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Small molecule ISRIB suppresses the integrated stress response within a defined window of activation.

ABSTRACT: Activation of the integrated stress response (ISR) by a variety of stresses triggers phosphorylation of the α-subunit of translation initiation factor eIF2. P-eIF2α inhibits eIF2B, the guanine nucleotide exchange factor that recycles inactive eIF2•GDP to active eIF2•GTP. eIF2 phosphorylation thereby represses translation. Persistent activation of the ISR has been linked to the development of several neurological disorders, and modulation of the ISR promises new therapeutic strategies. Recently, a small-molecule ISR inhibitor (ISRIB) was identified that rescues translation in the presence of P-eIF2α by facilitating the assembly of more active eIF2B. ISRIB enhances cognitive memory processes and has therapeutic effects in brain-injured mice without displaying overt side effects. While using ISRIB to investigate the ISR in picornavirus-infected cells, we observed that ISRIB rescued translation early in infection when P-eIF2α levels were low, but not late in infection when P-eIF2α levels were high. By treating cells with varying concentrations of poly(I:C) or arsenite to induce the ISR, we provide additional proof that ISRIB is unable to inhibit the ISR when intracellular P-eIF2α concentrations exceed a critical threshold level. Together, our data demonstrate that the effects of pharmacological activation of eIF2B are tuned by P-eIF2α concentration. Thus, ISRIB can mitigate undesirable outcomes of low-level ISR activation that may manifest neurological disease but leaves the cytoprotective effects of acute ISR activation intact. The insensitivity of cells to ISRIB during acute ISR may explain why ISRIB does not cause overt toxic side effects in vivo.

SUBMITTER: Rabouw HH

PROVIDER: S-EPMC6369741 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The small molecule ISRIB reverses the effects of eIF2α phosphorylation on translation and stress granule assembly.

Project description:Previously, we identified ISRIB as a potent inhibitor of the integrated stress response (ISR) and showed that ISRIB makes cells resistant to the effects of eIF2α phosphorylation and enhances long-term memory in rodents (Sidrauski et al., 2013). Here, we show by genome-wide in vivo ribosome profiling that translation of a restricted subset of mRNAs is induced upon ISR activation. ISRIB substantially reversed the translational effects elicited by phosphorylation of eIF2α and induced no major changes in translation or mRNA levels in unstressed cells. eIF2α phosphorylation-induced stress granule (SG) formation was blocked by ISRIB. Strikingly, ISRIB addition to stressed cells with pre-formed SGs induced their rapid disassembly, liberating mRNAs into the actively translating pool. Restoration of mRNA translation and modulation of SG dynamics may be an effective treatment of neurodegenerative diseases characterized by eIF2α phosphorylation, SG formation, and cognitive loss.

| S-EPMC4341466 | biostudies-literature

Suppression of MEHMO Syndrome Mutation in eIF2 by Small Molecule ISRIB.

Project description:Dysregulation of cellular protein synthesis is linked to a variety of diseases. Mutations in EIF2S3, encoding the γ subunit of the heterotrimeric eukaryotic translation initiation factor eIF2, cause MEHMO syndrome, an X-linked intellectual disability disorder. Here, using patient-derived induced pluripotent stem cells, we show that a mutation at the C terminus of eIF2γ impairs CDC123 promotion of eIF2 complex formation and decreases the level of eIF2-GTP-Met-tRNAiMet ternary complexes. This reduction in eIF2 activity results in dysregulation of global and gene-specific protein synthesis and enhances cell death upon stress induction. Addition of the drug ISRIB, an activator of the eIF2 guanine nucleotide exchange factor, rescues the cell growth, translation, and neuronal differentiation defects associated with the EIF2S3 mutation, offering the possibility of therapeutic intervention for MEHMO syndrome.

| S-EPMC7035991 | biostudies-literature

Targeting Gli transcription activation by small molecule suppresses tumor growth.

Project description:Targeted inhibition of Hedgehog signaling at the cell membrane has been associated with anticancer activity in preclinical and early clinical studies. Hedgehog signaling involves activation of Gli transcription factors that can also be induced by alternative pathways. In this study, we identified an interaction between Gli proteins and a transcription coactivator TBP-associated factor 9 (TAF9), and validated its functional relevance in regulating Gli transactivation. We also describe a novel, synthetic small molecule, FN1-8, that efficiently interferes with Gli/TAF9 interaction and downregulate Gli/TAF9-dependent transcriptional activity. More importantly, FN1-8 suppresses cancer cell proliferation in vitro and inhibits tumor growth in vivo. Our results suggest that blocking Gli transactivation, an important control point of multiple oncogenic pathways, may be an effective anticancer strategy.

| S-EPMC3947751 | biostudies-literature

The Small Molecule ISRIB Reverses the Effects of eIF2α Phosphorylation on Translation and Stress Granule Assembly

Project description:We recently identified ISRIB as a potent inhibitor of the integrated stress response (ISR). ISRIB renders cells resistant to the effects of eIF2α phosphorylation and enhances long-term memory in rodents (10.7554/eLife.00498). Here we show by genome-wide in vivo ribosome profiling that translation of a restricted subset of mRNAs is induced upon ISR activation. ISRIB substantially reversed the translational effects elicited by phosphorylation of eIF2α and induced no major changes in translation or mRNA levels in unstressed cells. eIF2α phosphorylation-induced stress granule (SG) formation was blocked by ISRIB. Strikingly, ISRIB addition to stressed cells with pre-formed SGs induced their rapid disassembly, liberating mRNAs into the actively translating pool. Restoration of mRNA translation and modulation of SG dynamics may be an effective treatment of neurodegenerative diseases characterized by eIF2α phosphorylation, SG formation and cognitive loss. Ribosome profiling with paired RNA-seq

2015-02-27 | E-GEOD-65778 | biostudies-arrayexpress

The Small Molecule ISRIB Reverses the Effects of eIF2α Phosphorylation on Translation and Stress Granule Assembly

2015-02-27 | GSE65778 | GEO

The small molecule ISRIB rescues the stability and activity of Vanishing White Matter Disease eIF2B mutant complexes.

Project description:eIF2B is a dedicated guanine nucleotide exchange factor for eIF2, the GTPase that is essential to initiate mRNA translation. The integrated stress response (ISR) signaling pathway inhibits eIF2B activity, attenuates global protein synthesis and upregulates a set of stress-response proteins. Partial loss-of-function mutations in eIF2B cause a neurodegenerative disorder called Vanishing White Matter Disease (VWMD). Previously, we showed that the small molecule ISRIB is a specific activator of eIF2B (Sidrauski et al., 2015). Here, we report that various VWMD mutations destabilize the decameric eIF2B holoenzyme and impair its enzymatic activity. ISRIB stabilizes VWMD mutant eIF2B in the decameric form and restores the residual catalytic activity to wild-type levels. Moreover, ISRIB blocks activation of the ISR in cells carrying these mutations. As such, ISRIB promises to be an invaluable tool in proof-of-concept studies aiming to ameliorate defects resulting from inappropriate or pathological activation of the ISR.

| S-EPMC5829914 | biostudies-literature

ISRIB Blunts the Integrated Stress Response by Allosterically Antagonising the Inhibitory Effect of Phosphorylated eIF2 on eIF2B.

Project description:The small molecule ISRIB antagonizes the activation of the integrated stress response (ISR) by phosphorylated translation initiation factor 2, eIF2(αP). ISRIB and eIF2(αP) bind distinct sites in their common target, eIF2B, a guanine nucleotide exchange factor for eIF2. We have found that ISRIB-mediated acceleration of eIF2B's nucleotide exchange activity in vitro is observed preferentially in the presence of eIF2(αP) and is attenuated by mutations that desensitize eIF2B to the inhibitory effect of eIF2(αP). ISRIB's efficacy as an ISR inhibitor in cells also depends on presence of eIF2(αP). Cryoelectron microscopy (cryo-EM) showed that engagement of both eIF2B regulatory sites by two eIF2(αP) molecules remodels both the ISRIB-binding pocket and the pockets that would engage eIF2α during active nucleotide exchange, thereby discouraging both binding events. In vitro, eIF2(αP) and ISRIB reciprocally opposed each other's binding to eIF2B. These findings point to antagonistic allostery in ISRIB action on eIF2B, culminating in inhibition of the ISR.

| S-EPMC7837216 | biostudies-literature

Small Molecule Modulation of the Integrated Stress Response Governs the Keratoconic Phenotype In Vitro.

Project description:PurposeThe degenerative corneal disease keratoconus is a leading indicator for corneal transplant with an unknown etiology. We recently identified the activation of the integrated stress response (ISR) in ex vivo human corneas and in vitro cell culture. Utilizing small molecules to modulate the ISR we sought to investigate the effects of stimulating the ISR in healthy cells to recapitulate aspects of the in vitro keratoconic phenotype and whether relieving the ISR signaling would recover the disease phenotype.MethodsCorneal fibroblasts were extracted from patients undergoing corneal transplant or unaffected cadaverous donor limbal rings. Cells were exposed to the DNA damage-inducible protein (GADD34) inhibitor SAL003 to stimulate the ISR, or Trans-ISRIB to relieve ISR signaling pathway. Collagen production was assessed through hydroxyproline production, Sirius Red incorporation, or quantitative (q)PCR. Western blotting, hydroxyproline, and qPCR were used to assess components of the ISR pathway and collagen production.ResultsISR stimulation through SAL003 resulted in significant decrease of hydroxyproline and COL1A1 transcription and eventual apoptosis in normal fibroblasts. Patient (KC) fibroblast production of hydroxyproline was increased in response to ISRIB, while matrix metalloproteinase (MMP)9 production was lowered. The prospective biomarker of keratoconus prolactin-inducible factor was also upregulated in KC fibroblast cultures in response to ISRIB. Inflammatory markers TNFα and IL-1β were unaffected.ConclusionsActivation of the ISR is sufficient to recapitulate many key aspects of the KC phenotype in unaffected cells in vitro. Inhibition of the ISR also relieves many of the hallmarks of KC in affected cells. Therefore, targeting of the ISR through small molecules is a potential therapeutic path for small molecule treatment of keratoconus.

| S-EPMC6686743 | biostudies-literature

Activation of the stress proteome as a mechanism for small molecule therapeutics.

Project description:Various small molecule pharmacologic agents with different known functions produce similar outcomes in diverse Mendelian and complex disorders, suggesting that they may induce common cellular effects. These molecules include histone deacetylase inhibitors, 4-phenylbutyrate (4PBA) and trichostatin A, and two small molecules without direct histone deacetylase inhibitor activity, hydroxyurea (HU) and sulforaphane. In some cases, the therapeutic effects of histone deacetylase inhibitors have been attributed to an increase in expression of genes related to the disease-causing gene. However, here we show that the pharmacological induction of mitochondrial biogenesis was necessary for the potentially therapeutic effects of 4PBA or HU in two distinct disease models, X-linked adrenoleukodystrophy and sickle cell disease. We hypothesized that a common cellular response to these four molecules is induction of mitochondrial biogenesis and peroxisome proliferation and activation of the stress proteome, or adaptive cell survival response. Treatment of human fibroblasts with these four agents induced mitochondrial and peroxisomal biogenesis as monitored by flow cytometry, immunofluorescence and/or western analyses. In treated normal human fibroblasts, all four agents induced the adaptive cell survival response: heat shock, unfolded protein, autophagic and antioxidant responses and the c-jun N-terminal kinase pathway, at the transcriptional and translational levels. Thus, activation of the evolutionarily conserved stress proteome and mitochondrial biogenesis may be a common cellular response to such small molecule therapy and a common basis of therapeutic action in various diseases. Modulation of this novel therapeutic target could broaden the range of treatable diseases without directly targeting the causative genetic abnormalities.

| S-EPMC3441123 | biostudies-literature

Small molecule inhibition of PAX3-FOXO1 through AKT activation suppresses malignant phenotypes of alveolar rhabdomyosarcoma.

Project description:Alveolar rhabdomyosarcoma comprises a rare highly malignant tumor presumed to be associated with skeletal muscle lineage in children. The hallmark of the majority of alveolar rhabdomyosarcoma is a chromosomal translocation that generates the PAX3-FOXO1 fusion protein, which is an oncogenic transcription factor responsible for the development of the malignant phenotype of this tumor. Alveolar rhabdomyosarcoma cells are dependent on the oncogenic activity of PAX3-FOXO1, and its expression status in alveolar rhabdomyosarcoma tumors correlates with worst patient outcome, suggesting that blocking this activity of PAX3-FOXO1 may be an attractive therapeutic strategy against this fusion-positive disease. In this study, we screened small molecule chemical libraries for inhibitors of PAX3-FOXO1 transcriptional activity using a cell-based readout system. We identified the Sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCA) inhibitor thapsigargin as an effective inhibitor of PAX3-FOXO1. Subsequent experiments in alveolar rhabdomyosarcoma cells showed that activation of AKT by thapsigargin inhibited PAX3-FOXO1 activity via phosphorylation. Moreover, this AKT activation appears to be associated with the effects of thapsigargin on intracellular calcium levels. Furthermore, thapsigargin inhibited the binding of PAX3-FOXO1 to target genes and subsequently promoted its proteasomal degradation. In addition, thapsigargin treatment decreases the growth and invasive capacity of alveolar rhabdomyosarcoma cells while inducing apoptosis in vitro. Finally, thapsigargin can suppress the growth of an alveolar rhabdomyosarcoma xenograft tumor in vivo. These data reveal that thapsigargin-induced activation of AKT is an effective mechanism to inhibit PAX3-FOXO1 and a potential agent for targeted therapy against alveolar rhabdomyosarcoma.

| S-EPMC3858449 | biostudies-literature

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