GSK2606414

PKR-like endoplasmic reticulum kinase (PERK) assay (HTRF Format)

GST-PERK cytoplasmic domain was purchased. 6-His-full-length human eIF2α was purified from baculovirus expression in Sf9 insect cells. The eIF2α protein was 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 was aliquoted and stored at -80°C. Quench solution was 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 were 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 was 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 was added to assay plates containing compounds and preincubated for 30 mins at room temperature. The reaction was initiated by the addition of ATP and eIF2α substrate solution. After 1 hr of incubation, the quench solution was added. The plates were covered for 2 hrs at room temperature prior to determination of signal. The resulting signal was quantified on a Viewlux reader. The APC signal is normalized to the europium signal by transforming the data through an APC/Eu calculation. The results for each compound were recorded as IC50 values.

Cell lines

A459 cells

Preparation method

The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20℃ for several months.

Reaction Conditions

0.003, 0.1 and 0.3 μM; 2 hrs

Applications

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

Animal models

BxPC3 human pancreatic xenograft model

Dosage form

~ 150 mg/kg; p.o.

Applications

In mice, rats and dogs, GSK2606414 exhibited high oral availability, and low to moderate blood clearance. In mice bearing pancreatic human BxPC3 tumors, GSK2606414 inhibited tumor growth in a dose-dependent manner.

Other notes

Please test the solubility of all compounds indoor, and the actual solubility may slightly differ with the theoretical value. This is caused by an experimental system error and it is normal.

References:

[1]. Axten JM, Medina JR, Feng Y, Shu A, Romeril SP, Grant SW, Li WH, Heerding DA, Minthorn E, Mencken T et al: Discovery of 7-methyl-5-(1-{[3-(trifluoromethyl)phenyl]acetyl}-2,3-dihydro-1H-indol-5-yl)-7H-p yrrolo[2,3-d]pyrimidin-4-amine (GSK2606414), a potent and selective first-in-class inhibitor of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK). J Med Chem, 55(16):7193-7207.