Salubrinal is a novel, potent, cell-permeable and selective inhibitor of eIF2 (eukaryotic translation initiation factor 2 subunit α) dephosphorylation that may have anticancer effects. With an EC50 of 15 μM in cell-free assays, it prevents ER stress-mediated apoptosis. In order to make proteins, the eukaryotic translation initiation factor 2 subunit α (eIF2α) is essential. Cells were largely shielded from apoptosis by eIF2α phosphorylation. The phosphatase complexes that dephosphorylate eIF-2α are selectively suppressed by salubrinal. By preventing the PP1/GADD34 complex from functioning, salubrinal prevents eIF2α dephosphorylation. By preventing eIF2α dephosphorylation, salubrinal blocks HSV replication with an IC50 of 3 μM.

Physicochemical Properties

Molecular Formula	C21H17CL3N4OS
Molecular Weight	479.81
Exact Mass	478.018
Elemental Analysis	C, 52.57; H, 3.57; Cl, 22.17; N, 11.68; O, 3.33; S, 6.68
CAS #	405060-95-9
Related CAS #	405060-95-9
PubChem CID	5717801
Appearance	Light brown to gray solid powder
Density	1.5±0.1 g/cm3
Index of Refraction	1.732
LogP	6.21
Hydrogen Bond Donor Count	3
Hydrogen Bond Acceptor Count	3
Rotatable Bond Count	5
Heavy Atom Count	30
Complexity	622
Defined Atom Stereocenter Count	0
SMILES	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
InChi Key	LCOIAYJMPKXARU-VAWYXSNFSA-N
InChi Code	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+
Chemical Name	(E)-3-phenyl-N-[2,2,2-trichloro-1-(quinolin-8-ylcarbamothioylamino)ethyl]prop-2-enamide
Synonyms	Salubrinal
HS Tariff Code	2934.99.9001
Storage	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.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	Dusp2; HSV-1
ln Vitro	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]
ln Vivo	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]
Enzyme Assay	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.
Cell Assay	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.
Animal Protocol	DMEM; 75μM; On cornea Eight-week-old male CD-1 outbred mice
References	[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.
Additional Infomation	Salubrinal is a member of the class of quinolines that is a mixed aminal resulting from the formal condensation oftrichloroacetaldehyde with the amide nitrogen of trans-cinnamamide and the primary amino group of 1-quinolin-8-ylthiourea. It is a selective inhibitor of cellular complexes that dephosphorylate eukaryotic translation initiation factor 2 subunit alpha (eIF2alpha). It is a member of quinolines, a member of thioureas, an aminal, an organochlorine compound and a secondary carboxamide. It is functionally related to a trichloroacetaldehyde and a trans-cinnamamide.

Solubility Data

Solubility (In Vitro)	DMSO: ~96 mg/mL (~200.1 mM) Water: <1 mg/mL (slightly soluble or insoluble) Ethanol: ~2 mg/mL (~4.2 mM)
Solubility (In Vivo)	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.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.0842 mL	10.4208 mL	20.8416 mL
	5 mM	0.4168 mL	2.0842 mL	4.1683 mL
	10 mM	0.2084 mL	1.0421 mL	2.0842 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.