ISR; eIF2α

ISRIB 抑制内源性 ATF4 的合成，而 XBP1 mRNA 剪接和生产仍在继续。通过阻碍 UPR PERK 分支的信号传导，ISRIB 使细胞无法恢复 ER 稳态并降低 ER 应激细胞的存活率。 [1]
ISRIB处理的细胞对eIF2α磷酸化具有抗性。 ISRIB是PERK分支特异性的，但不会损害PERK磷酸化。 ISRIB会损害对ER压力的适应。[1]

ISRIB 在体内表现出良好的生物利用度，并在药代动力学实验中表现出良好的特性。通过改善恐惧相关学习和空间学习，ISRIB（0.25 mg/kg ip）可改善小鼠的长期记忆。 [1]

---

eIF2α+/S51A（Eif2s1+/S51A）杂合子小鼠表现出增强的记忆，而脑锥体细胞中eIF2α激酶PKR的诱导会损害记忆（Costa-Mattioli等人，2007；Jiang等人，2010）。基于这些观察，我们想知道用ISRIB治疗小鼠是否会影响记忆。ISRIB在药代动力学分析实验中显示出良好的特性，表明体内研究具有足够的生物利用度。ISRIB在血浆中的半衰期为8小时（图6A），很容易穿过血脑屏障，迅速与中枢神经系统平衡（图6B）。单次腹腔注射后，我们在小鼠大脑中检测到ISRIB，其浓度比IC50高几倍（注射后24小时，ISRIB在大脑中的浓度约为60 nM）。为了探索ISRIB对记忆的影响，我们给小鼠腹腔注射了ISRIB，并测试了海马依赖的空间学习。为此，我们在Morris水迷宫中训练了小鼠，在这个迷宫中，动物学会了将视觉线索与水下隐藏平台的位置联系起来。因为我们在寻找记忆增强，所以我们使用了一个弱训练方案。如图6C所示，与赋形剂治疗的对照组（68.1±20秒，p<0.05）相比，ISRIB治疗的小鼠到达隐藏平台的速度明显更快（5天训练后的逃逸潜伏期=16.4±4.8秒）。在第3天和第4天，这种差异已经明显。与这些结果一致，在训练结束时进行的“探测测试”中，ISRIB治疗的小鼠明显更喜欢目标象限，其中平台从池中移除（p<0.05；图6D），平台位置的交叉增加（p<0.05；见图6E）。[1]

在 96 孔板中，用 100 nM 沙利度胺和 10μM 所选化合物处理表达 ATF4-dGFP-IRES-Cherry 报告基因的 U2OS 细胞 8 小时。 Hoechst 33,258 用于对细胞进行染色，并使用自动显微镜观察结果。 INCell Developer Toolbox 软件 1.9 版用于数据采集和图像分析。重新购买用于额外分析的化合物可防止 ATF4-dGFP 报告基因被诱导，不会阻止 IRES 下游 mCherry 的积累，并且根据通过细胞核计数的细胞数量判断为无毒。

将HEK293T细胞用或不用1μg/ml的衣霉素、衣霉素和ISRIB（200 nM）或ISRIB处理1小时。加入环己酰胺（CHX）（100μg/ml）2分钟，用冰冷的PBS（含100μg/ml的CHX）洗涤细胞，并在20mM Tris pH=7.4（RT）、200mM NaCl、15mM MgCl、1mM DTT、8%甘油、100μg/ml CHX、1%Triton和蛋白酶抑制剂中裂解。使用注射器（25G5/8）研制细胞，将裂解物以12000rpm澄清10分钟，将一半裂解物用于RNA提取，另一半用RNase I消化。基于通过分析多核糖体梯度分析的多核糖体坍塌至单体峰，对每个样品的RNase I的量和孵育时间进行优化。用SUPERaseIn猝灭反应，然后将消化的裂解物装载在800μl蔗糖缓冲液上（将1.7 g蔗糖溶解在3.9 ml不含Triton的裂解缓冲液中），并在TLA100.2转子中以70000rpm离心4小时。将沉淀重悬于10mM Tris pH=7（RT）中，提取RNA（苯酚/氯仿）[1]。

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

---

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.

---

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.

---

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

76.7Ų

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)

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

---

注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

View More

注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

---

口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

View More

口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

---

注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

---

请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。