Salubrinal, a phosphatases (PP1) inhibitor(IC50=1.7 μM), exhibits function on the eukaryotic translation initiation factor 2 subunit (eIF2α).

PP1:1.7 μM

在小鼠角膜感染的模型中,Salubrinal抑制HSV复制,降低感染动物眼拭子中病毒滴度.在脑室内给药Salubrinal,能够显著改变稳态睡眠响应.

Salubrinal（EC50=15 μM）以剂量依赖的方式抑制衣霉素诱导的ER应激,诱导细胞凋亡。Salubrinal（IC50=3 μM）通过抑制eIF2α去磷酸化,而抑制HSV复制。在C12细胞中,Salubrinal下调cyclin D1和上调GADD34和CHOP。

Phosphatase activities are determined on immunoprecipitates of the phosphatases. Briefly, 2×106 K562 cells are treated for 18 hr with Salubrinal (20 μM), PSI (10 nM), the combination of both drugs or okadaic acid (100 nM). After washing with PBS, cells are lysed for 15 min on ice either 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). Cell lysates containing 500 μg (PP1γ) or 300 μg (PP2A) protein are immunoprecipitated overnight at 4°C with 2-3 μg of the appropriate antibodies and then incubated with Protein A-Sepharose. 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 allowed to react with substrate for 1 hr at 37°C on an Eppendorf Thermoshaker, centrifuged and DiFMU fluorescence is measured on a BioTek Lambda Fluoro 320 microplate reader (360 nmex/460 nmem). Phosphatase activities are given as percent change relative to the control (DMSO treated cells)[1].

PC12 cells are plated in 384-well plates at 5000 cells per well in 40μL phenol red-freemedium containing 3μg/ml Tm to induce ER stress. 100 nL of the DiverSet E (5 mg/mlin DMSO) or National Cancer Institute's (NCI) Structural Diversity set and Open Collections (10 mM in DMSO) (NCI) are added to the wells by robotic pin transfer. After 48 hours, cell viability is assessed using a luminescence-based ATP assay. DMSO- and zVAD.fmk-treated wells on each plate served as negative and positive controls for rescue from ER stress-induced ATP loss, respectively.(Only for Reference)