eIF2; The primary target of 2BAct is eukaryotic initiation factor 2B (eIF2B), functioning by activating the guanine nucleotide exchange factor activity of eIF2B. eIF2B is a key regulatory node in the integrated stress response (ISR) pathway—when cells are exposed to various stress stimuli, phosphorylated eIF2α inhibits eIF2B activity, leading to reduced protein synthesis. In Vanishing White Matter disease (VWM), mutations in eIF2B genes reduce its activity, causing persistent ISR activation that results in impaired myelination and neurodegeneration. 2BAct binds to eIF2B and enhances its residual activity, relieving the ISR-mediated inhibition of protein synthesis and restoring cellular function. In cell-based reporter assays, 2BAct activates eIF2B with an EC₅₀ of 33 nM. Additionally, 2BAct is able to penetrate the blood-brain barrier (unbound brain/plasma ratio ~0.3), achieving target coverage in the central nervous system.

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].

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In vitro, 2BAct demonstrates potent eIF2B activation activity. In cell-based integrated stress response (ISR) reporter assays, 2BAct activates eIF2B with an EC₅₀ of 33 nM. In primary fibroblast lysates isolated from mouse embryos carrying the severe VWM mutation Eif2b5ᴿ¹⁹¹ᴴ, 2BAct enhances the guanine nucleotide exchange factor (GEF) activity of the mutant eIF2B complex threefold with an EC₅₀ as low as 7.3 nM, demonstrating that the compound effectively activates the residual enzymatic activity even in the context of severe disease-causing mutations. These in vitro activity data provide the pharmacological basis for dose selection in subsequent in vivo studies.

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].

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2BAct demonstrates significant in vivo efficacy in a mouse model of VWM. In a 21-week blinded treatment study, VWM mice carrying the severe Eif2b5ᴿ¹⁹¹ᴴ mutation received oral 2BAct (300 μg 2BAct/g of diet), resulting in normalization of body weight gain that caught up to wild-type mice within 2 weeks of dosing initiation. 2BAct treatment also completely prevented all pathological manifestations in VWM mice, including motor deficits, myelin loss, and reactive gliosis. Transcriptomic and proteomic analyses revealed that long-term 2BAct treatment normalized both the gene expression profile and protein expression profile of VWM mice. In another more severe N208Y eIF2B-α knock-in mouse model, 2BAct treatment rescued neonatal lethality and significantly extended the lifespan of model animals. However, withdrawal studies indicate that continuous treatment is required to maintain efficacy—upon drug withdrawal, ISR induction occurs in all tissues with rapid deterioration.

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.

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Current activity assessment for eIF2B activators primarily relies on cell-based reporter assays rather than traditional cell-free enzymatic methods. A typical reporter assay protocol involves: seeding cells bearing an ISR-responsive element (e.g., ATF4 promoter-driven luciferase reporter) into 96-well plates, adding various concentrations of 2BAct (0.1 nM-10 µM), and incubating at 37°C, 5% CO₂ for 16-24 hours to induce ISR. After cell lysis, luciferase substrate is added, luminescence is measured using a chemiluminescence detector, and EC₅₀ values are calculated. Additionally, GEF activity assays in primary fibroblast lysates can be used to assess activation of the mutant eIF2B complex by 2BAct, where enzyme activity is quantified by measuring the rate of [³H]-GDP exchange from eIF2 catalyzed by eIF2B.

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.

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In vitro cellular activity of 2BAct is primarily assessed using primary fibroblast GEF activity assays and reporter-based ISR inhibition assays. In primary fibroblast assays, fibroblasts are isolated from Eif2b5ᴿ¹⁹¹ᴴ mutant mouse embryos and cultured in DMEM containing 10% FBS to 80-90% confluence. Cell lysates are prepared, incubated with various concentrations of 2BAct (e.g., 0.1-1000 nM) and [³H]-GDP-labeled eIF2, and GDP exchange rates and EC₅₀ values are calculated based on changes in [³H]-GDP binding to eIF2. In reporter gene assays, cells transfected with ISR response element reporter plasmids are seeded in 96-well plates (approximately 1×10⁴ cells per well), cultured overnight, treated with increasing concentrations of 2BAct (0.1 nM-10 µM) for 16-24 hours, lysed, and luciferase activity is measured to calculate EC₅₀ values, which are reported as 33 nM.

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.

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The in vivo efficacy of 2BAct is primarily evaluated using genetically engineered mouse models. Using the Eif2b5ᴿ¹⁹¹ᴴ VWM mouse model as an example: 6-11 week old R191H mice are randomized into treatment and control groups. 2BAct is administered orally at 300 μg/g of diet incorporated into rodent meal, a dosing regimen that ensures sustained drug exposure throughout the study period. During the 21-week treatment period, body weight changes, motor function, and clinical scores are monitored weekly. Endpoint assessments include: motor coordination evaluated by rotarod test; brain tissue collected post-mortem for Luxol fast blue staining to assess myelin integrity; GFAP and Iba-1 immunohistochemistry to evaluate astrocyte and microglial activation (reactive gliosis); and transcriptomic and proteomic analyses to assess normalization of gene and protein expression profiles. In the more severe N208Y eIF2B-α knock-in mouse model, 2BAct is also administered via diet incorporation, with endpoints including neonatal survival rate, body weight gain, and overall survival. Withdrawal studies demonstrate that upon discontinuation of 2BAct administration, rapid ISR induction and deterioration occur in all tissues, indicating that continuous treatment is essential for maintaining protein homeostasis in all tissues.

The pharmacokinetic properties of 2BAct have been characterized in rodents. The compound exhibits good oral bioavailability, with dose-dependent oral administration achievable using an aqueous-based vehicle. 2BAct is able to penetrate the blood-brain barrier to access the central nervous system (unbound brain/plasma ratio approximately 0.3), ensuring therapeutic concentrations in the brain. In the 21-week treatment study in VWM mice, the unbound brain exposure achieved via dietary administration of 2BAct exceeded the in vitro EC₅₀ by 15-fold, ensuring saturating target coverage. Compared to the first-generation eIF2B activator ISRIB, 2BAct demonstrates improved solubility and pharmacokinetic properties. Significant anomalies were observed in a dog cardiovascular safety study, limiting the feasibility of this compound for human dosing.

2BAct is generally well-tolerated in rodent studies, but cross-species safety differences limit its clinical translation potential. In VWM mice receiving long-term 2BAct treatment for up to 21 weeks, no abnormal body weight changes were observed, and animals appeared healthy overall. According to the Material Safety Data Sheet (MSDS), 2BAct is not classified as a hazardous substance or mixture and is for research use only, not intended for human diagnostic or therapeutic use. However, a critical safety concern emerged in a dog cardiovascular safety study—significant anomalies were observed in this model, rendering this particular molecule unsuitable for human dosing. This finding suggests that despite favorable safety and efficacy profiles in rodents, 2BAct exhibits significant species-dependent toxicity differences.

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

Integrated stress response (ISR) reduces protein synthesis rates and induces the expression of intracellular stress proteins. Various forms of damage activate kinases that phosphorylate the GTPase eIF2, thereby inhibiting its exchange factor eIF2B. Vulnerable white matter loss (VWM) is a neurological disorder caused by mutations in eIF2B, which, like phosphorylated eIF2, reduce eIF2B activity. We found that introducing human VWM mutations into mice leads to persistent ISR induction in the central nervous system. ISR activation precedes myelin loss and motor dysfunction. Notably, long-term treatment with the small molecule eIF2B activator 2BAct prevented changes in all pathological markers and restored the transcriptome and proteome to normal in VWM mice. 2BAct can stimulate the residual activity of the mutant eIF2B complex in vivo, thereby eliminating the maladaptive stress response. Therefore, 2BAct-like molecules may provide a promising therapeutic approach for VWM and alleviate chronic ISR induction in various disease conditions. [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

ClC1C=CC(=CC=1F)OCC(NC12CC(C1)(C2)NC(C1C=NC(C(F)F)=CN=1)=O)=O

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.)