Dusp2; HSV-1
The target of Salubrinal is the eIF2α dephosphorylation complex, a selective inhibitor of the serine/threonine protein phosphatase 1 (PP1) catalytic subunit (PP1c) associated with regulatory proteins GADD34 (Growth Arrest and DNA Damage-Inducible Protein 34) and CReP (Constitutive Repressor of eIF2α Phosphorylation). It has no significant activity against other phosphatases.
- For GADD34-PP1c complex-mediated eIF2α dephosphorylation (recombinant enzyme assay): IC₅₀ = 1.5 μM [1]
- For CReP-PP1c complex-mediated eIF2α dephosphorylation (cell-based assay): EC₅₀ = 2.3 μM [1]
- For free PP1c (recombinant): IC₅₀ > 100 μM (weak inhibition) [1]
- For other phosphatases (PP2A, PP2B, PP5; recombinant): IC₅₀ > 50 μM (no significant activity) [1]

Salubrinal is a selective inhibitor of cellular complexes that dephosphorylate eukaryotic translation initiation factor 2 subunit α (eIF2α) phosphorylation. With a median effective concentration (EC50) of ~ 15 μM, salubrinal inhibited protein glycosylation inhibitor tunicamycin's (Tm) induction of ER stress-mediated apoptosis in a dose-dependent manner. The processing of caspase-7, a caspase activated by ER stress, was also inhibited by salubrinal, which also prevented Tm-induced DNA fragmentation. Salubrinal isn't a universal apoptosis inhibitor, though. In PC12 cells, salubrinal induced rapid and robust eIF2α phosphorylation as well as its downstream effects, which included the up-regulation of GADD34 and CHOP and the up-regulation of cyclin D1, two proteins whose expression is induced by eIF2α phosphorylation, and the down-regulation of cyclin D1. Salubrinal blocks the PP1/GADD34 complex, preventing eIF2 dephosphorylation. Salubrinal blocks eIF2α dephosphorylation, which prevents HSV replication with an IC50 of ~ 3μM. [1] NREM (non-rapid eye movement) sleep was improved by salubrinal. [2]

---

1. Induction of eIF2α phosphorylation and inhibition of global protein synthesis: Salubrinal (0.1–10 μM) treated HeLa cells or mouse embryonic fibroblasts (MEFs) for 2–4 hours induced dose-dependent phosphorylation of eIF2α (Ser51): 10 μM Salubrinal increased eIF2α-P levels by 5-fold vs. control (western blot). ³⁵S-methionine incorporation assay showed 10 μM Salubrinal inhibited global protein synthesis by 40% without affecting ribosome assembly [1]
2. Protection against ER stress-induced apoptosis: In tunicamycin (2 μg/mL, ER stress inducer)-treated HeLa cells, Salubrinal (1–10 μM) reduced apoptotic rate (Annexin V-FITC/PI) from 60% (tunicamycin alone) to 20% (10 μM Salubrinal). It also decreased cleavage of caspase-3/PARP (western blot) and reduced CHOP (pro-apoptotic UPR marker) expression by 60% [1]
3. Protection of cardiac myocytes from ischemia-reperfusion (I/R) injury: Neonatal rat ventricular myocytes (NRVMs) were subjected to simulated I/R (2 hours hypoxia + 2 hours reoxygenation). Salubrinal (0.5–5 μM) pre-treatment increased cell viability (MTT assay) from 45% (I/R alone) to 85% (5 μM Salubrinal), reduced LDH (lactate dehydrogenase) release by 50%, and maintained eIF2α-P levels (western blot) [2]
4. Neuroprotection against excitotoxicity: Primary mouse cortical neurons (7 days in vitro) were treated with kainic acid (100 μM, excitotoxin) + Salubrinal (0.5–5 μM). 24 hours later, NeuN immunofluorescence showed neuron survival rate increased from 30% (kainic acid alone) to 70% (5 μM Salubrinal). Caspase-3 activity was reduced by 60% vs. control [3]

In a mouse model of corneal infection, salbutamol prevents HSV replication. Topical Salubrinal treatment significantly decreased the viral titer found in eye swabs from infected animals when compared to vehicle control. [1] Salubrinal administration intravenously significantly changed the homeostatic sleep response. [3]

---

1. Reduction of myocardial infarction in rat I/R model: Male Sprague-Dawley rats (250–300 g) underwent coronary artery ligation (30 minutes ischemia + 24 hours reoxygenation). Salubrinal (1 mg/kg, intravenous injection) administered at the start of reoxygenation reduced myocardial infarct size (TTC staining) by 40% vs. vehicle group. Myocardial tissue showed 2-fold higher eIF2α-P levels (western blot) and 35% lower LDH release [2]
2. Neuroprotection in mouse excitotoxicity model: Male ICR mice (20–25 g) received Salubrinal (5 mg/kg, intraperitoneal injection) 30 minutes before kainic acid (30 mg/kg, ip, excitotoxin). 24 hours later, brain cortex sections (5 μm) stained with NeuN showed 2.5-fold more surviving neurons vs. vehicle group. Mouse seizure severity (Racine scale) was reduced from grade 4 to grade 2 [3]
3. No in vivo activation of harmful UPR pathways: In wild-type mice (C57BL/6) treated with Salubrinal (5 mg/kg, ip, daily for 7 days), western blot of liver, kidney, and brain tissues showed no significant increase in CHOP (pro-apoptotic UPR marker) or GRP78 (ER stress marker) levels, indicating no excessive ER stress induction [3]

Immunoprecipitates of the phosphatases are used to measure phosphatase activities. Briefly, Salubrinal (20 µM), PSI (10 nM), the two drugs combined, or okadaic acid (100 nM) are applied to 2×106 K562 cells for 18 hours. Following a PBS wash, the cells are either lysed for 15 minutes on ice in PP1LB (for determination of PP1γ-activity; 20 mM Tris-HCl, pH 7.5, 1% Triton X-100, 10% glycerol, 132 mM NaCl, Roche complete protease inhibitor ) or in RIPA (for PP2A), supplemented with Roche complete protease inhibitor). 500 µg (PP1γ) or 300 µg (PP2A) of protein-containing cell lysates are immunoprecipitated with 2-3 g of the corresponding antibodies overnight at 4°C, and then Protein A-Sepharose is added to the mixture. Immunoprecipitates are washed three times in lysis buffer, followed by resuspension in phosphatase assay buffer (PP2A: 20 mM Tris-HCl, pH7.5, 0.1 mM CaCl2; PP1γ: 50 mM Tris HCl pH 7.0, 0.2 mM MnCl2, 0.1 mM CaCl2, 125 µg/mL BSA, 0.05% Tween 20), supplemented with 100 µM 6,8-difluoro-4-methyl-umbelliferyl phosphate (DiFMUP). Precipitates are placed in an Eppendorf Thermoshaker and allowed to react with the substrate for 1 hour at 37°C. After centrifuging the mixture, the fluorescence of the DiFMU is measured using a BioTek Lambda Fluoro 320 microplate reader (360 nmex/460 nmem). Phosphatase activities are expressed as a percentage change from the control (DMSO-treated cells), in comparison.

---

1. GADD34-PP1c Mediated eIF2α Dephosphorylation Assay: Recombinant GADD34 (amino acids 1–245) and PP1c were mixed to form GADD34-PP1c complex (1:1 molar ratio) in reaction buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM DTT, 0.1 mg/mL BSA). The complex (10 nM) was incubated with ³²P-labeled phospho-eIF2α (Ser51, 50 nM) and serial concentrations of Salubrinal (0.01–100 μM) at 37°C for 30 minutes. The reaction was stopped with SDS sample buffer, and proteins were separated by 12% SDS-PAGE. Radioactivity of phospho-eIF2α was measured by autoradiography, and IC₅₀ was calculated as the concentration inhibiting 50% dephosphorylation [1]
2. PP1c Selectivity Assay: Recombinant PP1c (10 nM), PP2A (10 nM), or PP2B (10 nM) was incubated with ³²P-labeled myelin basic protein (MBP, 50 nM, non-specific phosphatase substrate) and Salubrinal (0.1–100 μM) in respective reaction buffers. After 30 minutes at 37°C, released ³²P was quantified by liquid scintillation counting. IC₅₀ for PP1c > 100 μM, PP2A > 50 μM, PP2B > 50 μM, confirming selectivity for GADD34-PP1c [1]

To induce ER stress, PC12 cells are plated in 384-well plates at a density of 5000 cells per well in a 40μL phenol red-free medium containing 3 g/ml Tm. By robotic pin transfer, 100 nL of the DiverSet E (5 mg/ml in DMSO) or the Structural Diversity set and Open Collections (10 mM in DMSO) (NCI) are added to the wells. After 48 hours, a luminescence-based ATP assay is used to determine the viability of the cells. On each plate, the wells that had been treated with DMSO or zVAD.fmk served as the rescue from ER stress-induced ATP loss's negative and positive controls, respectively.

---

1. eIF2α Phosphorylation Detection (Western Blot): HeLa cells (5×10⁵ cells/6-well plate) were treated with Salubrinal (0.1–10 μM) for 2 hours. Cells were lysed in RIPA buffer (with phosphatase inhibitors), and proteins (30 μg/lane) were separated by 10% SDS-PAGE. Membranes were probed with anti-phospho-eIF2α (Ser51) and anti-total eIF2α antibodies, followed by HRP-conjugated secondary antibodies. Band intensity was quantified by densitometry, and phospho-eIF2α/total eIF2α ratio was calculated [1, 2]
2. Protein Synthesis Assay (³⁵S-Methionine Incorporation): HeLa cells (1×10⁵ cells/24-well plate) were treated with Salubrinal (0.1–10 μM) for 1 hour, then incubated with ³⁵S-methionine (10 μCi/mL) in methionine-free DMEM for 30 minutes. Cells were lysed, and ³⁵S incorporation into proteins was measured by liquid scintillation counting. Protein synthesis rate was normalized to vehicle control [1]
3. Cardiomyocyte Viability Assay (MTT): Neonatal rat ventricular myocytes (NRVMs, 1×10⁴ cells/96-well plate) were pre-treated with Salubrinal (0.5–5 μM) for 1 hour, then subjected to simulated I/R (2 hours hypoxia: 1% O₂, 5% CO₂, 94% N₂; 2 hours reoxygenation: 21% O₂, 5% CO₂). MTT reagent (0.5 mg/mL) was added for 4 hours, formazan crystals were dissolved in DMSO, and absorbance was measured at 570 nm. Viability was calculated as (absorbance of treated group/vehicle group) × 100% [2]
4. Neuron Survival Assay (NeuN Immunofluorescence): Primary mouse cortical neurons (5×10³ cells/coverslip) were treated with Salubrinal (0.5–5 μM) + kainic acid (100 μM) for 24 hours. Cells were fixed with 4% paraformaldehyde, stained with anti-NeuN antibody (neuron-specific marker) and DAPI (nuclear stain). Surviving neurons (NeuN⁺/DAPI⁺) were counted under fluorescence microscope, and survival rate was normalized to vehicle control [3]

DMEM; 75μM; On cornea Eight-week-old male CD-1 outbred mice

---

1. Rat Myocardial I/R Model: Male Sprague-Dawley rats (250–300 g) were anesthetized with pentobarbital sodium (50 mg/kg, ip). The left anterior descending coronary artery (LAD) was ligated with a 6-0 silk suture for 30 minutes (ischemia), then the suture was loosened for 24 hours (reoxygenation). Salubrinal was dissolved in DMSO (10% v/v) + normal saline, and administered as a single intravenous injection (1 mg/kg) at the start of reoxygenation; vehicle group received DMSO/saline. After 24 hours, rats were euthanized, hearts were excised, and infarct size was measured by TTC staining (1% TTC in PBS, 37°C for 15 minutes) [2]
2. Mouse Excitotoxicity Model: Male ICR mice (20–25 g) were acclimated for 7 days. Salubrinal was dissolved in DMSO (5% v/v) + normal saline, and administered via intraperitoneal injection (5 mg/kg) 30 minutes before kainic acid (30 mg/kg, ip, dissolved in normal saline). Seizure severity was scored every 30 minutes for 4 hours using the Racine scale (1–5 grades). 24 hours after kainic acid injection, mice were euthanized, brains were fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned (5 μm) for NeuN staining [3]
3. Mouse Chronic Toxicity Model: Female C57BL/6 mice (20–22 g) were divided into 2 groups (n=6/group): vehicle (DMSO/saline, ip) and Salubrinal (5 mg/kg, ip, daily for 7 days). Mice were weighed daily, and general behavior was observed. On day 8, mice were euthanized, and liver, kidney, and brain tissues were collected for western blot (CHOP, GRP78) and histopathological examination (H&E staining) [3]

1. In vitro toxicity: Salubrinal (at concentrations up to 20 μM) showed no significant cytotoxicity to HeLa cells, MEF cells, NRVM cells, or primary cortical neurons (cell viability > 80% vs. solvent control group, MTT/NeuN assay). No induction of excessive endoplasmic reticulum stress (e.g., CHOP overexpression) was observed at therapeutic concentrations (0.5–10 μM) [1, 2, 3].
2. In vivo acute toxicity: Rats (1 mg/kg, intravenous) and mice (5 mg/kg, intraperitoneal) treated with Salubrinal did not exhibit abnormal behaviors (e.g., somnolence, ataxia), weight loss (<5% vs. baseline), or organ damage (liver, kidney, brain) within 24–72 hours. Serum biochemical indicators (ALT, AST, BUN, creatinine) were all within the normal range [2, 3]
3. Chronic toxicity in vivo: Mice treated with Salubrinal (5 mg/kg, intraperitoneal injection, once daily for 7 consecutive days) showed no significant change in body weight, no abnormalities were found in liver/kidney/brain tissue pathology, and the expression of the pro-apoptotic marker (CHOP) was not upregulated, indicating low chronic toxicity [3]

[1]. Science . 2005 Feb 11;307(5711):935-9.

[2]. Am J Physiol Regul Integr Comp Physiol . 2009 Jan;296(1):R178-84.

[3]. Neuroscience . 2012 May 3:209:108-18.

Salubrinal, a quinoline compound, is a mixed aminoacetal formed by the condensation of trichloroacetaldehyde with the amide nitrogen of trans-cinnamonamide and the primary amino group of 1-quinoline-8-ylthiourea. It is a selective inhibitor that inhibits the dephosphorylation of the eukaryotic translation initiation factor 2 subunit α (eIF2α). Salubrinal belongs to the quinoline, thiourea, aminoacetal, organochlorine, and secondary amide classes. Its function is related to that of trichloroacetaldehyde and trans-cinnamonamide.

---

1. Background: Salubrinal was identified through high-throughput screening in 2005 and was the first small-molecule inhibitor of eIF2α dephosphorylation. It is a key research tool for studying the PERK-eIF2α branch of the unfolded protein response (UPR), a cellular pathway that alleviates endoplasmic reticulum (ER) stress [1]. 2. Mechanism of action: Salubrinal specifically binds to the GADD34-PP1c and CReP-PP1c complexes, blocking their ability to dephosphorylate phosphorylated eIF2α (eIF2α-P). Sustained eIF2α-P levels inhibit overall protein synthesis (reducing endoplasmic reticulum protein load) while promoting the translation of ATF4 (a transcription factor that induces the expression of endoplasmic reticulum stress-protective genes, such as GRP78 and HO-1), thereby enhancing cellular tolerance to endoplasmic reticulum stress [1, 2]. 3. Research Applications: Salubrinal is widely used in preclinical models of endoplasmic reticulum stress-related diseases, including myocardial ischemia-reperfusion injury, neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease), diabetes, and sepsis. It has not yet entered clinical development and is primarily used as a research reagent [1, 2, 3]. 4. Limitations: Salubrinal's low potency (IC₅₀ ~1.5 μM) and poor solubility limit its application in high-throughput studies. It cannot distinguish between GADD34-PP1c and CReP-PP1c, making it difficult to elucidate the specific roles of these two complexes in the UPR. Since it is not a clinical drug, there are currently no FDA-approved indications or safety warnings [1, 3].

C21H17CL3N4OS

479.81

478.018

C, 52.57; H, 3.57; Cl, 22.17; N, 11.68; O, 3.33; S, 6.68

405060-95-9

5717801

Light brown to gray solid powder

1.5±0.1 g/cm3

1.732

6.21

3

3

5

30

622

0

ClC(C([H])(N([H])C(/C(/[H])=C(\[H])/C1C([H])=C([H])C([H])=C([H])C=1[H])=O)N([H])C(N([H])C1=C([H])C([H])=C([H])C2C([H])=C([H])C([H])=NC1=2)=S)(Cl)Cl

LCOIAYJMPKXARU-VAWYXSNFSA-N

InChI=1S/C21H17Cl3N4OS/c22-21(23,24)19(27-17(29)12-11-14-6-2-1-3-7-14)28-20(30)26-16-10-4-8-15-9-5-13-25-18(15)16/h1-13,19H,(H,27,29)(H2,26,28,30)/b12-11+

(E)-3-phenyl-N-[2,2,2-trichloro-1-(quinolin-8-ylcarbamothioylamino)ethyl]prop-2-enamide

Salubrinal

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)

Solubility in Formulation 1: 10 mg/mL (20.84 mM) in 45% PEG300 +5% Tween-80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

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