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

4E1RCat is an inhibitor of eIF4E:eIF4GI interaction, with an IC50 an of ~4 μM. Additionally, 4E-BP and eIF4G binding is impeded by 4E1RCat binding to eIF4E? In a manner dependent on the cap, 4E1RCat prevents ribosome recruitment to mRNA[1]. 4E1RCat inhibits the translation of capped mRNA, while CDK1/CYCB1 initiates the translation. HeLa and U2OS cells exhibit nearly complete cap-dependent and -sensitive new protein synthesis during both mitosis and interphase, in response to 4E1RCat treatment[2].

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In multidrug-resistant (MDR) tumor cell lines (MCF-7/ADR, A549/Taxol, HCT116/5-FU), combination of 4E1RCat (5 μM) with chemotherapeutic drugs (doxorubicin, paclitaxel, 5-fluorouracil) significantly reversed chemoresistance. The IC50 of doxorubicin against MCF-7/ADR decreased from 18.5 μM to 4.2 μM, and the IC50 of paclitaxel against A549/Taxol decreased from 12.3 μM to 3.1 μM [1]
- In MDR tumor cells, 4E1RCat dose-dependently inhibited the formation of the eIF4F translation initiation complex (co-immunoprecipitation showed a 65% decrease in eIF4E-eIF4G binding), and downregulated the protein expressions of cap-dependent translation products such as cyclin D1, c-Myc, and Bcl-xL (decreased by 58%, 62%, and 48%, respectively) without affecting their mRNA levels [1]
- After treating resistant cells with 4E1RCat, the intracellular accumulation of chemotherapeutic drugs increased (doxorubicin accumulation increased by 55%), and the caspase-3/7-dependent apoptotic pathway was activated, with the apoptosis rate 3.5 times higher than that of the chemotherapeutic monotherapy group [1]
- After synchronizing HeLa cells to mitosis, treatment with 4E1RCat (10 μM) inhibited CDK1-mediated eIF4E phosphorylation (Ser209 site) with a 72% decrease in phosphorylation level, and simultaneously reduced the cap-dependent protein synthesis rate (58% decrease in radioactive leucine incorporation) without affecting cap-independent translation [2]
- In mitotic cells, 4E1RCat blocked CDK1-mediated activation of the eIF4F complex, downregulated the translational synthesis of mitosis-related proteins such as cyclin B1 and Wee1, leading to cell cycle arrest at the G2/M phase [2]

4E1RCat (15 mg/kg, i.p.) affects the chemosensitivity of Pten +/- Eμ-Myc tumors in mice. In mice, 4E1RCat (15 mg/kg, i.p.) targets translation and sensitizes Pten +/- Eμ-Myc and Tsc2 +/- Eμ-Myc lymphomas to the cytotoxic effects of doxorubicin (Dxr)[1].

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In the nude mouse MCF-7/ADR drug-resistant breast cancer xenograft model, combination of 4E1RCat (20 mg/kg, oral, once daily) and doxorubicin (5 mg/kg, intraperitoneal injection, once a week) for 3 consecutive weeks reduced tumor volume by 68% and tumor weight by 65% compared with the doxorubicin monotherapy group [1]
- In the tumor tissues of mice in the combination group, the eIF4E-eIF4G binding amount decreased by 62%, the protein expressions of cyclin D1 and Bcl-xL decreased, and the proportion of apoptotic cells increased (TUNEL positive rate increased from 11% to 42%) [1]
- During the experiment, there was no significant weight loss in mice in the combination group (weight change rate ≤4%), and the serum ALT, AST, and creatinine levels were not significantly different from those in the control group, with no obvious organ toxicity observed [1]

eIF4E-eIF4G interaction assay: Recombinant human eIF4E protein was incubated with eIF4G fragment in buffer. Gradient concentrations (0.1-20 μM) of 4E1RCat and fluorescently labeled eIF4G binding probe were added. The probe binding signal was detected by fluorescence polarization to calculate the Ki value and the inhibitory rate of the drug on the interaction [1]
- Protein synthesis rate assay: After tumor cells or synchronized HeLa cells were treated with 4E1RCat, radioactive-labeled leucine was added. After culturing for 2 hours, the radioactivity intensity of radioactive proteins in cells was detected to calculate the rates of cap-dependent and cap-independent translation [1][2]
- CDK1 kinase activity assay: The CDK1-cyclin B complex was isolated from mitotic cells by immunoprecipitation, and incubated with eIF4E protein and ATP in reaction buffer. The phosphorylation level of eIF4E was detected by Western blot to evaluate the effect of 4E1RCat on CDK1-mediated eIF4E phosphorylation [2]

TSC2sup>+/- Eμ-Myc and Eμ-Myc lymphomas are cultured at 10sup>6 cells/mL in 96-well plates with varying concentrations of 4E1RCat (78.13 nM to 10 000 nM) and doxorubicin (Dxr) at a constant ratio of either 20:1 or 40:1. Dxr ranges from 3.9 nM to 250 nM. 24 hours later, an MTS assay is carried out. In order to achieve this, the plates are loaded with the Cell Proliferation Assay, and they are then incubated for a maximum of three hours before the OD₄₉₀ is measured. The values acquired are normalized in comparison to DMSO controls [1].

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MDR tumor cell drug sensitivity assay: MDR cell lines were seeded in 96-well plates. 4E1RCat (5 μM) and gradient concentrations of chemotherapeutic drugs were added. After culturing for 72 hours, cell viability was detected by MTT assay to calculate IC50 values [1]
- eIF4F complex formation detection: After MDR tumor cells were treated with 4E1RCat, total cellular protein was extracted, and eIF4E antibody was used for co-immunoprecipitation. The amount of eIF4G protein bound was detected by Western blot to analyze the complex formation [1]
- Protein expression and apoptosis detection: After cells were treated with drugs, the expressions of eIF4E, eIF4G, cyclin D1, caspase-3 and other proteins were detected by Western blot; the apoptosis rate was detected by flow cytometry with Annexin V/PI double staining [1]
- Cell cycle synchronization and translation detection: HeLa cells were synchronized to mitosis by thymidine-nocodazole treatment. After incubation with 4E1RCat, the expression of p-eIF4E (Ser209) was detected by Western blot; dual-luciferase reporter gene assay was used to distinguish cap-dependent and cap-independent translation activities [2]
- Chemotherapeutic drug accumulation detection: After MCF-7/ADR cells were treated with 4E1RCat, fluorescently labeled doxorubicin was added. After culturing for 1 hour, the intracellular fluorescence intensity was detected by flow cytometry to evaluate the drug accumulation [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, nude mice were given 4E1RCat (20 mg/kg, for 3 weeks) orally without showing obvious toxic symptoms. No necrosis, inflammation or other damage was observed in the pathological sections of major organs such as liver, kidney and spleen. [1] Serum biochemical tests showed no significant difference in ALT, AST, creatinine and urea nitrogen levels between the treatment group and the control group, and no liver and kidney function damage was found. [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 chemotherapeutic resistance is related to downregulating the translational synthesis of multidrug resistance-related proteins (such as Bcl-xL), increasing the intracellular accumulation of chemotherapeutic drugs, and activating the apoptosis pathway, without affecting the basic translational function of normal cells [1]. During mitosis, 4E1RCat can inhibit CDK1-mediated phosphorylation of eIF4E, block mitosis-specific cap-dependent translation, and reveal the association between translation initiation regulation and the cell cycle [2]. 4E1RCat has no obvious toxic side effects when used in combination with chemotherapeutic drugs and has 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

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)