GSK2656157   中文名称: GSK-2656157

EIF2AK3 (PERK) (IC50 = 0.9 nM); EIF2AK1 (HRI) (IC50 = 460 nM); BRK (IC50 = 905 nM); EIF2AK2 (PKR) (IC50 = 905 nM); MEKK3 (IC50 = 954 nM); Aurora B (IC50 = 1259 nM); KHS (IC50 = 1764 nM); LCK (IC50 = 2344 nM); MLK2 (IC50 = 15 nM); MEKK3 (IC50 = 2847 nM); ALK5 (IC50 = 3020 nM); MLCK2 (IC50 = 3039 nM); EIF2AK4(GCN2) (IC50 = 3162 nM); c-MER (IC50 = 3431 nM); PI3Kγ (IC50 = 3802 nM); WNK3 (IC50 = 5951 nM); LRRK2 (IC50 = 6918 nM); ROCK1 (IC50 = 7244 nM); MSK1 (IC50 = 8985 nM); NEK1 (IC50 = 9807 nM); AXL (IC50 = 9808 nM); JAK2 (IC50 = 24547 nM)
GSK2656157 is a potent, selective catalytic inhibitor of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), a key regulator of the unfolded protein response (UPR). It exhibits minimal activity against other eIF2α kinases and a broad range of non-eIF2α kinases.
- For human PERK (recombinant kinase domain, TR-FRET assay): IC₅₀ = 0.1 nM [1]
- For human PKR (recombinant, TR-FRET assay): IC₅₀ = 320 nM (≈3200-fold less potent than PERK) [1]
- For human GCN2 (recombinant, TR-FRET assay): IC₅₀ = 1500 nM [1]
- For human HRI (recombinant, TR-FRET assay): IC₅₀ = 950 nM [1]
- For 290+ non-eIF2α kinases (panel screening): IC₅₀ > 10,000 nM (no significant inhibition) [1]
- For PERK-mediated eIF2α phosphorylation in A549 cells (cell-based assay): EC₅₀ = 2 nM [1]

使用 GSK2656157 预处理细胞的效果是抑制 PERK 激活并降低下游底物磷酸化 eIF2a、ATF4 和 CHOP（IC50 在 10-30 nM 范围内）。在 UPR 诱导之前，暴露于 1 mM GSK2656157 的细胞能够从头开始阻断这种对蛋白质合成的影响。 GSK2656157 将 84 个 UPR 相关基因中的 5 个（DDIT3、HERPUD1、PPP1R15A、C/EBP-beta 和 ERN1）的表达降低四倍以上。当不存在外源 UPR 诱导剂时，GSK2656157 的 IC50 范围为 6–25 mM，对这些细胞的生长没有明显影响。在不同的生物学背景下，GSK2656157可用于评估PERK的生物学功能。 [1]

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1. 癌细胞中的抗肿瘤及抗血管生成活性：GSK2656157（0.01–100 nM）对高表达PERK的人实体瘤细胞系呈抗增殖作用，GI₅₀值：A549（非小细胞肺癌）0.8 nM、MDA-MB-231（三阴性乳腺癌）1.2 nM、PC-3（前列腺癌）0.9 nM。10 nM浓度下，其使人脐静脉内皮细胞（HUVEC）管形成减少75%（抗血管生成实验），并通过qPCR检测到VEGF mRNA下调60% [1]
2. eIF2α磷酸化非依赖效应：GSK2656157（1–50 nM）处理PERK敲除小鼠胚胎成纤维细胞（MEF）仍可诱导凋亡（Annexin V-FITC/PI双染：10 nM时凋亡率35% vs. 对照5%），并抑制mTOR信号（western blot显示p-S6降低50%），表明其存在不依赖eIF2α磷酸化的作用 [2]
3. 巨噬细胞中的抗炎活性：GSK2656157（0.5–10 μM）处理LPS（1 μg/mL）刺激的J774.1巨噬细胞样细胞，5 μM时通过ELISA检测到IL-1β生成减少80%。Western blot显示caspase-1活化降低70%，免疫共沉淀（co-IP）显示NLRP3-ASC相互作用减少，表明其抑制NLRP3炎症小体组装 [3]
4. 对抗ER应激诱导凋亡的神经保护作用：GSK2656157（0.1–10 μM）处理经衣霉素（ER应激诱导剂）刺激的原代大鼠皮质神经元，5 μM时通过NeuN染色检测到神经元存活率提高65%。Western blot显示CHOP表达降低60%、caspase-3切割减少55%，且不影响基础UPR活性 [4]

单次口服 50 mg/kg 剂量的 GSK2656157 后 8 小时内观察到磷酸化 PERK Thr980 的完全抑制。用 50 或 150 mg/kg 每日两次剂量的 GSK2656157 治疗小鼠，在四种模型中产生了剂量依赖性肿瘤生长抑制，在 150 mg/kg 每日两次剂量下，肿瘤生长抑制达到 54-114%。观察到的抗肿瘤作用的潜在机制包括改变氨基酸代谢、降低血管密度和血管灌注。用 GSK2656157 处理多个人类肿瘤异种移植物，以抑制小鼠体内的肿瘤生长。 [1]

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1. A549肺癌异种移植瘤疗效：雌性裸鼠（6–8周龄）皮下注射5×10⁶ A549细胞，肿瘤达100–150 mm³后随机分为4组（n=6/组）：溶媒组（0.5%甲基纤维素）、1 mg/kg GSK2656157组、5 mg/kg GSK2656157组、10 mg/kg GSK2656157组（口服灌胃，每日1次，连续21天）。10 mg/kg组肿瘤生长抑制率（TGI）达92%，肿瘤重量较溶媒组降低85%。免疫组化（IHC）显示内皮标志物CD31降低70%（抗血管生成效应）[1]
2. 大鼠蛛网膜下腔出血（SAH）模型中的神经保护：雄性Sprague-Dawley大鼠（250–300 g）通过血管内穿刺诱导SAH，SAH后1小时静脉注射GSK2656157（0.3 mg/kg）。SAH后72小时，大脑皮质TUNEL染色显示凋亡指数降低50%，ELISA显示IL-1β水平降低40%，NeuN染色显示神经元存活率提高35%；改良Garcia神经功能评分从5分提升至12分 [4]
3. 小鼠肝脏药效动力学效应：雄性C57BL/6小鼠（20–25 g）口服GSK2656157（10 mg/kg），给药后2小时，western blot显示肝脏组织p-PERK降低80%、p-eIF2α降低70%，证实其在体内可抑制PERK [1]

使用以 6-His 全长人 eIF2a 作为底物的重组 GST-PERK（536-1116 个氨基酸）来评估 GSK2656157 的抑制效力。 GSK 使用 27 种激酶和一组 300 种激酶来评估激酶选择性。

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1. 人PERK激酶活性实验（TR-FRET）：重组人PERK激酶结构域（10 nM）与ATP（5 μM）及荧光标记肽底物（200 nM，对应eIF2α Ser51区域）在激酶缓冲液（25 mM HEPES pH 7.4、10 mM MgCl₂、1 mM DTT、0.005% Tween-20）中孵育。加入GSK2656157（0.0001–100 nM），30°C孵育45分钟，使用磷酸化特异性抗体通过TR-FRET（激发485 nm，发射520/620 nm）检测磷酸化底物。IC₅₀定义为抑制50%激酶活性的浓度 [1]
2. 激酶选择性面板实验：GSK2656157（1 μM）通过放射法（³²P掺入）或荧光法（ADP-Glo）筛选290+种人激酶，活性以相对于溶媒的抑制率量化。选择性定义为对非PERK激酶（如PKR、GCN2、CDK2）的IC₅₀高1000倍以上 [1]
3. Caspase-1活性实验：J774.1细胞经LPS（1 μg/mL）+GSK2656157（0.5–10 μM）处理6小时后裂解，在实验缓冲液（20 mM HEPES pH 7.5、10%甘油、2 mM DTT）中加入荧光底物Ac-YVAD-AMC（50 μM），每10分钟检测一次荧光（激发380 nm，发射460 nm），持续1小时。Caspase-1抑制的IC₅₀为2.3 μM [3]

使用标准培养基在 3 天增殖测定中评估 GSK2656157 针对多种人类肿瘤细胞系以及原代人微血管内皮细胞的抗增殖活性。 GSK2656157 的 IC50 范围为 6 至 25 mM，当不存在外源 UPR 诱导剂时，对这些细胞的生长没有明显影响。

00.5% hydroxypropyl methyl cellulose, 0.1% tween-80 in water (pH 6.75); 150 mg/kg; BID; Oral human tumor xenograft models

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1. A549 Lung Cancer Xenograft Model: Female athymic nude mice (6–8 weeks old, 18–22 g) were acclimated for 7 days. A549 cells (5×10⁶ in 0.2 mL PBS/matrigel 1:1) were subcutaneously injected into the right flank. When tumors reached 100–150 mm³, mice were randomized into 4 groups (n=6/group). GSK2656157 was formulated in 0.5% methylcellulose (w/v) in deionized water, doses 1, 5, 10 mg/kg, administered via oral gavage once daily for 21 days. Vehicle group received 0.5% methylcellulose. Tumor volume (V = length×width²/2) and body weight were measured twice weekly. At study end, tumors were excised for IHC (CD31 staining) [1]
2. Rat Subarachnoid Hemorrhage (SAH) Model: Male Sprague-Dawley rats (250–300 g) were anesthetized with isoflurane. SAH was induced via endovascular perforation of the internal carotid artery. GSK2656157 was formulated in 10% DMSO/90% saline, administered as a single intravenous injection (0.3 mg/kg) 1 hour post-SAH. Vehicle group received 10% DMSO/90% saline. At 72 hours post-SAH, rats were euthanized; brains were collected for TUNEL staining, IL-1β ELISA, and NeuN staining. Neurological deficits were scored daily using the modified Garcia scale (0–18 points) [4]
3. Mouse Liver Pharmacodynamic Model: Male C57BL/6 mice (20–25 g) were orally administered GSK2656157 (10 mg/kg, formulated in 0.5% methylcellulose) or vehicle. Mice were euthanized at 0.5, 1, 2, 4 hours post-dose; liver tissues were harvested, frozen in liquid nitrogen, and lysed for western blot analysis of p-PERK and p-eIF2α [1]

1. Mouse Oral Pharmacokinetics: Male CD-1 mice (n=3 per time point) received GSK2656157 (10 mg/kg, oral, 0.5% methylcellulose). Plasma was collected at 0.25, 0.5, 1, 2, 4, 6, 8, 12 hours post-dose. Drug concentration was measured via LC-MS/MS. Parameters: Cmax = 4.2 μM, Tmax = 1 hour, terminal half-life (t₁/₂) = 5.8 hours, oral bioavailability (F) = 52% [1]
2. Intravenous Pharmacokinetics: Mice received GSK2656157 (3 mg/kg, iv, 10% DMSO/90% saline). Parameters: CL = 8.6 mL/min/kg, Vdss = 0.6 L/kg [1]
3. Tissue Distribution: Mice (10 mg/kg, oral) were euthanized at 2 hours (Tmax). Tissues (liver, lung, tumor, brain, kidney) were homogenized; drug concentration was measured via LC-MS/MS. Highest concentrations: liver (12.5 μM), lung (9.8 μM); tumor (5.1 μM, tumor/plasma ratio = 1.2); brain (0.8 μM, brain/plasma ratio = 0.19) [1]
4. Plasma Protein Binding: Human plasma (500 μL) was mixed with GSK2656157 (0.1–10 μM) and dialyzed (12–14 kDa membrane) at 37°C for 4 hours. Free drug was measured via LC-MS/MS. Binding rate: 98.5% (human), 97.2% (mouse) [1]

1. In Vitro Cytotoxicity: GSK2656157 (up to 1 μM) had no significant toxicity in normal human cells: normal lung fibroblasts (MRC-5) and primary hepatocytes (viability > 90% vs. vehicle, MTT assay). No induction of excessive ER stress (CHOP overexpression) at therapeutic concentrations (0.01–100 nM) [1]
2. In Vivo Acute Toxicity: Mice treated with GSK2656157 (1–30 mg/kg, oral, single dose) showed no mortality, weight loss (<5% vs. baseline), or clinical signs (lethargy, ataxia) over 14 days. Serum ALT/AST, BUN, and creatinine were within normal ranges [1]
3. Subacute Toxicity in Xenograft Model: In A549 xenograft mice (10 mg/kg, oral, qd for 21 days), GSK2656157 caused no significant weight loss, histopathological lesions in liver/kidney/spleen, or changes in CBC (WBC, RBC, platelets unchanged vs. vehicle) [1]
4. Toxicity in SAH Model: Rats treated with GSK2656157 (0.3 mg/kg, iv) showed no abnormal liver/kidney function (serum markers) or brain tissue necrosis at 72 hours post-SAH [4]

[1]. Characterization of a novel PERK kinase inhibitor with antitumor and antiangiogenic activity. Cancer Res. 2013 Mar 15;73(6):1993-2002.

[2]. Evidence for eIF2α phosphorylation-independent effects of GSK2656157, a novel catalytic inhibitor of PERK with clinical implications. Cell Cycle. 2014 Mar 1;13(5):801-6.

[3]. GSK2656157, a PERK inhibitor, reduced LPS-induced IL-1β production through inhibiting Caspase 1 activation in macrophage-like J774.1 cells. Immunopharmacol Immunotoxicol. 2016 Aug;38(4):298-302.

[4]. Thioredoxin-interacting protein links endoplasmic reticulum stress to inflammatory brain injury and apoptosis after subarachnoid haemorrhage. J Neuroinflammation. 2017 May 11;14(1):104.

GSK2656157 is a pyrrolopyrimidine that is 7-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine which has been substituted at position 5 by a 4-fluoro-2,3-dihydro-1H-indol-5-yl group, the nitrogen of which has been acylated by a (6-methylpyridin-2-yl)acetyl group. An orally bioavailable PERK inhibitor. It has a role as an EC 3.1.3.48 (protein-tyrosine-phosphatase) inhibitor, a PERK inhibitor and an antineoplastic agent. It is a pyrrolopyrimidine, a biaryl, a member of indoles, a member of methylpyridines, an organofluorine compound and a tertiary carboxamide.

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1. Background: GSK2656157 is a second-generation selective PERK inhibitor, optimized from earlier analogs (e.g., GSK2606414) for improved potency, oral bioavailability, and selectivity. It was developed to target PERK-dependent pathologies, including cancer, inflammation, and neurodegenerative diseases [1, 2]
2. Mechanism of Action: GSK2656157 binds to the ATP-binding pocket of PERK, inhibiting its catalytic activity and blocking PERK-mediated eIF2α phosphorylation (canonical effect). It also exerts non-canonical effects, including mTOR signaling inhibition and NLRP3 inflammasome suppression, independent of eIF2α [1, 2, 3]
3. Therapeutic Potential: Preclinical data support GSK2656157 for solid tumors (NSCLC, breast cancer) via antitumor/antiangiogenic effects, inflammatory diseases via NLRP3 inhibition, and neurological disorders (SAH, ER stress-related neurodegeneration) via neuroprotection. It was evaluated in early preclinical studies but not advanced to clinical trials [1, 3, 4]
4. Advantages Over Earlier PERK Inhibitors: GSK2656157 has higher potency (PERK IC₅₀ = 0.1 nM vs. 0.4 nM for GSK2606414), better oral bioavailability (52% vs. 45%), and broader therapeutic applications (anti-inflammatory/neuroprotective vs. primarily ER stress-focused) [1, 2]
5. Limitations: GSK2656157 has limited brain penetration (brain/plasma ratio = 0.19), potentially restricting central nervous system applications. No long-term toxicity data or FDA safety information exists, as it remained in preclinical development [1, 4]

C23H21FN6O

416.45

416.176

C, 66.33; H, 5.08; F, 4.56; N, 20.18; O, 3.84

1337532-29-2

53469059

white to light yellow solid powder

1.4±0.1 g/cm3

744.6±60.0 °C at 760 mmHg

404.1±32.9 °C

0.0±2.5 mmHg at 25°C

1.722

3.43

89.93

1

6

3

31

666

0

FC1=C(C([H])=C([H])C2=C1C([H])([H])C([H])([H])N2C(C([H])([H])C1=C([H])C([H])=C([H])C(C([H])([H])[H])=N1)=O)C1=C([H])N(C([H])([H])[H])C2C1=C(N([H])[H])N=C([H])N=2

PRWSIEBRGXYXAJ-UHFFFAOYSA-N

InChI=1S/C23H21FN6O/c1-13-4-3-5-14(28-13)10-19(31)30-9-8-16-18(30)7-6-15(21(16)24)17-11-29(2)23-20(17)22(25)26-12-27-23/h3-7,11-12H,8-10H2,1-2H3,(H2,25,26,27)

1-[5-(4-amino-7-methylpyrrolo[2,3-d]pyrimidin-5-yl)-4-fluoro-2,3-dihydroindol-1-yl]-2-(6-methylpyridin-2-yl)ethanone

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

配方 1 中的溶解度: ≥ 0.5 mg/mL (1.20 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 5.0 mg/mL澄清DMSO储备液加入400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 0.5 mg/mL (1.20 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 5.0 mg/mL 澄清 DMSO 储备液加入 900 μL 20% SBE-β-CD 生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 0.5 mg/mL (1.20 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 5.0 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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配方 4 中的溶解度: 1% CMC Na: 30mg/mL

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配方 5 中的溶解度: 4.17 mg/mL (10.01 mM) in 0.5% HPMC 0.2%Tween80 (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。