EIF2AK3 (PERK) (IC50 = 0.4 nM); EIF2AK1 (HRI) (IC50 = 420 nM); EIF2AK2 (PKR) (IC50 = 696 nM)

GSK2606414 inhibits PERK activation in cells[1].
GSK2606414 inhibited PK4 alone, with no effect on the other two Plasmodium eIF2α kinases that respond to alternative stress signals (Figure S6). Importantly, GSK2606414 inhibited Plasmodium eIF2α phosphorylation and abolished recrudescence post-ART therapy (Figure 5). Since the ER, and hence PERK, are absent in mature erythrocytes, GSK2606414 is most likely exerting its activity through the parasite. [2]

GSK2606414 shows low to moderate blood clearance and high oral availability in mouse, rat, and dog. Oral administration of GSK2606414 inhibits tumor growth in mice with pancreatic human BxPC3 tumors in a dose-dependent manner. [1]
Moreover, compound 38 (GSK2606414) was advanced to a human xenograft efficacy study in mice and demonstrated that small molecule inhibition of PERK can inhibit tumor growth in vivo, consistent with the slow growth of tumors derived from transformed PERK–/– mouse embryonic fibroblasts. Further optimization offering improvements to the physical properties and pharmacokinetics of 38 will be the subject of a future communication.[1]

The cytoplasmic domain of GST-PERK is bought. 6-Human eIF2α full-length is isolated from baculovirus expression in Sf9 insect cells. The eIF2α protein is buffer exchanged by dialysis into PBS, followed by chemical modification with NHS-LC-biotin and buffer exchanged into 50 mM Tris, pH 7.2, 250 mM NaCl, and 5 mM DTT. Aliquoted protein is kept at -80°C for storage. When the freshly prepared quench solution is added to the reaction, the final concentrations of 4 nM eIF2α phospho-ser51-antibody, 4 nM anti-rabbit IgG labeled with Eu-1024, 40 nM streptavidin Surelight APC, and 15 mM EDTA are obtained. At a final volume of 10 μL, reactions were carried out in black 384-well polystyrene low volume plates. 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 are all present in the reaction volume in final concentrations. The compounds being analyzed are dissolved in 1.0 mM DMSO and then serially diluted 1–3 with DMSO over the course of 11 dilutions. For every concentration, 0.1 μL is added to the appropriate well in an assay plate. This results in a final compound concentration range of 10 μM to 0.00017 μM. Assay plates containing compounds are preincubated for 30 minutes at room temperature before receiving the GST-PERK solution. The addition of ATP and the eIF2α substrate solution starts the reaction. The quench solution is added after an hour of incubation. Before the signal is determined, the plates are covered and left at room temperature for two hours. Using a Viewlux reader, the resulting signal is quantified. An APC/Eu calculation is used to transform the data and normalize the APC signal to the europium signal. The data reduction formula 100 × [1 – (U1 – C2)/(C1 – C2)] is used to plot the percentage inhibition against the compound concentration for concentration response curves. In this formula, U represents the unknown value, C1 represents the average control value obtained for 1% DMSO, and C2 represents the average control value obtained for 0.1 M EDTA. A, B, C, D, and x represent the compound concentration, IC50, slope factor, and minimum and maximum responses, respectively, on a curve that fits the data. As IC50 values, the outcomes are noted for every compound.

Thapsigargin was used to stimulate the cells after PERK inhibitor (GSK2606414, compound 38) incubation. The cells were lysed and examined for PERK autophosphorylation inhibition after two hours.
PERK Phosphorylation in A549 Cells[1]
Exponentially growing A549, a human lung adenocarcinoma cell line, was plated at 500 000 cells/well in a six-well dish in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and incubated overnight at 37 °C, 5% CO2. The following morning, cells were treated with DMSO or compound (3-fold dilutions 0.33, 0.11, 0.37 μM) and incubated as described above for 1 h. Cells were then stimulated with 1 μM thapsigargin for an additional hour to induce ER stress. Cells pellets were collected and lysed in cold RIPA buffer [150 mM NaCl, 50 mM Tris-HCl, pH 7.5, 0.25% sodium deoxycholate, 1% NP-40, protease and phosphatase inhibitors, and 100 mM sodium orthovanadate]. Clarified lysates were resolved by SDS–PAGE and transferred to nitrocellulose membrane using Invitrogen’s NuPAGE system. Blots were incubated with primary antibodies to total PERK and total eIF-2α. IRDye700DX-labeled goat anti-mouse IgG and IRDye800-CW donkey anti-goat IgG were used as secondary antibodies. Proteins were detected on the Odyssey infrared imager

Female nude mice have their right flank subcutaneously implanted with exponentially growing BxPC3 tumor cells (10×10⁶ cells/mouse) cultivated in cell culture. Mice with tumors measuring less than 200 mm³ are randomly assigned to one of eight treatment groups sixteen days post-implantation. The animals are given the compound at 50 or 150 mg/kg, b.i.d., for 21 days along with a vehicle (0.5% hydoxypropylmethylcellulose, 0.1% Tween 80 in water, pH 4.8). Using calipers, the tumor volume is measured twice a week and computed. When the dosage is finished, the results are expressed as a percent inhibition, or 100[1-(average growth of the drug-treated population)/(average growth of the vehicle-treated control population)]. In statistical analysis, the two-tailed t test is employed.
Pharmacokinetic Studies[1]
All studies were conducted after review by the Institutional Animal Care and Use Committee at GSK and in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals. Pharmacokinetics were studied in male Sprague–Dawley rats, male beagle dogs, and/or male CD-1 mice following single intravenous and/or oral administration. Absolute oral bioavailability was estimated using a crossover study design (n of 2 or 3), unless otherwise indicated. Blood samples were assayed using protein precipitation followed by LC/MS/MS analysis, and the resulting concentration–time data were analyzed by noncompartmental methods (WinNonlin Professional, version 4.1).[1]
BxPC3 Human Pancreatic Xenograft Model[1]
All studies were conducted after review by the Institutional Animal Care and Use Committee at GSK and in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals. Exponentially growing BxPC3 tumor cells (10 × 106 cells/mouse) from cell culture were implanted subcutaneously into the right flank of female nude mice (Charles River). Sixteen days after implantation, mice with ∼200 mm3 tumors were randomized into various treatment groups (n = 8 mice/group). Animals were orally treated with vehicle (0.5% hydoxypropylmethylcellulose, 0.1% Tween 80 in water, pH 4.8), compound 38 at 50 or 150 mg/kg, b.i.d. for 21 days. Tumor volume was measured twice weekly with calipers and calculated using the following equation: tumor volume (mm3) = (length × width)2/2. Results are represented as percent inhibition on completion of dosing, which is 100[1 – (average growth of drug-treated population)/(average growth of vehicle-treated control population)]. Statistical analysis was performed using a two-tailed t test.

[1]. Discovery of 7-methyl-5-(1-{[3-(trifluoromethyl)phenyl]acetyl}-2,3-dihydro-1H-indol-5-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine (GSK2606414), a potent and selective first-in-class inhibitor of protein kinase R (PKR)-like endoplasmic reticulu.

[2]. Inhibiting the Plasmodium eIF2α Kinase PK4 Prevents Artemisinin-Induced Latency. Cell Host Microbe. 2017 Dec 13;22(6):766-776.e4.

See also: Gsk2606414 (annotation moved to).

C24H20F3N5O

451.44

451.161

C, 63.85; H, 4.47; F, 12.63; N, 15.51; O, 3.54

1337531-36-8

53469448

white solid powder

1.4±0.1 g/cm3

720.3±60.0 °C at 760 mmHg

389.4±32.9 °C

0.0±2.3 mmHg at 25°C

1.668

4.53

1

7

3

33

721

0

FC(C1=C([H])C([H])=C([H])C(=C1[H])C([H])([H])C(N1C2C([H])=C([H])C(C3=C([H])N(C([H])([H])[H])C4C3=C(N([H])[H])N=C([H])N=4)=C([H])C=2C([H])([H])C1([H])[H])=O)(F)F

SIXVRXARNAVBTC-UHFFFAOYSA-N

InChI=1S/C24H20F3N5O/c1-31-12-18(21-22(28)29-13-30-23(21)31)15-5-6-19-16(11-15)7-8-32(19)20(33)10-14-3-2-4-17(9-14)24(25,26)27/h2-6,9,11-13H,7-8,10H2,1H3,(H2,28,29,30)

1-[5-(4-amino-7-methylpyrrolo[2,3-d]pyrimidin-5-yl)-2,3-dihydroindol-1-yl]-2-[3-(trifluoromethyl)phenyl]ethanone

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.54 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: 2.5 mg/mL (5.54 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (5.54 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 4: in 5% DMSO+45% PEG 300+ddH2O: 20mg/mL

---

Solubility in Formulation 5: 3.33 mg/mL (7.38 mM) in 0.5% HPMC 0.2%Tween80 (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.

---

 (Please use freshly prepared in vivo formulations for optimal results.)