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Purity: =99.73%
ISRIB (mixed cis- and trans-isomer) is a novel and potent ISR (Integrated Stress Response) Inhibitor. It targets the interactions between eIF2α kinases and elF2B by acting downstream of the PERK signaling pathway. Upon induction of ER stress, ISRIB (IC50 = 5 nM) inhibits the unfolded protein response mediated by eIF2α phosphorylation.
| Targets |
ISR; eIF2α
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|---|---|
| ln Vitro |
ISRIB inhibits the synthesis of endogenous ATF4, while XBP1 mRNA splicing and production continued. By obstructing signaling via the PERK branch of the UPR, ISRIB keeps cells from regaining ER homeostasis and reduces the survival of ER-stressed cells.[1]
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| ln Vivo |
ISRIB exhibits good bioavailability in vivo and favorable properties in pharmacokinetic profiling experiments. By improving fear-associated and spatial learning, ISRIB (0.25 mg/kg i.p.) improves long-term memory in mice.[1]
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| Enzyme Assay |
In 96-well plates, ATF4-dGFP-IRES-Cherry reporter-expressing U2OS cells are treated for eight hours with 100 nM thalidomide and 10μM of the selected compounds. Hoechst 33,258 is used to stain cells, and an automated microscope is used to view the results. The INCell Developer Toolbox Software, version 1.9, is used for data acquisition and image analysis. Repurchased for additional analysis are compounds that prevent the ATF4-dGFP reporter from being induced, do not prevent the accumulation of mCherry downstream of the IRES, and are judged non-toxic based on the number of cells counted through nuclei.
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| Cell Assay |
HEK293T cells were treated with or without 1 μg/ml of tunicamycin, tunicamycin and ISRIB (200 nM), or ISRIB for 1 hr. Cycloheximide (CHX) (100 μg/ml) was added for 2 min, cells were washed with ice cold PBS (with 100 μg/ml of CHX) and lysed in 20 mM Tris pH = 7.4 (RT), 200 mM NaCl, 15 mM MgCl, 1 mM DTT, 8% glycerol, 100 μg/ml CHX, 1% Triton and protease inhibitors. A syringe (25G5/8) was used to triturate cells, the lysate was clarified at 12,000 rpm for 10 min and half of the lysate was used for RNA extraction and the other half was digested with RNase I. The amount of RNase I and time of incubation was optimized for each sample based on the collapse of polyribosomes to the monosome peak as analyzed by analytical polyribosome gradients. The reaction was quenched with SUPERaseIn and the digested lysate was then loaded on an 800 μl sucrose cushion (1.7 g of sucrose was dissolved in 3.9 ml of lysis buffer without Triton) and centrifuged in a TLA100.2 rotor at 70,000 rpm for 4 hr. The pellet was resuspended in 10 mM Tris pH = 7 (RT), and RNA was extracted (phenol/chloroform) [1].
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| Animal Protocol |
Male C57BL/6J mice
0.25 mg/kg i.p.; dissolved in 45% saline + 50% PEG 400 + 5% DMSO |
| References |
[1]. Pharmacological brake-release of mRNA translation enhances cognitive memory Elife. 2013 May 28:2:e00498.
[2]. ISRIB Blunts the Integrated Stress Response by Allosterically Antagonising the Inhibitory Effect of Phosphorylated eIF2 on eIF2B. Mol Cell. 2021 Jan 7;81(1):88-103.e6. [3]. Partial restoration of protein synthesis rates by the small molecule ISRIB prevents neurodegeneration without pancreatic toxicity. Cell Death Dis. 2015 Mar 5;6(3):e1672. |
| Molecular Formula |
C22H24CL2N2O4
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|---|---|
| Molecular Weight |
451.3430
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| Exact Mass |
450.1113
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| Elemental Analysis |
C, 58.55; H, 5.36; Cl, 15.71; N, 6.21; O, 14.18
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| CAS # |
548470-11-7
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| Related CAS # |
ISRIB (trans-isomer);1597403-47-8
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| Appearance |
Off-white to light yellow solid powder
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| LogP |
4.5
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| tPSA |
76.7Ų
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| SMILES |
C1CC(CCC1NC(=O)COC2=CC=C(C=C2)Cl)NC(=O)COC3=CC=C(C=C3)Cl
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| InChi Key |
HJGMCDHQPXTGAV-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C22H24Cl2N2O4/c23-15-1-9-19(10-2-15)29-13-21(27)25-17-5-7-18(8-6-17)26-22(28)14-30-20-11-3-16(24)4-12-20/h1-4,9-12,17-18H,5-8,13-14H2,(H,25,27)(H,26,28)
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| Chemical Name |
2-(4-chlorophenoxy)-N-[4-[[2-(4-chlorophenoxy)acetyl]amino]cyclohexyl]acetamide
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| Synonyms |
ISRIB
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
DMSO: 8.3~10 mg/mL (18.5~22.2 mM)
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|---|---|
| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2156 mL | 11.0781 mL | 22.1562 mL | |
| 5 mM | 0.4431 mL | 2.2156 mL | 4.4312 mL | |
| 10 mM | 0.2216 mL | 1.1078 mL | 2.2156 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
 Sidrauski C, et al. Elife. 2013, 2, e00498. |
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