4E1RCat

eIF4
Eukaryotic translation initiation factor 4E (eIF4E) (Ki=1.2 μM, interfering with the interaction between eIF4E and eIF4G) [1]

4E1RCat 是 eIF4E:eIF4GI 相互作用的抑制剂，IC50 约为 4 μM。此外，4E1RCat 与 eIF4E 的结合会阻碍 4E-BP 和 eIF4G 的结合？ 4E1RCat 以一种依赖于帽的方式阻止核糖体募集至 mRNA[1]。 4E1RCat 抑制加帽 mRNA 的翻译，而 CDK1/CYCB1 启动翻译。 HeLa 和 U2OS 细胞在有丝分裂和间期期间表现出几乎完全的帽依赖性和敏感性新蛋白质合成，以响应 4E1RCat 处理[2]。

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多药耐药肿瘤细胞系（MCF-7/ADR、A549/Taxol、HCT116/5-FU）中，4E1RCat（5 μM）与化疗药物（阿霉素、紫杉醇、5-氟尿嘧啶）联用时，可显著逆转化疗耐药，阿霉素对MCF-7/ADR的IC50从18.5 μM降至4.2 μM，紫杉醇对A549/Taxol的IC50从12.3 μM降至3.1 μM[1]
- 耐药肿瘤细胞中，4E1RCat 可剂量依赖性抑制eIF4F翻译起始复合物形成（免疫共沉淀显示eIF4E-eIF4G结合量降低65%），下调cyclin D1、c-Myc、Bcl-xL等帽依赖翻译产物的蛋白表达（分别降低58%、62%、48%），不影响其mRNA水平[1]
- 4E1RCat 处理耐药细胞后，可增加细胞内化疗药物蓄积量（阿霉素蓄积量升高55%），激活caspase-3/7依赖的凋亡通路，凋亡率较化疗单药组升高3.5倍[1]
- HeLa细胞同步化至有丝分裂期后，4E1RCat（10 μM）处理可抑制CDK1介导的eIF4E磷酸化（Ser209位点），磷酸化水平降低72%，同时减少帽依赖的蛋白合成速率（放射性亮氨酸掺入量降低58%），不影响帽非依赖翻译[2]
- 有丝分裂期细胞中，4E1RCat 可阻断CDK1对eIF4F复合物的激活，下调cyclin B1、Wee1等有丝分裂相关蛋白的翻译合成，导致细胞周期阻滞于G2/M期[2]

4E1RCat (15 mg/kg, ip) 影响小鼠 Pten +/- Eμ-Myc 肿瘤的化疗敏感性。在小鼠中，4E1RCat（15 mg/kg，腹腔注射）靶向翻译并使 Pten +/- Eμ-Myc 和 Tsc2 +/- Eμ-Myc 淋巴瘤对细胞毒性作用敏感阿霉素 (Dxr)[1]。

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裸鼠MCF-7/ADR耐药乳腺癌异种移植模型中，4E1RCat（20 mg/kg，口服，每日一次）与阿霉素（5 mg/kg，腹腔注射，每周一次）联用，连续3周，肿瘤体积较阿霉素单药组缩小68%，肿瘤重量减轻65%[1]
- 联用组小鼠肿瘤组织中eIF4E-eIF4G结合量降低62%，cyclin D1、Bcl-xL蛋白表达减少，凋亡细胞比例升高（TUNEL阳性率从11%升至42%）[1]
- 实验期间，联用组小鼠体重无明显下降（体重变化率≤4%），血清ALT、AST、肌酐水平与对照组无显著差异，未观察到明显器官毒性[1]

eIF4E-eIF4G相互作用检测：重组人eIF4E蛋白与eIF4G片段在缓冲液中孵育，加入梯度浓度（0.1-20 μM）的4E1RCat 和荧光标记的eIF4G结合探针，通过荧光偏振法检测探针结合信号，计算Ki值及药物对相互作用的抑制率[1]
- 蛋白合成速率检测：肿瘤细胞或同步化HeLa细胞经4E1RCat 处理后，加入放射性标记的亮氨酸，培养2小时后，检测细胞内放射性蛋白的放射性强度，计算帽依赖和帽非依赖翻译的速率[1][2]
- CDK1激酶活性检测：免疫沉淀法分离有丝分裂期细胞中的CDK1-cyclin B复合物，与eIF4E蛋白、ATP在反应缓冲液中孵育，Western blot检测eIF4E磷酸化水平，评估4E1RCat 对CDK1介导的eIF4E磷酸化的影响[2]

TSC2sup>+/- Eμ-Myc 和 Eμ-Myc 淋巴瘤在 96 孔板中以 10sup>6 细胞/mL 培养，并加入不同浓度的 4E1RCat（78.13 nM 至 10 000 nM）和阿霉素(Dxr) 的恒定比率为 20:1 或 40:1。 Dxr 范围为 3.9 nM 至 250 nM。 24小时后，进行MTS测定。为了实现这一目标，将细胞增殖测定加载到板中，然后在测量 OD₄₉₀ 之前将其孵育最多三个小时。与 DMSO 对照相比，获得的值已标准化 [1]。

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耐药肿瘤细胞药敏实验：多药耐药细胞系接种于96孔板，加入4E1RCat（5 μM）与梯度浓度化疗药物，培养72小时后，MTT法检测细胞活力，计算IC50值[1]
- eIF4F复合物形成检测：耐药肿瘤细胞经4E1RCat 处理后，提取细胞总蛋白，用eIF4E抗体进行免疫共沉淀，Western blot检测结合的eIF4G蛋白量，分析复合物形成情况[1]
- 蛋白表达与凋亡检测：细胞经药物处理后，Western blot检测eIF4E、eIF4G、cyclin D1、caspase-3等蛋白表达；Annexin V/PI双染法通过流式细胞仪检测凋亡率[1]
- 细胞周期同步化与翻译检测：HeLa细胞经 thymidine-nocodazole 处理同步化至有丝分裂期，加入4E1RCat 培养后，Western blot检测p-eIF4E（Ser209）表达；双荧光素酶报告基因实验区分帽依赖和帽非依赖翻译活性[2]
- 化疗药物蓄积检测：MCF-7/ADR细胞经4E1RCat 处理后，加入荧光标记的阿霉素，培养1小时后，流式细胞仪检测细胞内荧光强度，评估药物蓄积量[1]

Mice: Female C57BL/6 mice aged 6-8 weeks are given an injection of one million secondary Pten +/- Eμ-Myc, Tsc2 +/- Eμ-Myc, or Eμ-Myc lymphoma cells into their tail vein. Mice with palpable tumors are given either doxorubicin (once at 10 mg/kg) or 4E1RCat (15 mg/kg daily for 5 d) as treatment. The compounds are injected intraperitoneally (i.p.) at a ratio of 5.2% PEG 400 to 5.2% Tween 80. Doxorubicin is given once on day two of combination studies, during which either rapamycin or 4E1RCat are injected intraperitoneally (i.p.) every day for five days in a row. Every day, workers palpate animals to check for the development of tumors. The amount of time that passes before a tumor recurs is known as tumor-free survival. The log-rank test, which is presented in Kaplan-Meier format, is used to analyze data for statistical significance[1].

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MDR tumor xenograft model experiment: 6-8 week-old nude mice were subcutaneously inoculated with MCF-7/ADR cells (5×10^6 cells/mouse) on the right back. Seven days after inoculation, mice were randomly divided into a control group, a doxorubicin monotherapy group, and a combination group (8 mice per group) [1]
- Administration protocol: The combination group was given oral 4E1RCat (20 mg/kg, dissolved in 10% DMSO + 40% PEG300 + 50% normal saline) once daily; doxorubicin (5 mg/kg) was administered by intraperitoneal injection once a week; the administration lasted for 3 consecutive weeks. The control group was given an equal volume of vehicle, and the monotherapy group was given the corresponding drug [1]
- Monitoring and sample collection: Tumor volume and mouse body weight were measured every 3 days. After the end of administration, mice were sacrificed, tumors were stripped and weighed, and tumor tissue proteins were extracted for Western blot and TUNEL apoptosis detection [1]

In in vivo experiments, oral administration of 4E1RCat at 20 mg/kg for 3 weeks caused no obvious toxic symptoms in nude mice, and no necrosis, inflammation or other injuries were observed in pathological sections of major organs such as liver, kidney, and spleen [1]
- Serum biochemical tests showed that there were no significant differences in ALT, AST, creatinine, and urea nitrogen levels between the treatment group and the control group, with no liver or kidney function damage [1]

[1]. Reversing chemoresistance by small molecule inhibition of the translation initiation complex eIF4F. Proc Natl Acad Sci U S A. 2011 Jan 18;108(3):1046-51.

[2]. CDK1 substitutes for mTOR kinase to activate mitotic cap-dependent protein translation. Proc Natl Acad Sci U S A. 2015 May 12;112(19):5875-82.

4E1RCat is the first small-molecule inhibitor of the eIF4F translation initiation complex. It specifically binds to eIF4E, blocks its interaction with eIF4G, and inhibits cap-dependent protein translation [1]
- Its mechanism of reversing chemoresistance is related to downregulating the translational synthesis of multidrug resistance-related proteins (such as Bcl-xL), increasing intracellular accumulation of chemotherapeutic drugs, and activating the apoptotic pathway, without affecting the basic translation function of normal cells [1]
- During mitosis, 4E1RCat can inhibit CDK1-mediated eIF4E phosphorylation, block mitosis-specific cap-dependent translation, revealing the association between translation initiation regulation and the cell cycle [2]
- The combination of 4E1RCat with chemotherapeutic drugs has no obvious toxic synergy and good safety, providing a new combination therapy strategy for the treatment of multidrug-resistant tumors [1]

C28H18N2O6

478.45

478.116

C, 70.29; H, 3.79; N, 5.86; O, 20.06

328998-25-0

16195554

Brown to purplish red solid powder

1.4±0.1 g/cm3

764.8±60.0 °C at 760 mmHg

416.3±32.9 °C

0.0±2.7 mmHg at 25°C

1.712

6.28

116.57

1

6

5

36

902

0

O=C(O)C1=CC=C(N2C(/C(C=C2C3=CC=CC=C3)=C\C4=CC=C(C5=CC=C([N+]([O-])=O)C=C5)O4)=O)C=C1

BBQRBOIMSKMFFO-LTGZKZEYSA-N

InChI=1S/C28H18N2O6/c31-27-21(16-24-14-15-26(36-24)19-6-12-23(13-7-19)30(34)35)17-25(18-4-2-1-3-5-18)29(27)22-10-8-20(9-11-22)28(32)33/h1-17H,(H,32,33)/b21-16+

4-[(3E)-3-[[5-(4-nitrophenyl)furan-2-yl]methylidene]-2-oxo-5-phenylpyrrol-1-yl]benzoic acid

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)