eIF2

Primary-generated fibrous voids from R191H exhibit a threefold increase in 2BAct activity (EC50=7.3 nM) and a lower GEF activity than WT voids [1].

2BAct (300 μg 2BAct/g food; 21 weeks) normalizes body weight growth in VWM mice [1]. 2BAct protects motor impairments, myelin loss, and reactive neural network insomnia in VWM [1]. Residual activity of 2BAct in the near-term eIF2B complex removes maladaptive near-term responses [1].

GEF assay[1]
The experiment was performed as previously described. Briefly, Bodipy-FL-GDP-loaded eIF2 was used as a substrate for lysates generated from WT and R191H MEFs. The assay was performed in 384-well plates. In a final assay volume of 10 µL/well, the following conditions were kept constant: 25 nM Bodipy-FL-GDP-loaded eIF2, 3 nM phospho-eIF2, 0.1 mM GDP, 1 mg/mL BSA, 0.1 mg/mL MEF lysate. 2BAct was dispensed from a 1 mM stock. For each run, triplicate measurements were made for each concentration of 2BAct. Reactions were read on a SpectraMax i3x plate reader using the following instrument parameters: plate temperature = 25°C; excitation wavelength = 485 nm (15 nm width); emission wavelength = 535 nm (25 nm width); read duration = 30 mins at 45 s intervals. Data were analyzed in Prism. GDP release half-lives were calculated by fitting single-exponential decay curves. EC50s were calculated by fitting log(inhibitor) vs response curves.

Western blots [1]
Cerebellum lysates were prepared in RIPA buffer + protease/phosphatase inhibitors. Tissues were lysed in a Qiagen TissueLyser II for 2 × 2 min intervals at 30 Hz. Lysates were incubated on ice for ten minutes and centrifuged (21,000 x g, 10 min, 4°C) to remove cellular debris. Protein concentrations were determined using a Pierce BCA assay and adjusted to 2 mg/mL using RIPA buffer. Lysates were aliquoted, flash-frozen and stored at −80°C. For western blots, samples were run on Mini-PROTEAN TGX 4–20% gradient gels and transferred using Trans-Blot Turbo Mini-PVDF Transfer packs on a Trans-Blot Turbo apparatus. Membranes were blocked with 5% milk in TBS-T and incubated overnight with primary antibody in the same blocking buffer at 4°C. After three washes of 15 min each in TBS-T, HRP-conjugated secondary antibodies were applied for 1 hr. Membranes were washed in TBS-T as before. Advansta WesternBright chemiluminescent substrate was applied to the membranes and images were obtained on a Bio-Rad ChemiDoc MP imaging system in signal accumulation mode.

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ATF4-luciferase reporter assay[1]
The experiment was performed as previously described (Wong et al., 2018). Briefly, HEK293T cells expressing an ATF4-luciferase reporter (Sidrauski et al., 2013) were seeded into 96-well plates and treated with 100 nM thapsigargin for 7 hr to induce ER stress. Cells were co-treated with 2BAct or ISRIB in dose response. Luminescence was measured using ONE-Glo Luciferase assay reagent (Promega) and a Molecular Devices SpectraMax i3x plate reader. Data were analyzed in Prism.

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

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

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.72 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (4.72 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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