GSK2606414

Kinase Assay: GST-PERK cytoplasmic domain is purchased. 6-His-full-length human eIF2α is purified from baculovirus expression in Sf9 insect cells. The eIF2α protein is buffer exchanged by dialysis into PBS, chemically modified by NHS-LC-biotin and then buffer exchanged by dialysis into 50 mM Tris, pH 7.2, 250 mM NaCl, 5 mM DTT. Protein is aliquoted and stored at −80 °C. Quench solution is freshly prepared and when added to the reaction gives final concentrations of 4 nM eIF2α phospho-ser51-antibody, 4 nM Eu-1024 labeled anti-rabbit IgG, 40 nM streptavidin Surelight APC, and 15 mM EDTA. Reactions were performed in black 384-well polystyrene low volume plates in a final volume of 10 μL. The reaction volume contains, in final concentrations, 10 mM HEPES, 5 mM MgCl₂, 5 μM ATP, 1 mM DTT, 2 mM CHAPS, 40 nM biotinylated 6-His-eIF2α, and 0.4 nM GST-PERK. Compounds under analysis are dissolved in DMSO to 1.0 mM and serially diluted 1–3 with DMSO through 11 dilutions. An amount of 0.1 μL of each concentration is transferred to the corresponding well of an assay plate. This creates a final compound concentration range from 0.00017 to 10 μM. GST-PERK solution is added to assay plates containing compounds and preincubated for 30 min at room temperature. The reaction is initiated by the addition of ATP and eIF2α substrate solution. After 1 h of incubation, the quench solution is added. The plates are covered for 2 h at room temperature prior to determination of signal. The resulting signal is quantified on a Viewlux reader. The APC signal is normalized to the europium signal by transforming the data through an APC/Eu calculation. The data for concentration response curves are plotted as % inhibition calculated with the data reduction formula 100 × [1 – (U1 – C2)/(C1 – C2)] versus concentration of compound where U is the unknown value, C1 is the average control value obtained for 1% DMSO, and C2 is the average control value obtained for 0.1 M EDTA. Data are fitted with a curve described by where A is the minimum response, B is the maximum response, D is the slope factor, x is the concentration of the compound, and C is the IC50. The results for each compound are recorded as IC50 values.

Cell Assay: In A459 cells, GSK2606414 inhibited PERK Autophosphorylation with the IC50 value of < 0.3 μM.