2BAct

eIF2

与 WT 空隙相比，R191H 初级生成的纤维空隙的 2BAct 活性增加了三倍 (EC50=7.3 nM)，而 GEF 活性较低 [1]。

2BAct（300 μg 2BAct/g 食物；21 周）使 VWM 小鼠的体重增长正常化 [1]。 2BAct 可以保护 VWM 中的运动障碍、髓磷脂缺失和反应性神经网络失眠 [1]。近期 eIF2B 复合体中 2BAct 的残余活性消除了适应不良的近期反应 [1]。

GEF测定[1]
按照前面所述进行实验。简言之，负载Bodipy FL GDP的eIF2用作WT和R191H MEFs产生的裂解物的底物。在384孔板中进行测定。在10µL/孔的最终测定体积中，以下条件保持恒定：25 nM Bodipy FL GDP负载的eIF2、3 nM磷酸-eIF2、0.1 mM GDP、1 mg/mL BSA、0.1 mg/mL MEF裂解物。2BAct是从1mM的储备中分配的。对于每次运行，对每种浓度的2BAct进行三次测量。使用以下仪器参数在SpectraMax i3x平板读取器上读取反应：平板温度=25°C；激发波长=485nm（15nm宽）；发射波长＝535nm（25nm宽度）；读取持续时间=30分钟，间隔45秒。数据在Prism中进行分析。GDP释放半衰期通过拟合单指数衰减曲线来计算。通过拟合log（抑制剂）与反应曲线来计算EC50。

蛋白质印迹[1]
在RIPA缓冲液+蛋白酶/磷酸酶抑制剂中制备小脑裂解物。在Qiagen TissueLyser II中以30Hz的频率裂解组织2×2分钟。将裂解物在冰上孵育10分钟，然后离心（21000 x g，10分钟，4°C）以去除细胞碎片。使用Pierce BCA测定法测定蛋白质浓度，并使用RIPA缓冲液调整至2 mg/mL。将裂解物等分，快速冷冻并在−80°C下储存。对于蛋白质印迹，样品在Mini-PROTEAN TGX 4–20%梯度凝胶上运行，并在Trans-Blot Turbo设备上使用Trans-Blot Turbo-Mini-PVDF转移包转移。用TBS-T中5%的牛奶封闭膜，并在4°C下与一级抗体在相同的封闭缓冲液中孵育过夜。在TBS-T中洗涤三次，每次15分钟后，施加HRP缀合的第二抗体1小时。如前所述，在TBS-T中洗涤膜。将Advansta WesternBright化学发光基质应用于膜，并在Bio-Rad ChemiDoc MP成像系统上以信号累积模式获得图像<小时> ATF4萤光素酶报告基因测定[1]
该实验如前所述进行（Wong等人，2018）。简言之，将表达ATF4萤光素酶报告基因的HEK293T细胞（Sidrauski等人，2013）接种到96孔板中，并用100nM thapsigargin处理7小时以诱导ER应激。在剂量反应中用2BAct或ISRIB共同处理细胞。使用ONE Glo萤光素酶测定试剂（Promega）和Molecular Devices SpectraMax i3x平板读取器测量发光。数据在Prism中进行分析。

2BAct microsuspension preparation [1]

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An aqueous suspension of 2BAct was prepared by suspending the drug in 0.5% hydroxypropyl methylcellulose (HPMC; Hypromellose 2910, 4000 mPa) in water. The suspending vehicle was first prepared by adding 5 g of HPMC to 500 mL of miliQ water heated to 60°C. This mixture was allowed to stir until all of HPMC was dispersed. This solution was then transferred to a volumetric flask with two additional rinses of the original container. Sufficient quantity of water was then added to prepare 1 L of vehicle and allowed to stir overnight to obtain a clear suspension. The vehicle was kept refrigerated and allowed to come to room temperature before each use. Fresh vehicle was prepared every month. For preparation of the aqueous suspension of 2BAct, the compound was weighed into an appropriately sized mortar and levigated with a pestle using a small amount of the vehicle. This was then collected into an appropriately sized glass vial, previously marked with a q.s. line. The mortar was rinsed five times, adding each rinse into the glass vial. Additional vehicle was added to the glass vial until q.s. line was reached and entire suspension mixed by vortexing for 10 s.[1]

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2BAct pharmacokinetics[1]
Six- to eight-week-old CD1 male mice were dosed with 2BAct at 1 mg/kg or 30 mg/kg orally at a dosing volume of 10 mL/kg. For dosing, 2BAct was micronized and suspended in 0.5% hydroxypropyl methylcellulose (HPMC) (see Microsuspension preparation above). Blood was drawn into EDTA charged capillary tubes via the tail vein at the following timepoints: 0.25, 0.5, 1, 3, 6, 9, 12 and 24 hr (N = 3 measurements per timepoint, mice bled at each timepoint, and combined in pairs for extraction). Blood was centrifuged at 3000 rpm and plasma harvested. Plasma samples and standards were extracted by protein precipitation with acetonitrile containing internal standards. The supernatant was diluted with 0.1% formic acid in water before injection into an HPLC-MS/MS system for separation and quantitation. The analytes were separated from matrix components using reverse phase chromatography (30 × 2.1 mm, 5 µm Fortis Pace C18) using gradient elution at a flow rate of 0.8 mL/min. The tandem mass spectrometry analysis was carried out on SCIEX triple quadrupole mass spectrometer with an electrospray ionization interface, in positive ion mode. Data acquisition and evaluation were performed using Analyst software (SCIEX).[1]

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Preparation of 2BAct in diet[1]
2BAct was administered orally by providing mice with the compound incorporated in rodent meal (2014, Teklad Global 14% Protein Rodent Maintenance Diet). For this, the compound was weighed, added to a mortar with small amount of powdered meal, and ground with a pestle until homogenous. This was further mixed with additional powdered meal in HDPE bottles by either geometric mixing with hand agitation or using a Turbula mixer set at 48 rpm for 15 min or contract manufactured at Envigo to achieve a 2BAct concentration of 300 ppm (300 µg 2BAct/g of meal). Teklad 2014 without added compound was offered as the placebo diet.[1]

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Generation of mouse models[1]
The Eif2b5R191H/R191H knock-in mutant mouse model was generated in the background strain C57BL/6J as a service by genOway. Briefly, a targeting vector was designed against the Eif2b5 locus to simultaneously insert: (1) a Flp-excisable neomycin resistance cassette between exons 2 and 3; (2) a CGC - > CAC codon substitution in exon 4 (changing residue Arg191 to His); (3) loxP sites flanking exons 3 and 7 (Figure 1—figure supplement 2). Successful homologous recombination in ES cells was verified by PCR and Southern Blotting. Chimeras were generated by blastocyst injection, which were then mated to WT C57BL/6J mice to identify F1 heterozygous Eif2b5+/R191H;FRT-neo (flox) progeny. The neomycin resistance cassette was removed by mating of heterozygous mice to Flp deleter mice. The resulting Eif2b5+/R191H (flox) mice were used as colony founders. Experiments were performed using homozygous mutant mice and their WT littermates as controls. The Eif2b5R132H/R132H mouse model was generated in a similar manner.

[1]. eIF2B activator prevents neurological defects caused by a chronic integrated stress response. Elife. 2019;8:e42940. Published 2019 Jan 9.

The integrated stress response (ISR) attenuates the rate of protein synthesis while inducing expression of stress proteins in cells. Various insults activate kinases that phosphorylate the GTPase eIF2 leading to inhibition of its exchange factor eIF2B. Vanishing White Matter (VWM) is a neurological disease caused by eIF2B mutations that, like phosphorylated eIF2, reduce its activity. We show that introduction of a human VWM mutation into mice leads to persistent ISR induction in the central nervous system. ISR activation precedes myelin loss and development of motor deficits. Remarkably, long-term treatment with a small molecule eIF2B activator, 2BAct, prevents all measures of pathology and normalizes the transcriptome and proteome of VWM mice. 2BAct stimulates the remaining activity of mutant eIF2B complex in vivo, abrogating the maladaptive stress response. Thus, 2BAct-like molecules may provide a promising therapeutic approach for VWM and provide relief from chronic ISR induction in a variety of disease contexts. [1]

C19H16CLF3N4O3

440.803553581238

440.09

C, 51.77; H, 3.66; Cl, 8.04; F, 12.93; N, 12.71; O, 10.89

2143542-28-1

2143542-28-1

132091799

Light yellow to light brown solid powder

2.2

93.2Ų

HYQJXXCYOYRNMP-UHFFFAOYSA-N

InChI=1S/C19H16ClF3N4O3/c20-11-2-1-10(3-12(11)21)30-6-15(28)26-18-7-19(8-18,9-18)27-17(29)14-5-24-13(4-25-14)16(22)23/h1-5,16H,6-9H2,(H,26,28)(H,27,29)

N-[3-[[2-(4-chloro-3-fluorophenoxy)acetyl]amino]-1-bicyclo[1.1.1]pentanyl]-5-(difluoromethyl)pyrazine-2-carboxamide

2BAct; 2B Act; 2143542-28-1; N-(3-(2-(4-Chloro-3-fluorophenoxy)acetamido)bicyclo[1.1.1]pentan-1-yl)-5-(difluoromethyl)pyrazine-2-carboxamide; N-{3-[2-(4-chloro-3-fluorophenoxy)acetamido]bicyclo[1.1.1]pentan-1-yl}-5-(difluoromethyl)pyrazine-2-carboxamide; 2BAct?; SCHEMBL19556524; SCHEMBL22956678; HYQJXXCYOYRNMP-UHFFFAOYSA-N; 2B-Act

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 本产品在运输和储存过程中需避光。

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

DMSO: ~250 mg/mL (~567.2 mM)

配方 1 中的溶解度: ≥ 2.08 mg/mL (4.72 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.08 mg/mL (4.72 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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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；
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