Sal003, an effective cell-permeable analog, inhibitis the eIF2α phosphatase.

In mouse embryonic fibroblasts, Sal003 induced depolymerization of polysaccharides by increasing the phosphorylation of eIF2a. Sal003 increased eIF2a phosphorylation by blocking eIF2a phosphodiesterase activity, and Sal003 enhanced the apoptotic signaling pathway induced by cytotoxicity by induced phosphorylation of eIF2α.

In mouse embryonic fibroblasts, Sal003 induced depolymerization of polysaccharides by increasing the phosphorylation of eIF2a. Sal003 increased eIF2a phosphorylation by blocking eIF2a phosphodiesterase activity, and Sal003 enhanced the apoptotic signaling pathway induced by cytotoxicity by induced phosphorylation of eIF2α.

Catalytic assay: MEK5 protein isolated from the baculovirus expression system is used to measure kinase activity utilizing PKLight ATP Detection Reagent. The assay is performed using 15 nM GST-MEK5 and 0.75 μM ATP in assay buffer consisting of 25 mM Hepes, pH 7.5, 10 mM MgCl2, 50 mM KCl, 0.2% BSA, 0.01% CHAPS, 100 μM Na3VO4, 0.5 mM DTT and 1% DMSO in the presence of varying concentrations of BIX02189. The kinase reaction mixture is incubated for 90 minutes at room temperature followed by addition of 10 μL of ATP detection reagent for 15 minutes. The relative light unit (RLU) signal is measured and the RLU signals are converted to percent of control (POC) values for the determination of IC50 value.