GSK2606414   中文名称: GSK-2606414

EIF2AK3 (PERK) (IC50 = 0.4 nM); EIF2AK1 (HRI) (IC50 = 420 nM); EIF2AK2 (PKR) (IC50 = 696 nM)
GSK2606414 is a potent and selective inhibitor of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), a key kinase in the unfolded protein response (UPR). It exhibits high selectivity over other eIF2α kinases and a broad panel of kinases.
- For human PERK (recombinant kinase domain, radiometric assay): IC₅₀ = 0.4 nM [1]
- For human PKR (recombinant, radiometric assay): IC₅₀ = 420 nM (≈1000-fold less potent than PERK) [1]
- For human GCN2 (recombinant, radiometric assay): IC₅₀ = 1800 nM [1]
- For human HRI (recombinant, radiometric assay): IC₅₀ = 1000 nM [1]
- For 300+ other kinases (panel screening): IC₅₀ > 10,000 nM (no significant inhibition) [1]
- For Plasmodium falciparum PK4 (PfPK4, a parasite eIF2α kinase, radiometric assay): IC₅₀ = 23 nM [2]

GSK2606414 抑制细胞中 PERK 激活[1]。
GSK2606414单独抑制PK4，对响应替代应激信号的其他两种疟原虫eIF2α激酶没有影响（图S6）。重要的是，GSK2606414抑制了疟原虫eIF2α磷酸化，并消除了ART治疗后的复发（图5）。由于ER和PERK在成熟红细胞中不存在，GSK2606414很可能通过寄生虫发挥其活性。

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1. 抑制PERK激活及下游UPR信号：GSK2606414（0.01–100 nM）处理人胚胎肾细胞（HEK293）或小鼠胚胎成纤维细胞（MEF）1–4小时，呈剂量依赖性抑制毒胡萝卜素（ER应激诱导剂）诱导的PERK自磷酸化（p-PERK），10 nM时抑制率达90%（western blot）。100 nM时减少eIF2α磷酸化（p-eIF2α）80%，并下调UPR靶基因（CHOP、GADD34）70%（qPCR）[1]
2. 减轻ER应激诱导的细胞死亡：在衣霉素处理的HeLa细胞中，GSK2606414（1–100 nM）可使细胞活力（MTT法）从30%（仅衣霉素组）升至100 nM时的85%，并降低caspase-3/7活性60% [1]
3. 抑制恶性疟原虫PfPK4并阻止青蒿素诱导的潜伏：GSK2606414（10–1000 nM）剂量依赖性抑制恶性疟原虫裂解物中PfPK4介导的eIF2α磷酸化（IC₅₀ = 45 nM，western blot）。在青蒿素（100 nM）处理的恶性疟原虫培养物中，100 nM GSK2606414使休眠寄生虫（休眠子）数量减少90%，并阻止药物撤除后的寄生虫复发 [2]
4. 对寄生虫与宿主细胞的选择性：GSK2606414（最高1 μM）对与恶性疟原虫共培养的HeLa细胞中人类PERK活性无显著影响，表明治疗浓度下可选择性抑制PfPK4 [2]

GSK2606414（50 和 150 mg/kg，口服）可防止小鼠肿瘤异种移植物生长[1]。
此外，化合物38（GSK2606414）被推进到小鼠的人类异种移植物功效研究中，并证明小分子抑制PERK可以抑制体内肿瘤生长，这与转化的PERK-/-小鼠胚胎成纤维细胞衍生的肿瘤生长缓慢一致。对38的物理性质和药代动力学进行进一步优化将是未来交流的主题。[1]

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1. 降低小鼠肝脏ER应激标志物：C57BL/6小鼠（雄性，20–25 g）在衣霉素（1 mg/kg，腹腔注射，ER应激诱导剂）前1小时给予GSK2606414（30 mg/kg，口服灌胃）。6小时后，肝组织中p-PERK降低70%，p-eIF2α降低60%（western blot），显著低于溶媒+衣霉素组 [1]
2. 在伯氏疟原虫小鼠疟疾模型中的疗效：感染伯氏疟原虫ANKA的雌性CD-1小鼠（20–22 g）经GSK2606414（50 mg/kg，腹腔注射，每日1次）+青蒿素（10 mg/kg，腹腔注射，每日1次）处理3天。第4天疟原虫血症（流式细胞术）降低95%，显著高于青蒿素单药组（60%）。联合组至第14天无复发，而80%的青蒿素单药组小鼠在第10天前出现复发 [2]
3. 在小鼠脑中的药效动力学效应：给予GSK2606414（30 mg/kg，口服）的小鼠在给药后2小时，大脑皮质p-eIF2α水平降低50%（western blot），证实其可穿透中枢神经系统 [1]

GST-PERK 胞质结构域是购买的。 6-His 全长人 eIF2 从 Sf9 昆虫细胞中的杆状病毒表达中纯化。 eIF2 蛋白通过透析到 PBS 中进行缓冲液交换，通过 NHS-LC-生物素进行化学修饰，然后通过透析到 50 mM Tris、pH 7.2、250 mM NaCl 和 5 mM DTT 中进行缓冲液交换。将蛋白质等分并保存在 80°C。将新鲜配制的淬灭溶液添加到反应中后，会产生 4 nM eIF2 磷酸 Ser51 抗体、已用 Eu-1024 标记的 4 nM 抗兔 IgG、40 nM 链霉亲和素 Surelight APC 和 15 mM 的终浓度乙二胺四乙酸。反应在黑色 384 孔聚苯乙烯低体积板中进行，最终体积为 10 μL。将所研究的化合物溶解在 DMSO 中至浓度为 1.0 mM，然后在 DMSO 中连续稀释 1-3 至 11 倍。将每个浓度以 0.1 μL 的量转移至测定板的相应孔中。结果产生 0.00017 至 10 μM 的最终化合物浓度范围。为了测定含有化合物的板，在室温下预孵育 30 分钟后添加 GST-PERK 溶液。添加 ATP 和 eIF2 底物溶液以启动反应。孵育1小时后添加淬灭溶液。在信号检测之前，将板在室温下覆盖 2 小时。在 Viewlux 阅读器上，产生的信号被量化。通过使用 APC/Eu 计算转换数据，APC 信号被归一化为铕信号。浓度响应曲线的信息绘制为使用数据缩减公式 100 × [1 – (U1 – C2)/(C1 – C2)] 与化合物浓度计算的抑制百分比，其中 U 是未知值，C1 是平均值C2 是 1% DMSO 获得的对照值，C2 是 0.1 M EDTA 获得的平均对照值。使用最小响应、最大响应、斜率因子、化合物浓度和抑制浓度 (IC50) 将数据拟合到曲线。每种化合物的结果均以 IC50 值列出。

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1. 人PERK激酶实验：重组人PERK激酶结构域（20 nM）与ATP（10 μM）及生物素化肽底物（500 nM，对应eIF2α Ser51区域序列）在激酶缓冲液（50 mM HEPES pH 7.5、10 mM MgCl₂、1 mM DTT、0.01% BSA）中孵育。加入GSK2606414（0.001–1000 nM），30°C孵育60分钟。通过链霉亲和素偶联磁珠及抗磷酸化eIF2α抗体，采用时间分辨荧光共振能量转移（TR-FRET）检测磷酸化底物。IC₅₀定义为抑制50%激酶活性的浓度 [1]
2. PfPK4激酶实验：重组恶性疟原虫PK4（50 nM）与[γ-³²P]ATP（5 μM）及重组恶性疟原虫eIF2α（100 nM）在寄生虫激酶缓冲液（25 mM Tris-HCl pH 7.4、5 mM MgCl₂、1 mM EGTA、0.1 mM Na₃VO₄）中孵育。加入GSK2606414（0.1–1000 nM），37°C反应30分钟，用SDS样品缓冲液终止反应。SDS-PAGE后通过放射自显影检测eIF2α中的³²P掺入量，从剂量-反应曲线确定IC₅₀ [2]
3. 激酶选择性面板：GSK2606414（1 μM）通过放射或荧光实验筛选317种人激酶，活性以相对于溶媒的抑制率表示，选择性定义为对非PERK激酶的IC₅₀高100倍以上 [1]

PERK 抑制剂（GSK2606414，化合物 38）孵育后，用毒胡萝卜素刺激细胞。两小时后裂解细胞并检查 PERK 自磷酸化抑制。
A549细胞中的PERK磷酸化[1]
将指数生长的A549，一种人肺腺癌细胞系，在补充有10%胎牛血清（FBS）的RPMI-1640培养基中的六孔培养皿中以500000个细胞/孔的速度接种，并在37°C、5%CO2下孵育过夜。第二天早上，用DMSO或化合物（3倍稀释液0.33、0.11、0.37μM）处理细胞，并如上所述孵育1小时。然后用1μM thapsigargin刺激细胞额外1小时，以诱导ER应激。收集细胞颗粒并在冷RIPA缓冲液[150mM NaCl、50mM Tris-HCl、pH 7.5、0.25%脱氧胆酸钠、1%NP-40、蛋白酶和磷酸酶抑制剂以及100mM原钒酸钠]中裂解。通过SDS–PAGE解析澄清的裂解物，并使用Invitrogen的NuPAGE系统将其转移到硝化纤维素膜上。将印迹与总PERK和总eIF-2α的初级抗体一起孵育。使用IRDye700DX标记的山羊抗小鼠IgG和IRDye800CW驴抗山羊IgG作为二抗。奥德赛红外成像仪检测到蛋白质.

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1. PERK/UPR信号检测（western blot）：HEK293细胞（5×10⁵细胞/6孔板）经GSK2606414（0.01–100 nM）预处理1小时，再用毒胡萝卜素（1 μM）刺激2小时。用含磷酸酶抑制剂的RIPA缓冲液裂解细胞，30 μg蛋白经10% SDS-PAGE分离后转膜，用抗p-PERK（Thr980）、抗p-eIF2α（Ser51）及总蛋白抗体孵育，后续结合HRP标记二抗，通过光密度法定量条带强度 [1]
2. ER应激下的细胞活力（MTT实验）：HeLa细胞（1×10⁴细胞/96孔板）经GSK2606414（1–100 nM）预处理1小时，再暴露于衣霉素（5 μg/mL）24小时。加入MTT试剂（0.5 mg/mL）孵育4小时，DMSO溶解甲臜后在570 nm处测吸光度，活力以溶媒处理细胞为对照进行标准化 [1]
3. 恶性疟原虫休眠子实验：恶性疟原虫3D7株在人红细胞（2%血细胞比容）中培养，用青蒿素（100 nM）+GSK2606414（10–1000 nM）处理48小时。通过吉姆萨染色（致密、圆形形态）鉴定休眠子，光学显微镜下计数。药物撤除后监测14天以评估复发情况 [2]

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.

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1. Mouse ER Stress Model: Male C57BL/6 mice (20–25 g) were acclimated for 7 days. GSK2606414 was formulated in 0.5% methylcellulose + 0.1% Tween 80, administered via oral gavage (30 mg/kg) 1 hour before tunicamycin (1 mg/kg, ip, dissolved in saline). Control groups received vehicle + tunicamycin or vehicle alone. Mice were euthanized 6 hours post-tunicamycin, and liver/brain tissues were collected for western blot analysis of p-PERK and p-eIF2α [1]
2. P. berghei Malaria Model: Female CD-1 mice (20–22 g) were infected with P. berghei ANKA (1×10⁶ parasitized erythrocytes, ip). When parasitemia reached 5–7% (day 3 post-infection), mice were randomized into 4 groups (n=6/group): vehicle (5% DMSO + 95% saline, ip), GSK2606414 alone (50 mg/kg, ip), artemisinin alone (10 mg/kg, ip), and combination. Treatments were given once daily for 3 days. Parasitemia was measured daily via flow cytometry (staining with SYBR Green I), and recrudescence was monitored for 14 days [2]

1. Mouse Pharmacokinetics: In male CD-1 mice, single oral administration of GSK2606414 (30 mg/kg) showed: Cmax = 2.8 μM, Tmax = 1 hour, terminal half-life (t₁/₂) = 4.2 hours, oral bioavailability (F) = 45%. Intravenous administration (10 mg/kg) showed CL = 12 mL/min/kg, Vd = 0.8 L/kg [1]
2. Tissue Distribution: Mice (30 mg/kg, oral) sacrificed at 2 hours post-dose had tissue concentrations: liver (5.2 μM), brain (1.1 μM), kidney (3.8 μM), plasma (2.5 μM), with brain/plasma ratio = 0.44 [1]
3. Plasma Protein Binding: Human plasma (90% v/v) was incubated with GSK2606414 (1 μM) and dialyzed using a 10 kDa cutoff membrane. Free drug fraction was 2.3%, indicating high plasma protein binding (97.7%) [1]

1. In vitro cytotoxicity: GSK2606414 (up to 1 μM) had no significant effect on viability of HEK293, HeLa, or primary human hepatocytes (viability > 90% vs. vehicle, MTT assay) [1]
2. In vivo acute toxicity: Mice treated with GSK2606414 (30–100 mg/kg, oral, single dose) showed no mortality, weight loss (<5%), or clinical signs (lethargy, ataxia) over 7 days. Serum ALT, AST, and creatinine levels were within normal ranges [1]
3. Combination toxicity in malaria model: Mice treated with GSK2606414 (50 mg/kg) + artemisinin (10 mg/kg) for 3 days had no significant changes in body weight or hematological parameters (RBC, WBC, platelets) compared to vehicle group [2]

[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).

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1. Background: GSK2606414 is the first-in-class selective PERK inhibitor, developed to probe the role of PERK in the UPR and ER stress-related diseases. It was identified via structure-based drug design, optimizing for potency and selectivity over other eIF2α kinases [1]
2. Mechanism of action: GSK2606414 binds to the ATP-binding pocket of PERK, inhibiting its kinase activity and preventing PERK-mediated eIF2α phosphorylation. This blocks the PERK branch of the UPR, reducing ER stress-induced pro-apoptotic signaling (e.g., CHOP) while preserving other UPR branches (IRE1, ATF6) [1]
3. Therapeutic potential: Preclinical studies suggest GSK2606414 may be useful in ER stress-related disorders (e.g., neurodegenerative diseases, diabetes) and malaria (by preventing artemisinin-induced parasite latency). It is a research tool and has not entered clinical trials [1, 2]
4. Limitations: GSK2606414 has moderate oral bioavailability (45%) and brain penetration (brain/plasma ratio 0.44), potentially limiting central nervous system applications. Its selectivity for PfPK4 expands its utility but requires further optimization for malaria therapy [1, 2]

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

77.04

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

注意： 本产品在运输和储存过程中需避光。

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: ≥ 2.5 mg/mL (5.54 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 25.0 mg/mL澄清DMSO储备液加入到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: 2.5 mg/mL (5.54 mM) 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), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 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: ≥ 2.5 mg/mL (5.54 mM) (饱和度未知) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
*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 4 中的溶解度: in 5% DMSO+45% PEG 300+ddH2O: 20mg/mL

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配方 5 中的溶解度: 3.33 mg/mL (7.38 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。