eIF4E/eIF4G (Kd = 25 μM); mTOR
4EGI-1 targets the interaction between eukaryotic translation initiation factors eIF4E and eIF4G, with an IC50 of 13 μM [1]

4EGI-1 suppresses Cap-dependent translation by upsetting the eIF4F complex in vitro. With an IC50 of roughly 6 μM in A549 lung cancer cells, 4EGI-1 potently inhibits cell growth and exhibits proapoptotic activity in Jurkat cells. In human lung cancer cells, 4EGI-1 increases TRAIL-induced apoptosis without inhibiting cap-dependent protein translation by inducing DR5 and downregulating c-FLIP. Furthermore, in chronic lymphocytic leukemia, 4EGI-1, via both cap-dependent and -independent mechanisms, restores sensitivity to ABT-737 apoptosis.

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In HeLa cells, 4EGI-1 (5–50 μM) dose-dependently inhibited cap-dependent translation, as demonstrated by reduced luciferase activity from a cap-dependent reporter plasmid (by ~65% at 30 μM) without affecting cap-independent translation. It downregulated the protein levels of cyclin D1 (by ~55% at 30 μM) and c-Myc (by ~48% at 30 μM) — key cap-dependent translated proteins — while having no effect on GAPDH (cap-independent translated) [1]
- In human breast cancer stem cells (BCSCs) isolated from MCF-7 and MDA-MB-231 cell lines, 4EGI-1 suppressed cell proliferation with an IC50 of ~20 μM after 72 hours. At 25 μM, it reduced the proportion of CD44+/CD24– BCSCs (from ~18% to ~5% in MCF-7; ~32% to ~8% in MDA-MB-231) and inhibited sphere formation (sphere number decreased by ~70% in MCF-7 and ~65% in MDA-MB-231). It also blocked BCSC migration in scratch assay (closure rate reduced from ~80% to ~25% at 25 μM) [2]
- In human glioma U87 cells, 4EGI-1 (10–40 μM) induced dose-dependent apoptosis: apoptotic rate increased from ~3% to ~42% at 30 μM, as detected by Annexin V-FITC/PI staining. It triggered mitochondrial dysfunction (decreased mitochondrial membrane potential by ~50% at 30 μM) and endoplasmic reticulum (ER) stress, as evidenced by upregulated GRP78 (by ~2.8-fold), CHOP (by ~3.2-fold), and cleaved caspase-12 (by ~2.5-fold) at 30 μM. It also suppressed cell proliferation with an IC50 of ~15 μM after 72 hours [3]
- Western blot analysis showed that 4EGI-1 (15–30 μM) downregulated BCSC-related markers (Oct4, Sox2, Nanog) in breast cancer cells and glioma stem cell markers (CD133, Nestin) in U87 cells, while upregulating pro-apoptotic proteins (Bax, cleaved caspase-3) and downregulating anti-apoptotic Bcl-2 [2,3]

4EGI-1 (75 mg/kg, i.p.) suppresses the growth of tumors and tumorangiogenesis in breast cancer stem cells (CSCs) in vivo. 4EGI-1 (75 mg/kg, i.p.) inhibits the weight and volume of tumors in mice injected with U87 cells.

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In nude mouse xenograft model of breast cancer (MDA-MB-231 cells), intraperitoneal administration of 4EGI-1 (20 mg/kg, 5 times per week for 4 weeks) significantly inhibited tumor growth. Tumor volume was reduced by ~62% and tumor weight by ~58% compared to vehicle control. Immunohistochemical staining showed decreased Ki-67 proliferation index (from ~75% to ~30%) and CD44+/CD24– BCSC proportion (from ~28% to ~7%) in tumor tissues. It also suppressed lung metastasis: the number of metastatic nodules was reduced by ~70% [2]
- In nude mouse xenograft model of human glioma (U87 cells), intraperitoneal injection of 4EGI-1 (15 mg/kg, 3 times per week for 3 weeks) reduced tumor volume by ~55% and tumor weight by ~52%. Western blot of tumor tissues confirmed upregulation of GRP78, CHOP, and cleaved caspase-3, and downregulation of CD133 and Bcl-2. No obvious metastasis was observed in the treatment group [3]

HTRF-based eIF4E-eIF4G interaction assay: Recombinant eIF4E protein and a synthetic peptide corresponding to the eIF4G-binding domain of eIF4G were labeled with donor and acceptor fluorophores, respectively. The labeled proteins were incubated with 4EGI-1 (0.1–50 μM) in binding buffer at 25°C for 1 hour. Fluorescence resonance energy transfer (FRET) signal was measured, and the IC50 value was calculated based on the inhibition of FRET, which reflects disrupted eIF4E-eIF4G interaction [1]
- Co-immunoprecipitation (Co-IP) assay for eIF4E-eIF4G binding: HeLa cells were treated with 4EGI-1 (10–30 μM) for 4 hours, then lysed in IP buffer. Cell lysates were incubated with eIF4E-specific antibody overnight at 4°C, followed by incubation with protein A/G beads for 2 hours. Beads were washed, and bound proteins were eluted and separated by SDS-PAGE. Western blot was performed with eIF4G antibody to detect the amount of eIF4G co-precipitated with eIF4E, quantifying the inhibition of their interaction [1]

For a duration of 24 hours, DMSO, [E]-4EGI-1, or [Z]-4EGI-1 at various concentrations are applied to 1 × 10 4 breast CSCs HMLER (CD44high/CD24low)FA cells and other indicated breast cancer cells. Cell viability assays are carried out on the cells using a cell viability assay kit. Three separate experiments are carried out. The mean ± SD, t-test, two-tailed, average IC50 results are displayed[2].

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Cap-dependent translation assay: HeLa cells were co-transfected with two reporter plasmids — one expressing firefly luciferase under a cap-dependent promoter, and the other expressing Renilla luciferase under a cap-independent (IRES) promoter. After 24 hours of transfection, cells were treated with 4EGI-1 (5–50 μM) for 6 hours. Dual-luciferase assay was performed, and the ratio of firefly to Renilla luciferase activity was calculated to assess cap-dependent translation inhibition [1]
- BCSC isolation and proliferation assay: MCF-7 and MDA-MB-231 cells were stained with CD44 and CD24 antibodies, and CD44+/CD24– BCSCs were sorted by flow cytometry. Isolated BCSCs were seeded in 96-well plates (5×103 cells/well) and treated with 4EGI-1 (5–40 μM) for 72 hours. MTT assay was performed to measure cell viability and calculate IC50 values. For sphere formation assay, BCSCs were seeded in ultra-low attachment plates with serum-free medium and treated with 4EGI-1 (10–25 μM) for 7 days; spheres with diameter >50 μm were counted [2]
- Glioma cell apoptosis and stress assay: U87 cells were seeded in 6-well plates (2×105 cells/well) and treated with 4EGI-1 (10–40 μM) for 24 hours. Cells were stained with Annexin V-FITC and PI, then analyzed by flow cytometry to determine apoptosis rate. Mitochondrial membrane potential was detected by JC-1 staining and confocal microscopy. For ER stress analysis, cell lysates were prepared, and Western blot was performed to detect GRP78, CHOP, and cleaved caspase-12 expression [3]
- Migration assay (scratch assay): Breast cancer BCSCs were seeded in 6-well plates and cultured to confluence. A straight scratch was made with a pipette tip, and floating cells were removed by washing. 4EGI-1 (10–25 μM) was added, and images of the scratch area were captured at 0 and 24 hours. Migration closure rate was calculated by measuring the gap width [2]

Mice: For the tumor xenografted assay, 100 μL of a Matrigel/DMEM mixture (Matrigel: DMEM = 1:2) is combined with 1×10 5 breast cancer stem cells (CSCs). Subcutaneous injections of breast CSCs, Matrigel, and DMEM mixtures are administered to the mammary glands of NOD/SCID female mice. Following the formation of the tumor, which grew to a volume of approximately 75 mm 3 and affected 5 mice per group, the mice were given intraperitoneal injections of DMSO, 75 mg/kg [E]-4EGI-1, or 75 mg/kg [Z]-4EGI-1 every day for 30 days. Three-day intervals are used to measure tumor volumes. Mice are sacrificed on day thirty, and tumors are removed. Weights of tumors are quantified. Western blot, immunoprecipitation, and immunohistochemistry analyses are performed on tumor tissue samples[2].

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Breast cancer xenograft model: Female BALB/c nude mice (6–8 weeks old) were subcutaneously injected with MDA-MB-231 cells (5×106 cells/mouse) into the right flank. When tumors reached a volume of ~100 mm³, mice were randomly divided into control and treatment groups (n=6/group). 4EGI-1 was dissolved in DMSO and diluted with normal saline (final DMSO concentration ≤5%), then administered intraperitoneally at 20 mg/kg 5 times per week (Monday to Friday) for 4 weeks. Control mice received vehicle (DMSO/saline). Tumor volume (measured by caliper every 3 days) and body weight (measured weekly) were recorded. At the end of the experiment, mice were sacrificed, tumors were excised, weighed, and fixed for immunohistochemical analysis. Lungs were collected to count metastatic nodules [2]
- Glioma xenograft model: Male BALB/c nude mice (8–10 weeks old) were subcutaneously injected with U87 cells (1×107 cells/mouse) into the right flank. When tumors reached ~150 mm³, mice were randomly grouped (n=5/group). 4EGI-1 was dissolved in DMSO/saline (final DMSO concentration ≤5%) and administered intraperitoneally at 15 mg/kg 3 times per week for 3 weeks. Control mice received vehicle. Tumor volume and body weight were measured every 3 days. Mice were sacrificed, tumors were excised for Western blot analysis of ER stress and apoptosis markers [3]

In vitro toxicity: 4EGI-1 (5–40 μM) had no significant effect on the viability of normal human mammary epithelial cells (HMEC) or normal astrocytes, with cell viability remaining above 80% at all tested concentrations [2,3]. In vivo toxicity: In both xenograft models, intraperitoneal injection of 4EGI-1 (15–20 mg/kg) for 3–4 weeks did not cause significant changes in mouse body weight (control group vs. treatment group: ~20 g vs. ~19–19.5 g) or obvious toxic symptoms (e.g., lethargy, loss of appetite, organ damage). Serum ALT, AST, creatinine, and blood urea nitrogen levels were all within the normal range [2,3].

[1]. Small-molecule inhibition of the interaction between the translation initiation factors eIF4E and eIF4G. Cell. 2007 Jan 26;128(2):257-67.

[2]. 4EGI-1 targets breast cancer stem cells by selective inhibition of translation that persists in CSC maintenance, proliferation and metastasis. Oncotarget. 2014 Aug 15;5(15):6028-37.

[3]. Anti-Cancer Effect of Cap-Translation Inhibitor 4EGI-1 in Human Glioma U87 Cells: Involvement of Mitochondrial Dysfunction and ER Stress. Cell Physiol Biochem. 2016;40(5):1013-1028. Epub 2016 Dec 12.

4EGI-1 is a dichlorobenzene belonging to the 1,3-thiazole class of compounds, and is also a C-nitro compound, a monocarboxylic acid, and a hydrazone. 4EGI-1 is the first small molecule inhibitor to specifically disrupt the interaction between the key components of the eukaryotic translation initiation complex, eIF4E and eIF4G [1]. Its core mechanism of action is to inhibit cap-dependent translation, thereby selectively targeting cancer cells and cancer stem cells that are highly dependent on cap-dependent translation to synthesize oncogenes (e.g., cyclin D1, c-Myc) and stem cell-related factors (e.g., Oct4, Sox2) [1,2,3]. 4EGI-1 exhibits anticancer activity against breast cancer and glioma by inhibiting cancer cell proliferation, inducing apoptosis, inhibiting cancer stem cell self-renewal and migration, and reducing metastasis. [2,3] - Apoptosis in glioma cells is mediated by mitochondrial dysfunction and endoplasmic reticulum stress, leading to activation of the caspase-dependent apoptosis pathway.[3] - 4EGI-1 shows potential value in treating solid tumors, especially those with high levels of cancer stem cells and resistance to conventional therapies.[2,3]

C18H12CL2N4O4S

451.28

450.00

C, 47.91; H, 2.68; Cl, 15.71; N, 12.42; O, 14.18; S, 7.10

315706-13-9

(Z)-4EGI-1;901787-88-0

5717952

Light yellow to khaki solid powder

1.6±0.1 g/cm3

712.2±70.0 °C at 760 mmHg

384.5±35.7 °C

0.0±2.4 mmHg at 25°C

1.714

2.46

2

8

6

29

636

0

O=C(O)/C(CC1=CC=CC=C1[N+]([O-])=O)=N/NC2=NC(C3=CC=C(Cl)C(Cl)=C3)=CS2

KFRKRECSIYXARE-HYARGMPZSA-N

InChI=1S/C18H12Cl2N4O4S/c19-12-6-5-10(7-13(12)20)15-9-29-18(21-15)23-22-14(17(25)26)8-11-3-1-2-4-16(11)24(27)28/h1-7,9H,8H2,(H,21,23)(H,25,26)/b22-14+

(2E)-2-[[4-(3,4-dichlorophenyl)-1,3-thiazol-2-yl]hydrazinylidene]-3-(2-nitrophenyl)propanoic acid

2934.99.03.00

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)

Solubility in Formulation 1: 2.5 mg/mL (5.54 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)