GSK2606414 is a selective inhibitor of PERK with IC50 value of 0.4nM.[1]
PERK (PRKR-like endoplasmic reticulum kinase or protein kinase R (PKR)-like endoplasmic reticulum kinase) is also known as EIF2AK3 (Eukaryotic translation initiation factor 2-alpha kinase 3). PERK is encoded by EIF2AK3 gene. It belongs to? type I membrane protein family. It located in the ER (endoplasmic reticulum) and is induced by ER stress which is caused from malfolded proteins. It causes the inactive of EIF2 (eukaryotic translation-initiation factor 2) by phosphorylating the alpha subunit. PERK can lead to repression of global protein synthesis and a rapid reduction of translational initiation. PERK has been identified to interact with NFE2L2 and DNAJC3. PERK mutation is related to WRS (Wolcott-Rallison syndrome) which is a autosomal recessive disorder with multiple epiphyseal dysplasia,infancy-onset diabetes mellitus, osteopenia, mental retardation, and hepatic and renal dysfunction. [2]
GSK2606414 inhibits the activity of PERK with IC50 of 0.4 nM by measuring the cytoplasmic PERK domain phosphorylation. GSK2606414 directly bind to PERK as measured in X-ray structure. GSK2606414 completely inhibit PERK phosphorylation at 30 nM in A549 cells. GSK2606414 has selective inhibition effect on PERK compared to a panel of 294 kinases. There only 20 protein kinases except PERK inhibited >85% by GSK2606414 at 10 μM. GSK2606414 inhibited tumor growth with a dose-dependent manner from 50 to 150 mg/kg in mice bearing pancreatic human BxPC3 tumors.[1]
GSK2606414是一种选择性PERK抑制剂，其IC50值为0.4nM[1]。 PERK（PRKR-like endoplasmic reticulum kinase或蛋白质激酶R（PKR）-like endoplasmic reticulum kinase）也称为EIF2AK3（真核翻译起始因子2α激酶3）。PERK由EIF2AK3基因编码。它属于I型跨膜蛋白家族，位于内质网（ER）中，受由不良折叠蛋白引起的ER应激诱导。它通过磷酸化α亚单位来使EIF2（真核翻译起始因子2）失活。PERK会导致全局蛋白质合成的抑制和翻译起始的快速降低。PERK已被鉴定为与NFE2L2和DNAJC3相互作用。PERK突变与WRS（Wolcott-Rallison综合征）相关，该综合征是一种常染色体隐性遗传疾病，伴有多发性肢骨发育不良、婴儿期发病的糖尿病、骨量减少、智力障碍以及肝肾功能障碍[2]。
GSK2606414通过测量胞质PERK域的磷酸化来抑制PERK活性，其IC50值为0.4 nM。GSK2606414可直接结合PERK，这一点可以通过X射线结构测定来证实。在A549细胞中，GSK2606414在30 nM时可以完全抑制PERK磷酸化。GSK2606414对294种蛋白激酶进行筛选，只有除PERK外的20种蛋白激酶在10 μM时被抑制了>85%。GSK2606414以剂量依赖的方式抑制胰腺人BxPC3肿瘤小鼠的肿瘤生长，剂量范围为50至150 mg/kg[1]。
参考文献:
[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.
[2].?? ?Shi Y, An J, Liang J, Hayes SE, Sandusky GE, Stramm LE, Yang NN: Characterization of a mutant pancreatic eIF-2alpha kinase, PEK, and co-localization with somatostatin in islet delta cells. J Biol Chem 1999, 274(9):5723-5730.
| Kinase experiment [1]: | |
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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 experiment [1]: | |
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Cell lines |
A459 cells |
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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. |
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Reaction Conditions |
0.003, 0.1 and 0.3 μM; 2 hrs |
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Applications |
In A459 cells, GSK2606414 inhibited PERK Autophosphorylation with the IC50 value of < 0.3 μM. |
| Animal experiment [1]: | |
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Animal models |
BxPC3 human pancreatic xenograft model |
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Dosage form |
~ 150 mg/kg; p.o. |
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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. |
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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. |
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参考文献: [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. | |
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