Inhibits the interaction between eIF4E (cap-binding protein) and eIF4G (large scaffolding protein), as well as the interaction between eIF4E and 4E-BP1. In a TR-FRET assay monitoring eIF4E-eIF4GI interaction, IC50 = 13.5 μM [1]
Does not compete with 5′ mRNA cap structures for binding to eIF4E [1]

4E2RCat suppresses cap-dependent translation by interfering with the interaction between the major scaffolding protein eIF4G and the cap-binding protein eIF4E. It dramatically lowers the amount of infected cells and intracellular and extracellular infectious viral titers caused by the human coronavirus 229E (HCoV-229E). Cap-dependent translation is inhibited by 4E2RCat in a dose-dependent manner. Cap-dependent FF translation is inhibited by 4E2RCat, whereas EMCV IRES-driven Ren translation is unaffected. Coronavirus replication is inhibited by 4E2RCat in a time- and dose-dependent manner [1].

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Inhibits cap-dependent translation in a dose-dependent manner in Krebs extract programmed with FF/HCV/Ren bicistronic mRNA; at 25 μM, inhibits FF (cap-dependent) while HCV IRES-driven Ren translation is not affected (even increased due to ribosome availability); at 100 μM, some off-target effects on HCV IRES are observed [1]
- In the same system with FF/EMCV/Ren bicistronic mRNA, 4E2RCat inhibits cap-dependent FF translation but not EMCV IRES-driven Ren translation [1]
- In pulldown assays using recombinant eIF4E and GST-eIF4GI517-606, 100 μM 4E2RCat blocks their interaction [1]
- Also blocks interaction between eIF4E and GST-eIF4GII555-658, and between eIF4E and GST-4E-BP1 at 100 μM [1]
- In m7GTP pull-down experiments using ribosome salt wash (RSW), 25 μM 4E2RCat disrupts preformed eIF4F complex: eIF4GI and eIF4A co-purification with eIF4E is significantly reduced, while eIF4E recovery remains unchanged [1]
- Inhibits protein synthesis in L132 cells in a dose- and time-dependent manner as measured by [35S]methionine metabolic labeling: at 4 h treatment, relative protein synthesis is ~80% at 6.25 μM, ~60% at 12.5 μM, ~50% at 25 μM, ~40% at 50 μM; at 24 h treatment, relative synthesis is ~60% at 6.25 μM, ~40% at 12.5 μM, ~30% at 25 μM, ~15% at 50 μM [1]
- In HCoV-229E-infected L132 cells (MOI 0.1), 4E2RCat at 12.5 μM completely blocks extracellular infectious virus production (below limit of detection) at 24 h and 48 h post-infection; at 6.25 μM, extracellular virus is very low at 48 h (<10 TCID50/ml) [1]
- Intracellular infectious virus titers are also significantly reduced: at 24 h and 48 h post-infection in the presence of 12.5 μM 4E2RCat, titers are <10 TCID50/ml and <20 TCID50/ml respectively, representing a nearly 10^6-fold reduction compared to DMSO control [1]
- Reduces the percentage of HCoV-229E S protein-positive cells in a dose-dependent manner: at 6.25 μM, ~2.4-fold reduction; at 12.5 μM, ~4-fold reduction compared to control [1]
- At 50 μM, 4E2RCat has no effect on poliovirus protein synthesis in HeLa cells infected with type 1 poliovirus (Mahoney strain) at 2 PFU/cell, despite significantly reducing host protein synthesis at this concentration [1]
- Structure-activity relationship (SAR) analysis of 19 analogs identified four compounds with some activity, but none were as potent as 4E2RCat in preventing eIF4E-eIF4G interaction or blocking cellular protein synthesis [1]

The body's inhibition of protein synthesis by 4E2RCat does not stem from a rise in cell death [1].

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Ultrahigh-throughput screening for eIF4E-eIF4G inhibitors used a time-resolved fluorescence resonance energy transfer (TR-FRET)-based assay miniaturized to a 1,536-well format. The assay monitors the interaction between eIF4E and eIF4GI. A library of 217,341 compounds was screened, and 4E2RCat was identified. The 8-point dose-response curve of 4E2RCat in this TR-FRET assay gave an IC50 of 13.5 μM [1]
- For pulldown experiments to assess inhibition of eIF4E binding partners: GST-eIF4GI517-606 or bovine serum albumin (BSA) was coupled to Affi-Gel-10 beads. Recombinant eIF4E was preincubated with 100 μM 4E2RCat or DMSO for 1 hour at room temperature, then mixed with Affi-Gel-bound proteins and incubated for another hour. Beads were washed, resolved by SDS-PAGE, and eIF4E detected by Western blotting. Similarly, pulldowns with GST-eIF4GII555-658 and GST-4E-BP1 were performed using 2.5 μg GST fusion proteins and 0.25 μg eIF4E, incubated with 25 μM 4E2RCat in binding buffer, then glutathione beads, eluted, and analyzed by Western blot [1]
- For assessing effect on preformed eIF4F complex: ribosome salt wash (RSW) was incubated with 25 μM 4E2RCat or DMSO for 1 hour at 30°C, then 50 μl of 50% m7GTP Sepharose beads were added and rotated for 2 hours at 4°C. Beads were washed, proteins eluted with m7GTP, and eluents analyzed by SDS-PAGE and Western blot using anti-eIF4E, anti-eIF4GI, and anti-eIF4A antibodies [1]
- In silico computational solvent mapping of eIF4E three-dimensional structures identified five shallow pockets as potential binding hot spots. Docking of 4E2RCat using AutoDock Vina 1.1.0 showed that 4E2RCat binds into four of the five pockets, which is predicted to clash with eIF4E-eIF4G and eIF4E-4E-BP1 interaction [1]

In vivo metabolic labeling: L132 cells were seeded at 60,000 cells/well in 24-well plates 24 hours prior to treatment. Cells were treated with increasing concentrations of 4E2RCat for 4 or 24 hours. For the last hour, medium was replaced with methionine-free DMEM supplemented with 10% dialyzed serum, and for the last 15 minutes cells were labeled with [35S]methionine (150-225 μCi/ml). Cells were washed, lysed in RIPA buffer, samples precipitated with trichloroacetic acid, and radioactivity determined by scintillation counting. Protein concentrations were measured and used to standardize counts [1]
- Cell viability/apoptosis assay: L132 cells (200,000 cells/well in 6-well plates) were treated with 12.5 μM 4E2RCat for indicated times (4, 8, 24, 48, 72 hours). Cells were collected (including supernatant), washed, and resuspended in annexin V binding buffer, then stained with annexin V/propidium iodide and analyzed by flow cytometry. The fraction of apoptotic cells relative to DMSO vehicle control (set to 1) showed no increase compared to control at any time point [1]
- Coronavirus infection and titer determination: L132 cells were infected with HCoV-229E at MOI 0.1 for 2 hours at 33°C, washed, then incubated in medium containing DMSO or 4E2RCat at indicated concentrations for up to 48 hours. Infectious virus titers (intra- and extracellular) were quantified by immunoperoxidase assay using monoclonal antibody 5-11H.6 against the S protein of HCoV-229E, with secondary horseradish peroxidase-conjugated antibody, and detection with diaminobenzidine and hydrogen peroxide. Titers calculated by Karber method [1]
- Immunofluorescence for viral S protein: Infected cells were fixed and stained with mouse IgG1 MAb 5-11H.6 followed by AlexaFluor-488 anti-mouse goat antibody (green), and nuclei stained with DAPI (blue). Percentage of S protein-positive cells was determined [1]
- Poliovirus infection: HeLa cells (3×10^5 per well in 6-well plates) were infected with Mahoney strain of poliovirus type 1 at 2 PFU/cell for 30 minutes at room temperature, washed, then incubated with vehicle (1% DMSO) or 50 μM 4E2RCat for 4 hours at 37°C. For the last 30 minutes, [35S]methionine (150 μCi/ml) was added. Cells were lysed and analyzed by SDS-PAGE and autoradiography [1]

At 12.5 μM 4E2RCat, no induction of cell death/apoptosis was observed in L132 cells up to 72 hours of treatment, as determined by annexin V/propidium iodide staining and flow cytometry (apoptotic fraction relative to DMSO control was set to 1 with no increase) [1]
- At concentrations that significantly reduced host protein synthesis (e.g., 50 μM), 4E2RCat had no effect on poliovirus replication, suggesting no nonspecific antiviral state induction at the cellular level [1]

[1]. Blocking eIF4E-eIF4G interaction as a strategy to impair coronavirus replication. J Virol. 2011 Jul;85(13):6381-9.

4E2RCat is an organic molecular entity.

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Coronaviruses (HCoV-229E) replication requires cap-dependent translation of subgenomic mRNAs. Overexpression of 4E-BP1 (which sequesters eIF4E) impairs coronavirus replication, supporting that eIF4F is a vulnerability. 4E2RCat blocks eIF4E-eIF4G interaction and thereby inhibits coronavirus replication [1]
- 4E2RCat is more potent than 4E1RCat (another eIF4E-eIF4G interaction inhibitor) in reducing coronavirus infectious virus production, although both have similar IC50s in the TR-FRET assay. 4E2RCat also appears more potent than the eIF4A inhibitors hippuristanol and silvestrol in this system [1]
- In silico modeling predicts that 4E2RCat binds to four of five hot spots in the eIF4G/4E-BP binding region of eIF4E. Extending the compound into the fifth unoccupied hot spot (magenta in Fig. 3) could improve affinity [1]
- The compound was identified from an ultrahigh-throughput screen of 217,341 compounds from the Molecule Library Screening Centers Network [1]

C22H14CLNO4S2

455.93

455.005

432499-63-3

2287238

Light yellow to yellow solid powder

1.6±0.1 g/cm3

659.0±65.0 °C at 760 mmHg

352.3±34.3 °C

0.0±2.1 mmHg at 25°C

1.760

5.09

1

6

5

30

724

0

C1=CC=C(C=C1)CN2C(=O)/C(=C\C3=CC=C(O3)C4=CC(=C(C=C4)Cl)C(=O)O)/SC2=S

WOBPZFKXPCYOLU-YBFXNURJSA-N

InChI=1S/C22H14ClNO4S2/c23-17-8-6-14(10-16(17)21(26)27)18-9-7-15(28-18)11-19-20(25)24(22(29)30-19)12-13-4-2-1-3-5-13/h1-11H,12H2,(H,26,27)/b19-11+

5-[5-[(E)-(3-Benzyl-4-oxo-2-sulfanylidene-1,3-thiazolidin-5-ylidene)methyl]furan-2-yl]-2-chlorobenzoic acid

4E2R Cat 4E2RCat4E2R-Cat

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)

DMSO : ~23.33 mg/mL (~51.17 mM)

Solubility in Formulation 1: 2.33 mg/mL (5.11 mM) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% 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 23.3 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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