedback inhibition of the unfolded protein response by GADD34-mediated dephosphorylation of eIF2α.
      ,
      • Ma Y.
      • Hendershot L.M.
      Delineation of a negative feedback regulatory loop that controls protein translation during endoplasmic reticulum stress.
      ). Through this transcriptional activity, PERK promotes cellular adaptation and survival in response to acute insults.
      However, severe or chronic ER stress promotes pro-apoptotic signaling downstream of PERK (
      • Hetz C.
      • Papa F.R.
      The unfolded protein response and cell fate control.
      ). One mechanism by which PERK promotes apoptosis is through the up-regulation of the transcription factor C/EBP-homologous protein 10 (CHOP) (
      • Marciniak S.J.
      • Yun C.Y.
      • Oyadomari S.
      • Novoa I.
      • Zhang Y.
      • Jungreis R.
      • Nagata K.
      • Harding H.P.
      • Ron D.
      CHOP induces death by promoting protein synthesis and oxidation in the stressed endoplasmic reticulum.
      ). CHOP induces the expression of multiple pro-apoptotic factors, including Bcl2-like protein 11 (BIM) and DR5 to activate intrinsic apoptotic signaling cascades and caspase activation (
      • Hetz C.
      • Papa F.R.
      The unfolded protein response and cell fate control.
      ,
      • Lu M.
      • Lawrence D.A.
      • Marsters S.
      • Acosta-Alvear D.
      • Kimmig P.
      • Mendez A.S.
      • Paton A.W.
      • Paton J.C.
      • Walter P.
      • Ashkenazi A.
      Opposing unfolded-protein-response signals converge on death receptor 5 to control apoptosis.
      ,
      • Puthalakath H.
      • O'Reilly L.A.
      • Gunn P.
      • Lee L.
      • Kelly P.N.
      • Huntington N.D.
      • Hughes P.D.
      • Michalak E.M.
      • McKimm-Breschkin J.
      • Motoyama N.
      • Gotoh T.
      • Akira S.
      • Bouillet P.
      • Strasser A.
      ER stress triggers apoptosis by activating BH3-only protein Bim.
      ,
      • Zhang L.
      • Lopez H.
      • George N.M.
      • Liu X.
      • Pang X.
      • Luo X.
      Selective involvement of BH3-only proteins and differential targets of Noxa in diverse apoptotic pathways.
      ,
      • Yamaguchi H.
      • Wang H.G.
      CHOP is involved in endoplasmic reticulum stress-induced apoptosis by enhancing DR5 expression in human carcinoma cells.
      ). PERK can also promote cell death through other mechanisms, including increased oxidative stress associated with the recovery of protein synthesis following translational attenuation, increased expression of microRNAs that disrupt cellular metabolism, and the suppression of antiapoptotic factors, such as X-linked inhibitor of apoptosis (XIAP) (
      • Hetz C.
      • Papa F.R.
      The unfolded protein response and cell fate control.
      ,
      • Han J.
      • Back S.H.
      • Hur J.
      • Lin Y.H.
      • Gildersleeve R.
      • Shan J.
      • Yuan C.L.
      • Krokowski D.
      • Wang S.
      • Hatzoglou M.
      • Kilberg M.S.
      • Sartor M.A.
      • Kaufman R.J.
      ER-stress-induced transcriptional regulation increases protein synthesis leading to cell death.
      ,
      • Hiramatsu N.
      • Messah C.
      • Han J.
      • LaVail M.M.
      • Kaufman R.J.
      • Lin J.H.
      Translational and posttranslational regulation of XIAP by eIF2α and ATF4 promotes ER stress-induced cell death during the unfolded protein response.
      ,
      • Hiramatsu N.
      • Chiang K.
      • Aivati C.
      • Rodvold J.J.
      • Lee J.M.
      • Han J.
      • Chea L.
      • Zanetti M.
      • Koo E.H.
      • Lin J.H.
      PERK-mediated induction of microRNA-483 disrupts cellular ATP homeostasis during the unfolded protein response.
      ). Interestingly, this pro-apoptotic signaling downstream of PERK coordinates with other UPR signaling to dictate cell fate in response to severe ER insults. For example, the CHOP-regulated, pro-apoptotic DR5 mRNA is a target of the RIDD pathway, which functions to suppress apoptotic signaling (
      • Lu M.
      • Lawrence D.A.
      • Marsters S.
      • Acosta-Alvear D.
      • Kimmig P.
      • Mendez A.S.
      • Paton A.W.
      • Paton J.C.
      • Walter P.
      • Ashkenazi A.
      Opposing unfolded-protein-response signals converge on death receptor 5 to control apoptosis.
      ). In response to prolonged ER stress, RIDD activity declines, releasing this “break” on apoptosis and allowing DR5-mediated apoptotic signaling. This type of integration provides a sophisticated mechanism to regulate cell fate in response to varying levels of ER stress.
      Considering the importance of PERK in dictating both adaptation and survival in response to ER stress, it is not surprising that both increases and decreases in PERK activity are implicated in diverse types of disease. For example, mutations in EIF2AK3 (the gene encoding PERK) that reduce or eliminate signaling through this UPR pathway are associated with Wolcott–Rallison syndrome and progressive supranuclear palsy (Fig. 3B) (
      • Delepine M.
      • Nicolino M.
      • Barrett T.
      • Golamaully M.
      • Lathrop G.M.
      • Julier C.
      EIF2AK3, encoding translation initiation factor 2-α kinase 3, is mutated in patients with Wolcott-Rallison syndrome.
      ,
      • Yuan S.H.
      • Hiramatsu N.
      • Liu Q.
      • Sun X.V.
      • Lenh D.
      • Chan P.
      • Chiang K.
      • Koo E.H.
      • Kao A.W.
      • Litvan I.
      • Lin J.H.
      Tauopathy-associated PERK alleles are functional hypomorphs that increase neuronal vulnerability to ER stress.
      ,
      • Höglinger G.U.
      • Melhem N.M.
      • Dickson D.W.
      • Sleiman P.M.
      • Wang L.S.
      • Klei L.
      • Rademakers R.
      • de Silva R.
      • Litvan I.
      • Riley D.E.
      • van Swieten J.C.
      • Heutink P.
      • Wszolek Z.K.
      • Uitti R.J.
      • Vandrovcova J.
      • et al.
      Identification of common variants influencing risk of the tauopathy progressive supranuclear palsy.
      ). In contrast, overactivity of PERK is observed in clinical samples and mouse models of many neurodegenerative diseases, including prion disease, Alzheimer's disease, and frontotemporal dementia (
      • Hughes D.
      • Mallucci G.R.
      The unfolded protein response in neurodegenerative disorders—therapeutic modulation of the PERK pathway.
      ). Because of this, there has been significant interest in establishing new strategies to pharmacologically inhibit or activate PERK signaling for different human diseases.

      Inhibiting PERK autophosphorylation using kinase inhibitors

      One of the earliest strategies to modulate PERK focused on targeting its kinase active site to inhibit the PERK autophosphorylation step required for activation. The initial compound that emerged from this approach was GSK2606414, which was shown to have >100-fold selectivity for the PERK kinase domain relative to other kinases tested (Fig. 3C) (
      • Axten J.M.
      • Medina J.R.
      • Feng Y.
      • Shu A.
      • Romeril S.P.
      • Grant S.W.
      • Li W.H.
      • Heerding D.A.
      • Minthorn E.
      • Mencken T.
      • Atkins C.
      • Liu Q.
      • Rabindran S.
      • Kumar R.
      • Hong X.
      • Goetz A.
      • et al.
      Discovery of 7-methyl-5-(1-{[3-(trifluoromethyl)phenyl]acetyl}-2,3-dihydro-1H-indol-5-yl)-7H-p yrrolo[2,3-d]pyrimidin-4-amine (GSK2606414), a potent and selective first-in-class inhibitor of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK).
      ,
      • Axten J.M.
      • Romeril S.P.
      • Shu A.
      • Ralph J.
      • Medina J.R.
      • Feng Y.
      • Li W.H.
      • Grant S.W.
      • Heerding D.A.
      • Minthorn E.
      • Mencken T.
      • Gaul N.
      • Goetz A.
      • Stanley T.
      • Hassell A.M.
      • Gampe R.T.
      • Atkins C.
      • Kumar R.
      Discovery of GSK2656157: an optimized PERK inhibitor selected for preclinical development.
      ). However, more recent studies have indicated that GSK2606414 and its close analog GSK2656157 can inhibit other kinases, such as receptor-interacting serine/threonine kinase (RIPK) and receptor protein tyrosine kinase (KIT), reflecting off-target activity often associated with targeting kinase active sites and potential complications when interpreting the direct cause of physiological changes from treatment by these compounds (
      • Mahameed M.
      • Wilhelm T.
      • Darawshi O.
      • Obiedat A.
      • Tommy W.S.
      • Chintha C.
      • Schubert T.
      • Samali A.
      • Chevet E.
      • Eriksson L.A.
      • Huber M.
      • Tirosh B.
      The unfolded protein response modulators GSK2606414 and KIRA6 are potent KIT inhibitors.
      ,
      • Rojas-Rivera D.
      • Delvaeye T.
      • Roelandt R.
      • Nerinckx W.
      • Augustyns K.
      • Vandenabeele P.
      • Bertrand M.J.M.
      When PERK inhibitors turn out to be new potent RIPK1 inhibitors: critical issues on the specificity and use of GSK2606414 and GSK2656157.
      ). New analogs of these two compounds, such as AMG52, and novel quinoline-based PERK inhibitors, such as AMG44, have been recently engineered to decrease RIPK inhibition. However, these compounds show reduced activity for inhibiting PERK signaling in cellular models as compared with GSK2606414 (Fig. 3C) (
      • Smith A.L.
      • Andrews K.L.
      • Beckmann H.
      • Bellon S.F.
      • Beltran P.J.
      • Booker S.
      • Chen H.
      • Chung Y.A.
      • D'Angelo N.D.
      • Dao J.
      • Dellamaggiore K.R.
      • Jaeckel P.
      • Kendall R.
      • Labitzke K.
      • Long A.M.
      • Materna-Reichelt S.
      • Mitchell P.
      • et al.
      Discovery of 1H-pyrazol-3(2H)-ones as potent and selective inhibitors of protein kinase R-like endoplasmic reticulum kinase (PERK).
      ). Regardless, PERK kinase inhibitors (e.g. GSK2606414) have been widely used both in vitro and in vivo to define the pathologic and therapeutic implications of PERK signaling in diverse diseases. For example, administration of PERK kinase inhibitors reduced tumorigenesis in pancreatic and multiple myeloma tumor xenograft models and improved outcomes in mouse models of neurodegenerative diseases, including prion disease and frontotemporal dementia (
      • Axten J.M.
      • Medina J.R.
      • Feng Y.
      • Shu A.
      • Romeril S.P.
      • Grant S.W.
      • Li W.H.
      • Heerding D.A.
      • Minthorn E.
      • Mencken T.
      • Atkins C.
      • Liu Q.
      • Rabindran S.
      • Kumar R.
      • Hong X.
      • Goetz A.
      • et al.
      Discovery of 7-methyl-5-(1-{[3-(trifluoromethyl)phenyl]acetyl}-2,3-dihydro-1H-indol-5-yl)-7H-p yrrolo[2,3-d]pyrimidin-4-amine (GSK2606414), a potent and selective first-in-class inhibitor of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK).
      ,
      • Atkins C.
      • Liu Q.
      • Minthorn E.
      • Zhang S.Y.
      • Figueroa D.J.
      • Moss K.
      • Stanley T.B.
      • Sanders B.
      • Goetz A.
      • Gaul N.
      • Choudhry A.E.
      • Alsaid H.
      • Jucker B.M.
      • Axten J.M.
      • Kumar R.
      Characterization of a novel PERK kinase inhibitor with antitumor and antiangiogenic activity.
      ,
      • Radford H.
      • Moreno J.A.
      • Verity N.
      • Halliday M.
      • Mallucci G.R.
      PERK inhibition prevents tau-mediated neurodegeneration in a mouse model of frontotemporal dementia.
      ,
      • Moreno J.A.
      • Halliday M.
      • Molloy C.
      • Radford H.
      • Verity N.
      • Axten J.M.
      • Ortori C.A.
      • Willis A.E.
      • Fischer P.M.
      • Barrett D.A.
      • Mallucci G.R.
      Oral treatment targeting the unfolded protein response prevents neurodegeneration and clinical disease in prion-infected mice.
      ). However, these compounds are still somewhat limited in their in vivo application as they have demonstrated dose-dependent defects, including weight loss and pancreatic toxicity (
      • Halliday M.
      • Radford H.
      • Sekine Y.
      • Moreno J.
      • Verity N.
      • Le Quesne J.
      • Ortori C.A.
      • Barrett D.A.
      • Fromont C.
      • Fischer P.M.
      • Harding H.P.
      • Ron D.
      • Mallucci G.R.
      Partial restoration of protein synthesis rates by the small molecule ISRIB prevents neurodegeneration without pancreatic toxicity.
      ,
      • Yu Q.
      • Zhao B.
      • Gui J.
      • Katlinski K.V.
      • Brice A.
      • Gao Y.
      • Li C.
      • Kushner J.A.
      • Koumenis C.
      • Diehl J.A.
      • Fuchs S.Y.
      Type I interferons mediate pancreatic toxicities of PERK inhibition.
      ). Regardless, PERK kinase inhibitors have proven valuable for defining the therapeutic potential for inhibiting PERK in models of disease and continue to be explored for clinical use.

      Pharmacologic activation of eIF2B to inhibit PERK signaling

      As opposed to the direct targeting of the kinase active site, other strategies have employed unbiased cell-based phenotypic screens to identify compounds that inhibit PERK signaling. The most prominent compound to emerge from this approach is ISRIB (Fig. 3D). ISRIB was identified from an HTS, where >100,000 compounds were screened for inhibition of ER stress–dependent activation of a cell-based luciferase reporter for ATF4 translation (
      • Sidrauski C.
      • Acosta-Alvear D.
      • Khoutorsky A.
      • Vedantham P.
      • Hearn B.R.
      • Li H.
      • Gamache K.
      • Gallagher C.M.
      • Ang K.K.
      • Wilson C.
      • Okreglak V.
      • Ashkenazi A.
      • Hann B.
      • Nader K.
      • Arkin M.R.
      • et al.
      Pharmacological brake-release of mRNA translation enhances cognitive memory.
      ). ISRIB showed high potency for selectively inhibiting PERK-regulated transcriptional and translational signaling without significantly affecting other arms of the UPR (
      • Sidrauski C.
      • Acosta-Alvear D.
      • Khoutorsky A.
      • Vedantham P.
      • Hearn B.R.
      • Li H.
      • Gamache K.
      • Gallagher C.M.
      • Ang K.K.
      • Wilson C.
      • Okreglak V.
      • Ashkenazi A.
      • Hann B.
      • Nader K.
      • Arkin M.R.
      • et al.
      Pharmacological brake-release of mRNA translation enhances cognitive memory.
      ). Interestingly, ISRIB did not reduce PERK-dependent phosphorylation of eIF2α, indicating that this compound worked downstream of PERK kinase activity and likely functions by desensitizing cells to eIF2α phosphorylation. A consequence of this specific mechanism is that ISRIB can block eIF2α phosphorylation-dependent signaling induced by other stress-regulated eIF2α kinases, including general control nonderepressible 4 (GCN4), activated in response to nutrient deprivation, heme-regulated inhibitor (HRI), activated by oxidative or mitochondrial stress, and protein kinase R (PKR), activated by viral infection (
      • Costa-Mattioli M.
      • Walter P.
      The integrated stress response: From mechanism to disease.
      ). This means care must be taken when assigning ISRIB-sensitive phenotypes specifically to PERK signaling under conditions where these other kinases may be active.
      The biological target of ISRIB was identified using genetic screening approaches that showed disruption of specific subunits of eIF2B-desensitized cells to ISRIB (
      • Sidrauski C.
      • Tsai J.C.
      • Kampmann M.
      • Hearn B.R.
      • Vedantham P.
      • Jaishankar P.
      • Sokabe M.
      • Mendez A.S.
      • Newton B.W.
      • Tang E.L.
      • Verschueren E.
      • Johnson J.R.
      • Krogan N.J.
      • Fraser C.S.
      • Weissman J.S.
      • et al.
      Pharmacological dimerization and activation of the exchange factor eIF2B antagonizes the integrated stress response.
      ,
      • Sekine Y.
      • Zyryanova A.
      • Crespillo-Casado A.
      • Fischer P.M.
      • Harding H.P.
      • Ron D.
      Stress responses: mutations in a translation initiation factor identify the target of a memory-enhancing compound.
      ). Two subsequent cryo-EM structures of eIF2B bound to ISRIB showed that this molecule binds eIF2B at a regulatory site localized between its β and γ subunits, stabilizing the decameric enzyme complex in its active conformation (
      • Zyryanova A.F.
      • Weis F.
      • Faille A.
      • Alard A.A.
      • Crespillo-Casado A.
      • Sekine Y.
      • Harding H.P.
      • Allen F.
      • Parts L.
      • Fromont C.
      • Fischer P.M.
      • Warren A.J.
      • Ron D.
      Binding of ISRIB reveals a regulatory site in the nucleotide exchange factor eIF2B.
      ,
      • Tsai J.C.
      • Miller-Vedam L.E.
      • Anand A.A.
      • Jaishankar P.
      • Nguyen H.C.
      • Renslo A.R.
      • Frost A.
      • Walter P.
      Structure of the nucleotide exchange factor eIF2B reveals mechanism of memory-enhancing molecule.
      ). This binding increases eIF2B dimerization and subsequent activation, allowing eIF2B to remain active even in the presence of phosphorylated eIF2α. This structural elucidation of ISRIB binding to eIF2B has enabled structure-based drug design to establish next-generation ISRIB analogs with improved efficacy and potency for biological applications.
      Unlike PERK kinase inhibitors, ISRIB does not induce off-target activity, such as weight loss or pancreatic toxicity (
      • Halliday M.
      • Radford H.
      • Sekine Y.
      • Moreno J.
      • Verity N.
      • Le Quesne J.
      • Ortori C.A.
      • Barrett D.A.
      • Fromont C.
      • Fischer P.M.
      • Harding H.P.
      • Ron D.
      • Mallucci G.R.
      Partial restoration of protein synthesis rates by the small molecule ISRIB prevents neurodegeneration without pancreatic toxicity.
      ). This observation can be explained by the fact that ISRIB functions as a partial inhibitor of eIF2α phosphorylation-dependent signaling, with efficacy varying, depending on the amount and extent of eIF2α phosphorylation (
      • Halliday M.
      • Radford H.
      • Sekine Y.
      • Moreno J.
      • Verity N.
      • Le Quesne J.
      • Ortori C.A.
      • Barrett D.A.
      • Fromont C.
      • Fischer P.M.
      • Harding H.P.
      • Ron D.
      • Mallucci G.R.
      Partial restoration of protein synthesis rates by the small molecule ISRIB prevents neurodegeneration without pancreatic toxicity.
      ,
      • Rabouw H.H.
      • Langereis M.A.
      • Anand A.A.
      • Visser L.J.
      • de Groot R.J.
      • Walter P.
      • van Kuppeveld F.J.M.
      Small molecule ISRIB suppresses the integrated stress response within a defined window of activation.
      ). In response to mild to moderate acute insults, ISRIB is effective at inhibiting eIF2α-mediated signaling. However, chronic or severe insults show reduced sensitivity to ISRIB-dependent inhibition of eIF2α signaling. As a consequence, ISRIB can mitigate pathologic outcomes associated with moderate increases in eIF2α phosphorylation, while allowing for protective signaling through this pathway in tissues such as the pancreas that experience high levels of ER stress.
      The relatively low toxicity associated with ISRIB has allowed this compound to be widely used to probe the importance of PERK activity and/or eIF2α phosphorylation in diverse in vivo and cellular models of disease. For example, ISRIB has been shown to improve cognitive function in mice, reduce stress granule formation, promote cytotoxicity in xenograft models of prostate cancer, and protect against neurodegeneration in rodent models of prion disease and vanishing white matter disease (
      • Halliday M.
      • Radford H.
      • Sekine Y.
      • Moreno J.
      • Verity N.
      • Le Quesne J.
      • Ortori C.A.
      • Barrett D.A.
      • Fromont C.
      • Fischer P.M.
      • Harding H.P.
      • Ron D.
      • Mallucci G.R.
      Partial restoration of protein synthesis rates by the small molecule ISRIB prevents neurodegeneration without pancreatic toxicity.
      ,
      • Sidrauski C.
      • Acosta-Alvear D.
      • Khoutorsky A.
      • Vedantham P.
      • Hearn B.R.
      • Li H.
      • Gamache K.
      • Gallagher C.M.
      • Ang K.K.
      • Wilson C.
      • Okreglak V.
      • Ashkenazi A.
      • Hann B.
      • Nader K.
      • Arkin M.R.
      • et al.
      Pharmacological brake-release of mRNA translation enhances cognitive memory.
      ,
      • Wong Y.L.
      • LeBon L.
      • Basso A.M.
      • Kohlhaas K.L.
      • Nikkel A.L.
      • Robb H.M.
      • Donnelly-Roberts D.L.
      • Prakash J.
      • Swensen A.M.
      • Rubinstein N.D.
      • Krishnan S.
      • McAllister F.E.
      • Haste N.V.
      • O'Brien J.J.
      • Roy M.
      • et al.
      eIF2B activator prevents neurological defects caused by a chronic integrated stress response.
      ,
      • Sidrauski C.
      • McGeachy A.M.
      • Ingolia N.T.
      • Walter P.
      The small molecule ISRIB reverses the effects of eIFα phosphorylation on translation and stress granule assembly.
      ,
      • Wong Y.L.
      • LeBon L.
      • Edalji R.
      • Lim H.B.
      • Sun C.
      • Sidrauski C.
      The small molecule ISRIB rescues the stability and activity of vanishing white matter disease eIF2B mutant complexes.
      ). Further, ISRIB has proven invaluable for probing the importance of eIF2α phosphorylation-mediated signaling in regulating diverse biological processes, including ER proteostasis and mitochondrial regulation (
      • Lebeau J.
      • Saunders J.M.
      • Moraes V.W.R.
      • Madhavan A.
      • Madrazo N.
      • Anthony M.C.
      • Wiseman R.L.
      The PERK arm of the unfolded protein response regulates mitochondrial morphology during acute endoplasmic reticulum stress.
      ,
      • Quiros P.M.
      • Prado M.A.
      • Zamboni N.
      • D'Amico D.
      • Williams R.W.
      • Finley D.
      • Gygi S.P.
      • Auwerx J.
      Multi-omics analysis identifies ATF4 as a key regulator of the mitochondrial stress response in mammals.
      ,
      • Romine I.C.
      • Wiseman R.L.
      PERK signaling regulates extracellular proteostasis of an amyloidogenic protein during endoplasmic reticulum stress.
      ). Taken together, the development of ISRIB has proven transformative for improving our understanding of both the pathologic and potential therapeutic implications of eIF2α-dependent signaling in the context of health and disease.
      Despite the promise of ISRIB, a limiting factor in the further translational development of this compound is its poor solubility. Although new analogs of ISRIB, such as 2BAct, that show improved solubility and chemical properties continue to be developed (
      • Wong Y.L.
      • LeBon L.
      • Basso A.M.
      • Kohlhaas K.L.
      • Nikkel A.L.
      • Robb H.M.
      • Donnelly-Roberts D.L.
      • Prakash J.
      • Swensen A.M.
      • Rubinstein N.D.
      • Krishnan S.
      • McAllister F.E.
      • Haste N.V.
      • O'Brien J.J.
      • Roy M.
      • et al.
      eIF2B activator prevents neurological defects caused by a chronic integrated stress response.
      ), there is significant interest in identifying other compounds that target the PERK signaling pathway in ways analogous to ISRIB. One potential alternative is the antidepressant selective serotonin uptake inhibitor trazodone (Fig. 3D). Trazodone was identified as a UPR modulator in a phenotypic screen of a >1000-compound library enriched for Food and Drug Administration–approved drugs that monitored ER stress–induced developmental delays in Caenorhabditis elegans (
      • Halliday M.
      • Radford H.
      • Zents K.A.M.
      • Molloy C.
      • Moreno J.A.
      • Verity N.C.
      • Smith E.
      • Ortori C.A.
      • Barrett D.A.
      • Bushell M.
      • Mallucci G.R.
      Repurposed drugs targeting eIF2α-P-mediated translational repression prevent neurodegeneration in mice.
      ). Trazodone, like ISRIB, was shown to block eIF2α transcriptional and translational signaling downstream of eIF2α phosphorylation. However, unlike ISRIB, which activates eIF2B, trazodone is suggested to prevent eIF2α phosphorylation–dependent reductions in ternary complex formation required for translation initiation, although this mechanism remains to be formally defined (
      • Halliday M.
      • Radford H.
      • Zents K.A.M.
      • Molloy C.
      • Moreno J.A.
      • Verity N.C.
      • Smith E.
      • Ortori C.A.
      • Barrett D.A.
      • Bushell M.
      • Mallucci G.R.
      Repurposed drugs targeting eIF2α-P-mediated translational repression prevent neurodegeneration in mice.
      ). Despite this, initial studies show that treatment with trazodone mimics benefits observed with ISRIB, including reductions in both neurodegeneration in models of prion disease and tumor metastasis (
      • Halliday M.
      • Radford H.
      • Zents K.A.M.
      • Molloy C.
      • Moreno J.A.
      • Verity N.C.
      • Smith E.
      • Ortori C.A.
      • Barrett D.A.
      • Bushell M.
      • Mallucci G.R.
      Repurposed drugs targeting eIF2α-P-mediated translational repression prevent neurodegeneration in mice.
      ,
      • Harvey R.F.
      • Poyry T.A.A.
      • Stoneley M.
      • Willis A.E.
      Signaling from mTOR to eIF2α mediates cell migration in response to the chemotherapeutic doxorubicin.
      ). Whereas additional studies are required to fully appreciate the potential of trazodone as an inhibitor of eIF2α phosphorylation–dependent signaling (e.g. the possibility of separating selective serotonin uptake inhibitor activity from its effects on signaling downstream of eIF2α), the identification of trazodone further highlights the potential for targeting PERK signaling downstream of eIF2α phosphorylation as a promising strategy to intervene in disease.

      Targeting protein phosphatases to enhance PERK-eIF2α signaling

      Apart from inhibiting PERK-mediated eIF2α signaling, enhancing activity through this pathway also offers opportunities to improve pathologic outcomes in human disease. A significant challenge in developing compounds that activate PERK is the pro-apoptotic signaling induced by this pathway. To avoid pro-apoptotic signaling, strategies to increase PERK activity have primarily focused on targeting downstream aspects of PERK signaling, most notably the phosphatases responsible for dephosphorylating eIF2α. Dephosphorylation of eIF2α is mediated by complexes of protein phosphatase 1 (PP1), G-actin, and one of two regulatory subunits, CreP or GADD34 (
      • Chen R.
      • Rato C.
      • Yan Y.
      • Crespillo-Casado A.
      • Clarke H.J.
      • Harding H.P.
      • Marciniak S.J.
      • Read R.J.
      • Ron D.
      G-actin provides substrate-specificity to eukaryotic initiation factor 2α holophosphatases.
      ,
      • Chambers J.E.
      • Dalton L.E.
      • Clarke H.J.
      • Malzer E.
      • Dominicus C.S.
      • Patel V.
      • Moorhead G.
      • Ron D.
      • Marciniak S.J.
      Actin dynamics tune the integrated stress response by regulating eukaryotic initiation factor 2α dephosphorylation.
      ). CreP (PPP1R15B) is constitutively expressed and functions to dephosphorylate eIF2α under basal conditions (
      • Jousse C.
      • Oyadomari S.
      • Novoa I.
      • Lu P.
      • Zhang Y.
      • Harding H.P.
      • Ron D.
      Inhibition of a constitutive translation initiation factor 2α phosphatase, CReP, promotes survival of stressed cells.
      ). In contrast, GADD34 (PPP1R15A) is a stress-activated eIF2α phosphatase regulatory subunit that is induced through transcriptional signaling activated by eIF2α phosphorylation and functions to dephosphorylate eIF2α as part of a negative feedback loop in the PERK signaling pathway (
      • Ma Y.
      • Hendershot L.M.
      Delineation of a negative feedback regulatory loop that controls protein translation during endoplasmic reticulum stress.
      ,
      • Novoa I.
      • Zhang Y.
      • Zeng H.
      • Jungreis R.
      • Harding H.P.
      • Ron D.
      Stress-induced gene expression requires programmed recovery from translational repression.
      ,
      • Brush M.H.
      • Weiser D.C.
      • Shenolikar S.
      Growth arrest and DNA damage-inducible protein GADD34 targets protein phosphatase 1 α to the endoplasmic reticulum and promotes dephosphorylation of the α subunit of eukaryotic translation initiation factor 2.
      ). Thus, targeting the activity of CreP or GADD34 offers opportunities to increase eIF2α phosphorylation–dependent signaling by changing the dynamics of its dephosphorylation.
      The first compound suggested to target these phosphatases was salubrinal, which was identified in a screen for compounds that block ER stress–induced apoptosis (Fig. 3E) (
      • Boyce M.
      • Bryant K.F.
      • Jousse C.
      • Long K.
      • Harding H.P.
      • Scheuner D.
      • Kaufman R.J.
      • Ma D.
      • Coen D.M.
      • Ron D.
      • Yuan J.
      A selective inhibitor of eIF2α dephosphorylation protects cells from ER stress.
      ). Treatment with salubrinal induced eIF2α phosphorylation and delayed dephosphorylation of eIF2α following acute stress. Although these results indicate that salubrinal impacts eIF2α dephosphorylation, its precise mechanism of action remains undefined. Despite this, salubrinal is widely used and has proven protective in cellular and in vivo models of diverse diseases, including viral infection (
      • Boyce M.
      • Bryant K.F.
      • Jousse C.
      • Long K.
      • Harding H.P.
      • Scheuner D.
      • Kaufman R.J.
      • Ma D.
      • Coen D.M.
      • Ron D.
      • Yuan J.
      A selective inhibitor of eIF2α dephosphorylation protects cells from ER stress.
      ), retinitis pigmentosa (
      • Yoshida T.
      • Ozawa Y.
      • Suzuki K.
      • Yuki K.
      • Ohyama M.
      • Akamatsu W.
      • Matsuzaki Y.
      • Shimmura S.
      • Mitani K.
      • Tsubota K.
      • Okano H.
      The use of induced pluripotent stem cells to reveal pathogenic gene mutations and explore treatments for retinitis pigmentosa.
      ,
      • Athanasiou D.
      • Aguila M.
      • Bellingham J.
      • Kanuga N.
      • Adamson P.
      • Cheetham M.E.
      The role of the ER stress-response protein PERK in rhodopsin retinitis pigmentosa.
      ), and familial ALS (
      • Saxena S.
      • Cabuy E.
      • Caroni P.
      A role for motoneuron subtype-selective ER stress in disease manifestations of FALS mice.
      ). However, treatment with salubrinal also has been shown to exacerbate neurotoxic eIF2α-dependent signaling in mouse models of prion disease, highlighting the challenges of activating this pathway in human disease (
      • Moreno J.A.
      • Radford H.
      • Peretti D.
      • Steinert J.R.
      • Verity N.
      • Martin M.G.
      • Halliday M.
      • Morgan J.
      • Dinsdale D.
      • Ortori C.A.
      • Barrett D.A.
      • Tsaytler P.
      • Bertolotti A.
      • Willis A.E.
      • Bushell M.
      • et al.
      Sustained translational repression by eIF2α-P mediates prion neurodegeneration.
      ).
      Based on the protection afforded by genetic reductions of GADD34 in models of ALS and Charcot-Marie-Tooth disease (
      • D'Antonio M.
      • Musner N.
      • Scapin C.
      • Ungaro D.
      • Del Carro U.
      • Ron D.
      • Feltri M.L.
      • Wrabetz L.
      Resetting translational homeostasis restores myelination in Charcot-Marie-Tooth disease type 1B mice.
      ,
      • Wang L.
      • Popko B.
      • Roos R.P.
      An enhanced integrated stress response ameliorates mutant SOD1-induced ALS.
      ), there has been significant interest in identifying compounds that selectively inhibit GADD34 activity to delay translational recovery following acute insult. One of the first compounds proposed to selectively target GADD34 was the α2-adrenergic agonist guanabenz (Fig. 3E). Both guanabenz and its close analog sephin-1, which lacks α2-adrenergic activity, were previously shown to delay translation recovery following acute ER insult (Fig. 3E) (
      • Das I.
      • Krzyzosiak A.
      • Schneider K.
      • Wrabetz L.
      • D'Antonio M.
      • Barry N.
      • Sigurdardottir A.
      • Bertolotti A.
      Preventing proteostasis diseases by selective inhibition of a phosphatase regulatory subunit.
      ,
      • Tsaytler P.
      • Harding H.P.
      • Ron D.
      • Bertolotti A.
      Selective inhibition of a regulatory subunit of protein phosphatase 1 restores proteostasis.
      ). Whereas initial biochemical studies suggested that this delay could be attributed to selective inhibition of GADD34 (
      • Das I.
      • Krzyzosiak A.
      • Schneider K.
      • Wrabetz L.
      • D'Antonio M.
      • Barry N.
      • Sigurdardottir A.
      • Bertolotti A.
      Preventing proteostasis diseases by selective inhibition of a phosphatase regulatory subunit.
      ,
      • Tsaytler P.
      • Harding H.P.
      • Ron D.
      • Bertolotti A.
      Selective inhibition of a regulatory subunit of protein phosphatase 1 restores proteostasis.
      ,
      • Carrara M.
      • Sigurdardottir A.
      • Bertolotti A.
      Decoding the selectivity of eIF2α holophosphatases and PPP1R15A inhibitors.
      ), other studies indicate that this compound does not inhibit GADD34-dependent phosphatase activity in vitro, casting doubt on this potential mechanism of action (
      • Crespillo-Casado A.
      • Claes Z.
      • Choy M.S.
      • Peti W.
      • Bollen M.
      • Ron D.
      A Sephin1-insensitive tripartite holophosphatase dephosphorylates translation initiation factor 2α.
      ,
      • Crespillo-Casado A.
      • Chambers J.E.
      • Fischer P.M.
      • Marciniak S.J.
      • Ron D.
      PPP1R15A-mediated dephosphorylation of eIF2α is unaffected by Sephin1 or Guanabenz.
      ). Further, the reductions in ER stress–associated signaling afforded by treatment with guanabenz and sephin-1 appear independent of eIF2α phosphorylation, suggesting that these compounds exert their effects through an alternative mechanism (
      • Crespillo-Casado A.
      • Chambers J.E.
      • Fischer P.M.
      • Marciniak S.J.
      • Ron D.
      PPP1R15A-mediated dephosphorylation of eIF2α is unaffected by Sephin1 or Guanabenz.
      ). Although the mechanism of action for these compounds remains to be established, guanabenz and sephin-1 are protective in mouse models of multiple diseases, including Charcot-Marie-Tooth, familial ALS, multiple sclerosis, and prion disease, reflecting their ability to improve pathologic outcomes in the context of different diseases (
      • Das I.
      • Krzyzosiak A.
      • Schneider K.
      • Wrabetz L.
      • D'Antonio M.
      • Barry N.
      • Sigurdardottir A.
      • Bertolotti A.
      Preventing proteostasis diseases by selective inhibition of a phosphatase regulatory subunit.
      ,
      • Tsaytler P.
      • Harding H.P.
      • Ron D.
      • Bertolotti A.
      Selective inhibition of a regulatory subunit of protein phosphatase 1 restores proteostasis.
      ,
      • Thapa S.
      • Abdelaziz D.H.
      • Abdulrahman B.A.
      • Schatzl H.M.
      Sephin1 reduces prion infection in prion-infected cells and animal model.
      ,
      • Chen Y.
      • Podojil J.R.
      • Kunjamma R.B.
      • Jones J.
      • Weiner M.
      • Lin W.
      • Miller S.D.
      • Popko B.
      Sephin1, which prolongs the integrated stress response, is a promising therapeutic for multiple sclerosis.
      ). That being said, it is important to use caution when interpreting the relationship between this protection and signaling through PERK and related pathways due to the lack of clarity regarding the mechanism of action for these compounds. Regardless, GADD34 remains an attractive therapeutic target for modulating eIF2α-dependent signaling in the context of health and disease.

      PERK activators

      Phenotypic screening has also been employed to identify compounds that activate the PERK pathway. Initial reports of a small molecule that selectively activates PERK signaling resulted from a high-throughput screen in human colon carcinoma cells focused on identifying compounds that halt cancer cell progression by activating the G1/S cell-cycle checkpoint (
      • Stockwell S.R.
      • Platt G.
      • Barrie S.E.
      • Zoumpoulidou G.
      • Te Poele R.H.
      • Aherne G.W.
      • Wilson S.C.
      • Sheldrake P.
      • McDonald E.
      • Venet M.
      • Soudy C.
      • Elustondo F.
      • Rigoreau L.
      • Blagg J.
      • Workman P.
      • et al.
      Mechanism-based screen for G1/S checkpoint activators identifies a selective activator of EIF2AK3/PERK signalling.
      ). The identification of compound CCT020312 by this method was then linked to PERK signaling by microarray transcriptional profiling, revealing similarities between this compound and other positive effectors of eIF2α phosphorylation signaling (Fig. 3F) (
      • Stockwell S.R.
      • Platt G.
      • Barrie S.E.
      • Zoumpoulidou G.
      • Te Poele R.H.
      • Aherne G.W.
      • Wilson S.C.
      • Sheldrake P.
      • McDonald E.
      • Venet M.
      • Soudy C.
      • Elustondo F.
      • Rigoreau L.
      • Blagg J.
      • Workman P.
      • et al.
      Mechanism-based screen for G1/S checkpoint activators identifies a selective activator of EIF2AK3/PERK signalling.
      ). CCT020312 did not appear to exhibit global UPR activation in cell culture models, and compound-dependent increases in eIF2α phosphorylation were shown to be PERK-dependent via RNAi depletion (
      • Stockwell S.R.
      • Platt G.
      • Barrie S.E.
      • Zoumpoulidou G.
      • Te Poele R.H.
      • Aherne G.W.
      • Wilson S.C.
      • Sheldrake P.
      • McDonald E.
      • Venet M.
      • Soudy C.
      • Elustondo F.
      • Rigoreau L.
      • Blagg J.
      • Workman P.
      • et al.
      Mechanism-based screen for G1/S checkpoint activators identifies a selective activator of EIF2AK3/PERK signalling.
      ). However, the mechanism of PERK activation by CCT020312 remains undefined. Another phenotypic screen using a FRET-based reporter of eIF2α phosphorylation also identified promising pyrazole-carboxylate derivatives, including Compound A, which elicited eIF2α phosphorylation and NRF2 activation in a PERK activity–dependent manner (Fig. 3F) (
      • Xie W.
      • Pariollaud M.
      • Wixted W.E.
      • Chitnis N.
      • Fornwald J.
      • Truong M.
      • Pao C.
      • Liu Y.
      • Ames R.S.
      • Callahan J.
      • Solari R.
      • Sanchez Y.
      • Diehl A.
      • Li H.
      Identification and characterization of PERK activators by phenotypic screening and their effects on NRF2 activation.
      ). Further, computational screening for compounds that activate the PERK kinase identified compound A4, which was subsequently shown to activate PERK in cell culture models and protect against HTT-associated toxicity in neuronal cells (
      • Wang H.
      • Blais J.
      • Ron D.
      • Cardozo T.
      Structural determinants of PERK inhibitor potency and selectivity.
      ,
      • Leitman J.
      • Barak B.
      • Benyair R.
      • Shenkman M.
      • Ashery U.
      • Hartl F.U.
      • Lederkremer G.Z.
      ER stress-induced eIF2-α phosphorylation underlies sensitivity of striatal neurons to pathogenic huntingtin.
      ). Subsequent biochemical studies indicated that this compound (and its more potent derivative MK-28) activates PERK signaling both in vitro and in vivo and protects against ER stress–associated insults (Fig. 3F) (
      • Ganz J.
      • Shacham T.
      • Kramer M.
      • Shenkman M.
      • Eiger H.
      • Weinberg N.
      • Iancovici O.
      • Roy S.
      • Simhaev L.
      • Da'adoosh B.
      • Engel H.
      • Perets N.
      • Barhum Y.
      • Portnoy M.
      • Offen D.
      • et al.
      A novel specific PERK activator reduces toxicity and extends survival in Huntington's disease models.
      ). Although the mechanism of action for MK-28 remains to be established, molecular modeling suggests that MK-28 could activate PERK kinase activity through allosteric regulation of the kinase active loop, suggesting a direct effect on the PERK kinase, although this remains to be formally tested (
      • Ganz J.
      • Shacham T.
      • Kramer M.
      • Shenkman M.
      • Eiger H.
      • Weinberg N.
      • Iancovici O.
      • Roy S.
      • Simhaev L.
      • Da'adoosh B.
      • Engel H.
      • Perets N.
      • Barhum Y.
      • Portnoy M.
      • Offen D.
      • et al.
      A novel specific PERK activator reduces toxicity and extends survival in Huntington's disease models.
      ).
      The identification of these PERK-activating compounds provides new opportunities to activate PERK signaling in cellular and in vivo models. Initial results using these activators have shown protection in mouse models of diverse diseases, including mitochondrial diseases, tauopathy, and Huntington's disease, through mechanisms linked to both increased eIF2α phosphorylation and NRF2 activation (
      • Ganz J.
      • Shacham T.
      • Kramer M.
      • Shenkman M.
      • Eiger H.
      • Weinberg N.
      • Iancovici O.
      • Roy S.
      • Simhaev L.
      • Da'adoosh B.
      • Engel H.
      • Perets N.
      • Barhum Y.
      • Portnoy M.
      • Offen D.
      • et al.
      A novel specific PERK activator reduces toxicity and extends survival in Huntington's disease models.
      ,
      • Bruch J.
      • Xu H.
      • Rosler T.W.
      • De Andrade A.
      • Kuhn P.H.
      • Lichtenthaler S.F.
      • Arzberger T.
      • Winklhofer K.F.
      • Muller U.
      • Hoglinger G.U.
      PERK activation mitigates tau pathology in vitro in vivo.
      ,
      • Balsa E.
      • Soustek M.S.
      • Thomas A.
      • Cogliati S.
      • Garcia-Poyatos C.
      • Martin-Garcia E.
      • Jedrychowski M.
      • Gygi S.P.
      • Enriquez J.A.
      • Puigserver P.
      ER and nutrient stress promote assembly of respiratory chain supercomplexes through the PERK-eIF2α axis.
      ). However, because the mechanism of action for these compounds and the selectivity of these compounds for PERK relative to other UPR or stress-responsive signaling pathways remain to be defined, care should be taken when relating biologic effects of these compounds specifically to PERK activation.

      The ATF6 arm of the UPR

      The last arm of the UPR to be identified was the ATF6 pathway. As with IRE1, humans encode two different ATF6 genes, ATF6α and ATF6β. ATF6β functions in a predominantly regulatory role, whereas ATF6α is the primary protein responsible for adapting cellular physiology in response to ER stress (
      • Yoshida H.
      • Okada T.
      • Haze K.
      • Yanagi H.
      • Yura T.
      • Negishi M.
      • Mori K.
      ATF6 activated by proteolysis binds in the presence of NF-Y (CBF) directly to the cis-acting element responsible for the mammalian unfolded protein response.
      ,
      • Haze K.
      • Okada T.
      • Yoshida H.
      • Yanagi H.
      • Yura T.
      • Negishi M.
      • Mori K.
      Identification of the G13 (cAMP-response-element-binding protein-related protein) gene product related to activating transcription factor 6 as a transcriptional activator of the mammalian unfolded protein response.
      ,
      • Yoshida H.
      • Okada T.
      • Haze K.
      • Yanagi H.
      • Yura T.
      • Negishi M.
      • Mori K.
      Endoplasmic reticulum stress-induced formation of transcription factor complex ERSF including NF-Y (CBF) and activating transcription factors 6α and 6β that activates the mammalian unfolded protein response.
      ,
      • Yamamoto K.
      • Sato T.
      • Matsui T.
      • Sato M.
      • Okada T.
      • Yoshida H.
      • Harada A.
      • Mori K.
      Transcriptional induction of mammalian ER quality control proteins is mediated by single or combined action of ATF6α and XBP1.
      ,
      • Thuerauf D.J.
      • Marcinko M.
      • Belmont P.J.
      • Glembotski C.C.
      Effects of the isoform-specific characteristics of ATF6 α and ATF6 β on endoplasmic reticulum stress response gene expression and cell viability.
      ,
      • Thuerauf D.J.
      • Morrison L.
      • Glembotski C.C.
      Opposing roles for ATF6α and ATF6β in endoplasmic reticulum stress response gene induction.
      ,
      • Forouhan M.
      • Mori K.
      • Boot-Handford R.P.
      Paradoxical roles of ATF6α and ATF6β in modulating disease severity caused by mutations in collagen X.
      ,
      • Pieper L.A.
      • Strotbek M.
      • Wenger T.
      • Olayioye M.A.
      • Hausser A.
      ATF6β-based fine-tuning of the unfolded protein response enhances therapeutic antibody productivity of Chinese hamster ovary cells.
      ). Thus, we primarily discuss ATF6α in this review, which will be referred to as ATF6 herein. ATF6 is a type II transmembrane protein comprising an N-terminal bZIP transcription factor domain and a C-terminal ER luminal domain (Fig. 4, A and B). Unlike IRE1 and PERK, which rely on oligomerization and autophosphorylation for activation, ATF6 is activated through a different mechanism. In the absence of ER stress, ATF6 exists as monomers and disulfide-bound dimers/oligomers that are maintained by protein disulfide isomerases (PDIs) localized to the ER lumen (Fig. 4A) (
      • Nadanaka S.
      • Okada T.
      • Yoshida H.
      • Mori K.
      Role of disulfide bridges formed in the luminal domain of ATF6 in sensing endoplasmic reticulum stress.
      ,
      • Nadanaka S.
      • Yoshida H.
      • Mori K.
      Reduction of disulfide bridges in the lumenal domain of ATF6 in response to glucose starvation.
      ,
      • Koba H.
      • Jin S.
      • Imada N.
      • Ishikawa T.
      • Ninagawa S.
      • Okada T.
      • Sakuma T.
      • Yamamoto T.
      • Mori K.
      Reinvestigation of disulfide-bonded oligomeric forms of the unfolded protein response transducer ATF6.
      ). Oxidized ATF6 is bound at its luminal domain by the ER HSP70 BiP and retained within the ER (Fig. 4A) (
      • Shen J.
      • Chen X.
      • Hendershot L.
      • Prywes R.
      ER stress regulation of ATF6 localization by dissociation of BiP/GRP78 binding and unmasking of Golgi localization signals.
      ,
      • Shen J.
      • Snapp E.L.
      • Lippincott-Schwartz J.
      • Prywes R.
      Stable binding of ATF6 to BiP in the endoplasmic reticulum stress response.
      ). In response to ER stress, ATF6 disulfides are reduced through a PDI-dependent mechanism, and BiP is released from the luminal domain, resulting in an increase in reduced monomeric ATF6. This reduced ATF6 monomer is then trafficked to the golgi and proteolytically processed by site 1 and site 2 proteases (S1P and S2P, respectively) (Fig. 4A) (
      • Nadanaka S.
      • Okada T.
      • Yoshida H.
      • Mori K.
      Role of disulfide bridges formed in the luminal domain of ATF6 in sensing endoplasmic reticulum stress.
      ,
      • Ye J.
      • Rawson R.B.
      • Komuro R.
      • Chen X.
      • Dave U.P.
      • Prywes R.
      • Brown M.S.
      • Goldstein J.L.
      ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs.
      ,
      • Haze K.
      • Yoshida H.
      • Yanagi H.
      • Yura T.
      • Mori K.
      Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress.
      ,
      • Oka O.B.
      • van Lith M.
      • Rudolf J.
      • Tungkum W.
      • Pringle M.A.
      • Bulleid N.J.
      ERp18 regulates activation of ATF6α during unfolded protein response.
      ,
      • Higa A.
      • Taouji S.
      • Lhomond S.
      • Jensen D.
      • Fernandez-Zapico M.E.
      • Simpson J.C.
      • Pasquet J.M.
      • Schekman R.
      • Chevet E.
      Endoplasmic reticulum stress-activated transcription factor ATF6α requires the disulfide isomerase PDIA5 to modulate chemoresistance.
      ). This releases the active, N-terminal ATF6 bZIP transcription factor domain, which dimerizes and localizes to the nucleus. This active ATF6 transcription factor elicits a transcriptional response that includes the up-regulation of multiple ER proteostasis factors (e.g. BiP) through binding ER stress–responsive elements (ERSEs) in target gene promoters (Fig. 4A) (
      • Shoulders M.D.
      • Ryno L.M.
      • Genereux J.C.
      • Moresco J.J.
      • Tu P.G.
      • Wu C.
      • Yates 3rd, J.R.
      • Su A.I.
      • Kelly J.W.
      • Wiseman R.L.
      Stress-independent activation of XBP1s and/or ATF6 reveals three functionally diverse ER proteostasis environments.
      ,
      • Yamamoto K.
      • Yoshida H.
      • Kokame K.
      • Kaufman R.J.
      • Mori K.
      Differential contributions of ATF6 and XBP1 to the activation of endoplasmic reticulum stress-responsive cis-acting elements ERSE, UPRE and ERSE-II.
      ,
      • Adachi Y.
      • Yamamoto K.
      • Okada T.
      • Yoshida H.
      • Harada A.
      • Mori K.
      ATF6 is a transcription factor specializing in the regulation of quality control proteins in the endoplasmic reticulum.
      ). Apart from ER proteostasis, ATF6 activation also transcriptionally regulates other aspects of cellular physiology, including cell growth and redox regulation, through the up-regulation of transcriptional targets, including RHEB and catalase, respectively (
      • Blackwood E.A.
      • Hofmann C.
      • Santo Domingo M.
      • Bilal A.S.
      • Sarakki A.
      • Stauffer W.
      • Arrieta A.
      • Thuerauf D.J.
      • Kolkhorst F.W.
      • Muller O.J.
      • Jakobi T.
      • Dieterich C.
      • Katus H.A.
      • Doroudgar S.
      • Glembotski C.C.
      ATF6 regulates cardiac hypertrophy by transcriptional induction of the mTORC1 activator, Rheb.
      ,
      • Jin J.K.
      • Blackwood E.A.
      • Azizi K.
      • Thuerauf D.J.
      • Fahem A.G.
      • Hofmann C.
      • Kaufman R.J.
      • Doroudgar S.
      • Glembotski C.C.
      ATF6 decreases myocardial ischemia/reperfusion damage and links ER stress and oxidative stress signaling pathways in the heart.
      ,
      • Allen D.
      • Seo J.
      ER stress activates the TOR pathway through Atf6.
      ,
      • Schewe D.M.
      • Aguirre-Ghiso J.A.
      ATF6α-Rheb-mTOR signaling promotes survival of dormant tumor cells in vivo.
      ). ATF6 transcriptional activity integrates with IRE1 signaling through multiple mechanisms, including the ATF6-dependent up-regulation of XBP1 and heterodimerization between the cleaved ATF6 transcription factor and XBP1s, which increases expression of genes involved in ER proteostasis, including ERAD factors (
      • Yoshida H.
      • Matsui T.
      • Yamamoto A.
      • Okada T.
      • Mori K.
      XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor.
      ,
      • Shoulders M.D.
      • Ryno L.M.
      • Genereux J.C.
      • Moresco J.J.
      • Tu P.G.
      • Wu C.
      • Yates 3rd, J.R.
      • Su A.I.
      • Kelly J.W.
      • Wiseman R.L.
      Stress-independent activation of XBP1s and/or ATF6 reveals three functionally diverse ER proteostasis environments.
      ,
      • Yamamoto K.
      • Sato T.
      • Matsui T.
      • Sato M.
      • Okada T.
      • Yoshida H.
      • Harada A.
      • Mori K.
      Transcriptional induction of mammalian ER quality control proteins is mediated by single or combined action of ATF6α and XBP1.
      ). Through this transcriptional activity, ATF6 functions to promote adaptive remodeling of cellular physiology following ER stress.
      Figure thumbnail gr4
      Figure 4Identification and targeting of the ATF6 UPR signaling pathway. A, mechanism of ATF6 activation and downstream transcriptional signaling. B, domain architecture of ATF6, including the N-terminal bZIP transcription factor domain, the transmembrane domain (TM), and the luminal domain. Upon ATF6 activation, the N-terminal cytosolic domain containing the leucine zipper motif is liberated via processing in the golgi as shown in A, resulting in release of the active ATF6 transcription factor. Specific inhibiting (red) or activating (blue) mutations in ATF6 implicated in achromatopsia are indicated. C, structures of ATF6 inhibitors, including the S1P inhibitors PF429242 and AEBSF and the selective ATF6 inhibitor Ceapin-A7. D, structures of ATF6-activating compounds BiX, AA147, and AA263.
      Significant mouse and human genetic evidence highlights the unique potential for targeting ATF6 to intervene in human disease. Unlike IRE1 and PERK, genetic deletion of Atf6α or Atf6β in mice does not result in any prominent phenotype (
      • Yamamoto K.
      • Sato T.
      • Matsui T.
      • Sato M.
      • Okada T.
      • Yoshida H.
      • Harada A.
      • Mori K.
      Transcriptional induction of mammalian ER quality control proteins is mediated by single or combined action of ATF6α and XBP1.
      ), although aged mice lacking Atf6α show rod and cone dysfunction in the eye (
      • Kohl S.
      • Zobor D.
      • Chiang W.C.
      • Weisschuh N.
      • Staller J.
      • Gonzalez Menendez I.
      • Chang S.
      • Beck S.C.
      • Garcia Garrido M.
      • Sothilingam V.
      • Seeliger M.W.
      • Stanzial F.
      • Benedicenti F.
      • Inzana F.
      • Heon E.
      • et al.
      Mutations in the unfolded protein response regulator ATF6 cause the cone dysfunction disorder achromatopsia.
      ) and increased sensitivity to ER stress (
      • Yamamoto K.
      • Sato T.
      • Matsui T.
      • Sato M.
      • Okada T.
      • Yoshida H.
      • Harada A.
      • Mori K.
      Transcriptional induction of mammalian ER quality control proteins is mediated by single or combined action of ATF6α and XBP1.
      ,
      • Wu J.
      • Rutkowski D.T.
      • Dubois M.
      • Swathirajan J.
      • Saunders T.
      • Wang J.
      • Song B.
      • Yau G.D.
      • Kaufman R.J.
      ATF6α optimizes long-term endoplasmic reticulum function to protect cells from chronic stress.
      ). However, combined deletion of both Atf6α and Atf6β is embryonic lethal, suggesting a potential overlap in developmental roles of these two ATF6 isoforms (
      • Yamamoto K.
      • Sato T.
      • Matsui T.
      • Sato M.
      • Okada T.
      • Yoshida H.
      • Harada A.
      • Mori K.
      Transcriptional induction of mammalian ER quality control proteins is mediated by single or combined action of ATF6α and XBP1.
      ). Regardless, these results indicate that reducing ATF6 activity does not significantly impact organismal physiology in the absence of stress. Similarly, overexpression of the active ATF6 transcription factor domain in various mouse tissues is well-tolerated and is not associated with tissue-specific toxicity. Instead, increased ATF6 transcriptional activity has been shown to be protective in cellular and rodent models of multiple diseases, including diabetes, protein-misfolding diseases, myocardial infarction, and stroke (
      • Shoulders M.D.
      • Ryno L.M.
      • Genereux J.C.
      • Moresco J.J.
      • Tu P.G.
      • Wu C.
      • Yates 3rd, J.R.
      • Su A.I.
      • Kelly J.W.
      • Wiseman R.L.
      Stress-independent activation of XBP1s and/or ATF6 reveals three functionally diverse ER proteostasis environments.
      ,
      • Jin J.K.
      • Blackwood E.A.
      • Azizi K.
      • Thuerauf D.J.
      • Fahem A.G.
      • Hofmann C.
      • Kaufman R.J.
      • Doroudgar S.
      • Glembotski C.C.
      ATF6 decreases myocardial ischemia/reperfusion damage and links ER stress and oxidative stress signaling pathways in the heart.
      ,
      • Martindale J.J.
      • Fernandez R.
      • Thuerauf D.
      • Whittaker R.
      • Gude N.
      • Sussman M.A.
      • Glembotski C.C.
      Endoplasmic reticulum stress gene induction and protection from ischemia/reperfusion injury in the hearts of transgenic mice with a tamoxifen-regulated form of ATF6.
      ,
      • Yu Z.
      • Sheng H.
      • Liu S.
      • Zhao S.
      • Glembotski C.C.
      • Warner D.S.
      • Paschen W.
      • Yang W.
      Activation of the ATF6 branch of the unfolded protein response in neurons improves stroke outcome.
      ,
      • Chen X.
      • Zhang F.
      • Gong Q.
      • Cui A.
      • Zhuo S.
      • Hu Z.
      • Han Y.
      • Gao J.
      • Sun Y.
      • Liu Z.
      • Yang Z.
      • Le Y.
      • Gao X.
      • Dong L.Q.
      • Gao X.
      • et al.
      Hepatic ATF6 increases fatty acid oxidation to attenuate hepatic steatosis in mice through peroxisome proliferator-activated receptor α.
      ,
      • Ozcan L.
      • Ghorpade D.S.
      • Zheng Z.
      • de Souza J.C.
      • Chen K.
      • Bessler M.
      • Bagloo M.
      • Schrope B.
      • Pestell R.
      • Tabas I.
      Hepatocyte DACH1 is increased in obesity via nuclear exclusion of HDAC4 and promotes hepatic insulin resistance.