Isrib

Manufactured by Merck Group

Sourced in United States, Germany, Canada

ISRIB is a small molecule compound produced by Merck Group. It functions as an inhibitor of the integrated stress response (ISR) pathway. ISRIB acts by modulating the activity of the eIF2B protein complex, which plays a key role in the ISR. This compound is primarily used in research applications to study the ISR and related cellular processes.

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Lab products found in correlation

Tunicamycin, by Merck Group (8 mentions) Cycloheximide (chx), by Merck Group (5 mentions) Dimethyl sulfoxide (dmso), by Merck Group (4 mentions) Thapsigargin, by Merck Group (4 mentions) Stf 083010, by Merck Group (3 mentions) Sodium arsenite, by Merck Group (3 mentions) Actinomycin d, by Merck Group (3 mentions) Gsk2656157, by Abcam (3 mentions) Antimycin a, by Merck Group (3 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions) Mg132, by Merck Group (2 mentions) Arsenite, by Merck Group (2 mentions) Accuri c6, by BD (2 mentions) Thapsigargin, by Thermo Fisher Scientific (2 mentions) Ionomycin, by Merck Group (2 mentions) Mouse prelid1 shrna, by Merck Group (2 mentions) Human prelid1 shrna, by Merck Group (2 mentions) Uk5099, by Merck Group (2 mentions) Fluoro carbonyl cyanide phenylhydrazone (fccp), by Agilent Technologies (2 mentions) Oligomycin, by Agilent Technologies (2 mentions)

Spelling variants of this product

ISRIB (SML0843) (2 citations)

73 protocols using isrib

Modulating UPR Activation in Neurons

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The ER stress pharmacological inducers tunicamycin (Tun) (Applichem) and thapsigargin (Thap) (Applichem), as well as the UPR modulators ISRIB (Sigma), 4μ8C (Sigma), GSK2606414 (GSK) (Toronto Research Chemicals, North York, Canada), and Sephin1 (Sigma), were dissolved in dimethyl sulfoxide (DMSO) (Sigma) and stored at −80 °C.
For survival and UPR activation experiments, neuronal cultures were supplemented with a single dose of ISRIB (0.1, 0.5, or 1 μM), 4μ8C (5 or 16 µM), or GSK (0.5, 2, 4, or 8 µM), and the highest equivalent dose of DMSO as a control 1 day post transfection.
To characterize UPR activation, HEK293 cells and primary cortical neurons were treated with Thap (100 nM) alone or in combination with the UPR modulators described above [ISRIB (500 nM), GSK (2 µM), or 4µ8C (5, 16, and 50 µM), and Sephin1 (10 µM), and DMSO as a control]. Cells were harvested for Western blot (WB) analysis at different time points after treatment (from 1.5, 3, 6, 18 to 24 h).

Bugallo R., Marlin E., Baltanás A., Toledo E., Ferrero R., Vinueza-Gavilanes R., Larrea L., Arrasate M, & Aragón T. (2020). Fine tuning of the unfolded protein response by ISRIB improves neuronal survival in a model of amyotrophic lateral sclerosis. Cell Death & Disease, 11(5), 397.

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Preparation of Stock Solutions for Cell Studies

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CB-5083 (S8101, Selleckchem, Houston, TX, USA), NMS-873 (S7285, Selleckchem), DBeQ (SML0031, Sigma-Aldrich, Saint Louis, MO, USA), STF-083010 (S7771, Selleckchem,), STF-083010 (SML0409, Sigma-Aldrich), ISRIB (SML0843, Sigma-Aldrich) and mifepristone (M8046, Sigma-Aldrich) were dissolved in dimethyl sulfoxide (DMSO) at 50 mM stock solution. Tunicamycin (T7765, Sigma-Aldrich) was dissolved in DMSO at 10 mM stock solution and ISRIB (SML0843, Sigma-Aldrich) was dissolved in DMSO at 5 mM stock solution. All stock solutions were aliquoted in small volumes and stored at −80 °C. Before use, appropriate concentrations of these compounds were prepared by dissolving the stock solution (or its subsequent dilution) into the appropriate growth media.

Bastola P., Leiserowitz G.S, & Chien J. (2022). Multiple Components of Protein Homeostasis Pathway Can Be Targeted to Produce Drug Synergies with VCP Inhibitors in Ovarian Cancer. Cancers, 14(12), 2949.

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Pharmacological Modulation of Cellular Stress

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ISRIB (SML0843 –Sigma) diluted in DMSO was used at concentrations of 200 nM and 500 nM, and treatments were performed after a change of transfection media at 4 hours post transfection (hpt) until cell lysis or fixation. Arsenite (S7400 –Sigma) diluted in sterile water was used at concentrations of 0.5 mM for 30 min in HEK293T, HEp-2 and A549 cells, and 1 mM for 1h in Vero E6 cells. puromycin was used at the final concentration of 10 μg/mL, and was incubated for 10 min for SUnSET assays, using co-treatment with cycloheximide (100 μg/mL) as a negative control [24 (link)]. For immunofluorescence, cells were incubated with puromycin (10 μg/mL) and MG132 (Abcam) (5 μM) for 2h. Additionally, MG132 treatments were performed for 2h (immunofluorescence) and overnight (4 hpt until cell lysis–for western blot) at 5 μM. The HSP70 inhibitor VER-155008 (Sigma) was used at concentrations of 2.5 μM and 5 μM, with similar treatments to ISRIB.

Scudero O.B., Santiago V.F., Palmisano G., Simabuco F.M, & Ventura A.M. (2023). The respiratory syncytial virus M2-2 protein is targeted for proteasome degradation and inhibits translation and stress granules assembly. PLOS ONE, 18(7), e0289100.

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Molecular Insights into Viral Stress Response

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Sodium arsenite (Sigma-Aldrich) was added to the cells at a concentration of 250 μM for 20 min prior to fixation or cell lysate collection. The PKR inhibitor C16 (Sigma-Aldrich) was added to infected cells at a concentration of 1 μM at 1 hpi, and cell lysates were collected at 12 hpi. The ISR inhibitor ISRIB (Sigma-Aldrich) was added at 1 hpi at 0.5 μM. Puromycin (Life Technologies) was added to cells at a concentration of 10 μg/ml at indicated times prior to cell lysate collection. Poly(I⋅C) (Sigma-Aldrich, catalog no. P1530) is a dsRNA analogue and was used as a positive control for immune activation. Poly(I⋅C) was used at a concentration of 20 μg/ml and added to the cell culture medium in combination with Lipofectamine 2000. MNV ORF1 plasmids were described and used previously (22 (link)).
Goat anti-eIF3η, goat anti-G3BP1, and goat anti-TIA-1 were all purchased from Santa Cruz Biotech. Rabbit anti-eIF2α was purchased from Invitrogen; Rabbit anti-actin from Sigma-Aldrich; Mouse anti-Puromycin was obtained from Kerafast, Inc. Mouse anti-G3BP1, mouse anti-GAPDH, rabbit anti-His, and rabbit anti-calnexin were obtained from Abcam, and rabbit anti-p-eIF2α (S52) and Alexa Fluor-conjugated species-specific IgG were purchased from Life Technologies. Rabbit anti-NS7 and rabbit anti-NS5 were manufactured and produced by Invitrogen.

Fritzlar S., Aktepe T.E., Chao Y.W., Kenney N.D., McAllaster M.R., Wilen C.B., White P.A, & Mackenzie J.M. (2019). Mouse Norovirus Infection Arrests Host Cell Translation Uncoupled from the Stress Granule-PKR-eIF2α Axis. mBio, 10(3), e00960-19.

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Peptide and Lipid Standards Preparation

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Angiotensin I (Sigma, A9650–1MG), QCAL Peptide Mix (Sigma, MSQC2) and Hela digest standard (Thermo Fisher Scientific, Catalog number: 88328) were dissolved into different concentrations with 50% acetonitrile (ACN) in 0.2% formic acid (FA). Lipid standards (product No. 330707) was purchased form Avanti. Drugs including deferoxamine mesylate salt (Product No. D9533), mTOR inhibitor torin2 (Product No. SML1224), integrated stress response inhibitor ISRIB (Product No. SML0843), proteasome inhibitor MG-132 (Product No. 474790) and SCD1 inhibitor A939572 (Product No. SML2356) were purchased from Sigma-Aldrich.

Jiang Y., Salladay-Perez I., Momenzadeh A., Covarrubias A.J, & Meyer J.G. (2023). Simultaneous Multi-Omics Analysis by Direct Infusion Mass Spectrometry (SMAD-MS). bioRxiv.

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T cell Cytokine Release Assay

Cited 1 time

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Following Th cell differentiation, the cells were seeded at 50,000 cells per well in 200 μL of culture medium in 96-well plates. Compounds were dissolved in DMSO at concentrations previously described in Cribbs et al. (35 (link)) and diluted to achieve the desired working solutions. Compound effects were compared with cells cultured in 0.1% DMSO alone whereas wells filled with media served as a background control. Following 24 h of compound treatment, media were harvested, and enzyme-linked immunosorbent assay (ELISA) was used to measure cytokine release. For ISRIB (Sigma) blocking experiments we cultured our cells using 5 nM. Small molecules that were able to modify cytokine expression by a twofold change and P < 0.05 were considered as significant.

Cribbs A.P., Terlecki-Zaniewicz S., Philpott M., Baardman J., Ahern D., Lindow M., Obad S., Oerum H., Sampey B., Mander P.K., Penn H., Wordsworth P., Bowness P., de Winther M., Prinjha R.K., Feldmann M, & Oppermann U. (2020). Histone H3K27me3 demethylases regulate human Th17 cell development and effector functions by impacting on metabolism. Proceedings of the National Academy of Sciences of the United States of America, 117(11), 6056-6066.

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Antibody-based Protein Detection Assay

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Anti-DUSP5 and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibodies were obtained from Abcam (Cambridge, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Antibodies directed against CHOP, spliced form of X-box-binding protein-1 (XBP1s), IRE1, and ATF6, phosphorylated ERK, ERK or cleaved caspase-3 were purchased from Cell Signaling Technology (Danvers, MA, USA). Horseradish peroxidase-linked anti-rabbit and anti-mouse IgGs were also provided from Cell Signaling Technology. TM, ISRIB, and other reagents were supplied from Sigma (St. Louis, MO, USA).

Jo H.J., Yang J.W., Park J.H., Choi E.S., Lim C.S., Lee S, & Han C.Y. (2019). Endoplasmic Reticulum Stress Increases DUSP5 Expression via PERK-CHOP Pathway, Leading to Hepatocyte Death. International Journal of Molecular Sciences, 20(18), 4369.

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Antioxidant and Stress Response Compounds

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Carnosic acid, carnosol, and rosmarinic acid were supplied by Nagase Co LTD. (Kobe, Japan). Piciferic acid was purchased from Tokyo Chemical Industry (Tokyo, Japan). Dimethylsulfoxide, actinomycin D, cycloheximide, tunicamycin, and hemin were obtained from Wako Pure Chemicals (Osaka, Japan). The tert-butylhydroquione (tBHQ) was obtained from Kanto Chemical (Tokyo, Japan). ISRIB was obtained from Sigma-Aldrich (St. Louis, MO, USA). Primary antibodies against ATF4 (CREB2) (C-20), Nrf2 (H-300), lamin B (M-20), and HSP90α/β (AC88) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA), and primary antibodies against eIF2α (9722S) and phospho-Ser51 eIF2α (9721S) were purchased from Cell Signaling Technology (Danvers, MA, USA). Secondary antibodies against rabbit and goat immunoglobulin G (IgG) were obtained from Life Technologies (Carlsbad, CA, USA) and Santa Cruz Biotechnology, respectively. hemin was purchased from ICN Biomedicals (Irvine, CA, USA). hemin stock solution (100×) was made by dissolving hemin in 20 mM NaOH.

Mimura J., Inose-Maruyama A., Taniuchi S., Kosaka K., Yoshida H., Yamazaki H., Kasai S., Harada N., Kaufman R.J., Oyadomari S, & Itoh K. (2019). Concomitant Nrf2- and ATF4-Activation by Carnosic Acid Cooperatively Induces Expression of Cytoprotective Genes. International Journal of Molecular Sciences, 20(7), 1706.

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Inhibition of Cellular Pathways

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CB-839 (S7655) was from Selleck Chemicals, bafilomycin A1 (88899-55-2), BPTES (19284) and compound 968 (17199) were from Cayman Chemical, and cycloheximide (C4859) and ISRIB (SML0843) were from Sigma-Aldrich. All chemical inhibitors were resuspended in DMSO. An equivalent amount of DMSO was added to control samples to control for any solvent-based effects.

Pavlova N.N., King B., Josselsohn R.H., Violante S., Macera V.L., Vardhana S.A., Cross J.R, & Thompson C.B. (2020). Translation in amino-acid-poor environments is limited by tRNAGln charging. eLife, 9, e62307.

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ISRIB Enhances Cognitive Function in Aged Rats

Cited 2 times

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(R,S)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid ((±)-CPP, Alomone Labs, C-175) and 3-((2-methyl-1,3-thiazol-4-yl)ethynyl)pyridine hydrochloride (MTEP hydrochloride, Abcam, ab120035) were prepared in distilled water and diluted with saline to the required concentration. Trans-N,N^′-(Cyclohexane-1,4-diyl)bis(2-(4-chlorophenoxy) acetamide (ISRIB, Sigma, SML0843) was dissolved in dimethyl sulfoxide (DMSO) with gentle warming in a 40°C water bath and vortexed until the solution became clear. Then the solution was diluted in polyethylene glycol 400 (PEG400) with gentle warming in a 40°C water bath and vortexed. The solution was prepared freshly and diluted in warm saline (37°C) before injection. 1:1 DMSO and PEG400 in saline was used as vehicle control. The choice of dose and timing of ISRIB administration was based on previous reports [42 (link)] and our study of the pharmacokinetics of ISRIB in live rats [44 (link)]. Four pair-housed aged (17-18-month-old) rats received a single daily injection of ISRIB (2.5 mg/kg, i.p.) on 3 consecutive days. Then the rats stayed pair-housed for 18 days after the third injection of ISRIB.

Zhang Y., Yang Y., Hu Z., Zhu M., Qin S., Yu P., Li B., Xu J., Ondrejcak T., Klyubin I., Rowan M.J, & Hu N.W. (2022). Long-Term Depression-Inducing Low Frequency Stimulation Enhances p-Tau181 and p-Tau217 in an Age-Dependent Manner in Live Rats. Journal of Alzheimer's Disease, 89(1), 335-350.

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