ISR; eIF2α

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]
ISRIB-treated cells are resistant to eIF2α phosphorylation. ISRIB is PERK-branch specific but does not impair PERK phosphorylation. ISRIB impairs adaptation to ER stress. [1]
ISRIB substantially reduced the translational effects elicited by stress and eIF2α phosphorylation. ISRIB prevents formation of stress granules exclusively triggered by eIF2α phosphorylation. ISRIB triggers rapid disassembly of stress granules and restores translation.[2]
ISRIB blocks production of endogenous ATF4, whereas XBP1 mRNA splicing and XBP1s production persisted. By blocking signaling through the PERK branch of the UPR, ISRIB prevents cells from re-establishing ER homeostasis and reduces the viability of cells that are experiencing ER-stress.[1]

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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eIF2α+/S51A (Eif2s1+/S51A) heterozygote mice display enhanced memory, while induction of the eIF2α kinase PKR in brain pyramidal cells impairs memory (Costa-Mattioli et al., 2007; Jiang et al., 2010). Based on these observations, we wondered whether treatment of mice with ISRIB would affect memory. ISRIB showed favorable properties in pharmacokinetic profiling experiments indicating sufficient bioavailability for in vivo studies. ISRIB displayed a half-life in plasma of 8 hr (Figure 6A) and readily crossed the blood-brain barrier, quickly equilibrating with the central nervous system (Figure 6B). After a single intraperitoneal injection, we detected ISRIB in the brain of mice at concentrations several fold higher than its IC50 (24 hr after injection, the ISRIB concentration in the brain was approximately 60 nM). To explore ISRIB's effects on memory, we injected mice intraperitoneally with ISRIB and tested hippocampus-dependent spatial learning. To this end, we trained mice in a Morris water maze, in which animals learn to associate visual cues with the location of a submerged hidden platform. Because we were looking for memory enhancement, we used a weak training protocol. As shown in Figure 6C, ISRIB-treated mice reached the hidden platform significantly faster (escape latency after 5 days of training = 16.4 ± 4.8 s) compared to vehicle treated controls (68.1 ± 20 s, p<0.05). The difference was already pronounced by days 3 and 4. In agreement with these results, ISRIB-treated mice significantly preferred the target quadrant in a ‘probe test’ conducted at the end of the training sessions, in which the platform was removed from the pool (p<0.05; Figure 6D) and showed increased crossing of the platform location (p<0.05; Figure 6E).[1]

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.

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].

Male C57BL/6J mice
0.25 mg/kg
i.p.; dissolved in 45% saline + 50% PEG 400 + 5% DMSO

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Morris water maze[1]
Mice were trained in a water pool of 100 cm diameter with a hidden platform of 10 cm diameter. Mice were handled daily for 3 days before the experiment, and the training protocol consisted of one swimming trial per day. Each mouse swam until it found the hidden platform or 120 s, when it was gently guided to the platform and stayed there for 10 s before being returned to the cage. Immediately after the swimming trial the mice were injected intraperitoneally with ISRIB (0.25 mg/kg in saline, 1% DMSO). For the probe test, the platform was removed and each mouse was allowed to swim for 60 s, while its swimming trajectory was monitored with a video tracking system.

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Contextual fear conditioning[1]
Mice were trained with a protocol that consisted of a 2-min period of context exploration, followed by a single foot shock of 0.35 mA for 1 s. Mice received a single injection of ISRIB (2.5 mg/kg in 50% DMSO, 50% PEG 400, IP) immediately after training and were returned to their home cage. One and 24 hr after training, the mice were tested for contextual fear memory by placing the animals in the conditioning context for a 4-min period. The incidence of freezing was scored in 5-s intervals as either ‘freezing’ or ‘not freezing’. Percent of freezing indicates the number of intervals in which freezing was observed divided by total number of 5-s intervals. Statistical analyses were done by Student’s t tests and one-way ANOVA followed by between-group comparisons using Tukey’s posthoc test.

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Cannulation and auditory fear conditioning[1]
Male Sprague Dawley rats (275–350 g) were used for cannulation as described in Migues et al., 2010 (Migues et al., 2010). ISRIB (0.05 mg/ml, 0.5 μl) was infused bilaterally into the amygdala immediately after auditory fear conditioning training. The infusion was performed with a microinjector (28 gauge) connected to a Hamilton syringe with plastic tubing at a rate of 0.25 μl/min. To allow for the solution containing ISRIB to diffuse from the tip of the cannula into the tissue, the microinjector stayed in the cannula for one additional minute. Training protocol for auditory fear conditioning consisted of a 2-min period of context A exploration, followed by one pairing of a tone (5000 Hz, 75 dB, 30 s) with a co-terminating foot shock (0.75 mA, 1 s). Rats were returned to their home cage 1 min after the shock. Test for auditory fear memory consisted of a 2 min acclimatizing period to the context B (pre-CS), followed by tone presentation (CS) (2800 Hz, 85 dB, 30 s). Freezing time was measured and percent of freezing was calculated. At the end of the experiment, cannula placement was checked by examining 50 μm brain sections stained with formal-thionin under a light microscope.
Mice: Female CD-1 mice that are 6-7 weeks old are given medication via the intra-peritoneal (ip) route. Groups of three mice/compound/route of administration receive a single dose of 5 mg/kg of animals. DMSO is used to dissolve ISRIB, which is then diluted 1:1 in super-refined PEG 400. Following dosing, blood (80 μL) is drawn from the saphenous vein in EDTA-containing collection tubes at intervals of 20 minutes, 1 hour, 3 hours, 8 hours, and 24 hours. Plasma is then prepared for analysis. Time-of-flight mass spectroscopy is used to find substances.

[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.

Phosphorylation of the α-subunit of initiation factor 2 (eIF2) controls protein synthesis by a conserved mechanism. In metazoa, distinct stress conditions activate different eIF2α kinases (PERK, PKR, GCN2, and HRI) that converge on phosphorylating a unique serine in eIF2α. This collection of signaling pathways is termed the 'integrated stress response' (ISR). eIF2α phosphorylation diminishes protein synthesis, while allowing preferential translation of some mRNAs. Starting with a cell-based screen for inhibitors of PERK signaling, we identified a small molecule, named ISRIB, that potently (IC50 = 5 nM) reverses the effects of eIF2α phosphorylation. ISRIB reduces the viability of cells subjected to PERK-activation by chronic endoplasmic reticulum stress. eIF2α phosphorylation is implicated in memory consolidation. Remarkably, ISRIB-treated mice display significant enhancement in spatial and fear-associated learning. Thus, memory consolidation is inherently limited by the ISR, and ISRIB releases this brake. As such, ISRIB promises to contribute to our understanding and treatment of cognitive disorders. DOI:http://dx.doi.org/10.7554/eLife.00498.001.[1]

C22H24CL2N2O4

451.3430

450.111

C, 58.55; H, 5.36; Cl, 15.71; N, 6.21; O, 14.18

548470-11-7

ISRIB (trans-isomer);1597403-47-8

1011240

Off-white to light yellow solid powder

1.3±0.1 g/cm3

719.0±60.0 °C at 760 mmHg

388.6±32.9 °C

0.0±2.3 mmHg at 25°C

1.603

4.49

2

4

8

30

493

0

ClC1C([H])=C([H])C(=C([H])C=1[H])OC([H])([H])C(N([H])C1([H])C([H])([H])C([H])([H])C([H])(C([H])([H])C1([H])[H])N([H])C(C([H])([H])OC1C([H])=C([H])C(=C([H])C=1[H])Cl)=O)=O

HJGMCDHQPXTGAV-UHFFFAOYSA-N

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)

2-(4-chlorophenoxy)-N-[4-[[2-(4-chlorophenoxy)acetyl]amino]cyclohexyl]acetamide

ISRIB; ISRIB; 1597403-47-8; trans-ISRIB; 548470-11-7; ISRIB (trans-isomer); 1597403-48-9; N,N'-(cis-Cyclohexane-1,4-diyl)bis(2-(4-chlorophenoxy)acetamide); ISRIB trans-isomer;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 8.3~10 mg/mL (18.5~22.2 mM)

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)