Cell experiment:

L132 cells are treated with 12.5 μM 4E2RCat for the indicated times and are processed for annexin V/propidium iodide staining. To this end, cell medium is collected. Cells are ished with 1 mL PBS, which is collected as well, and trypsinized in 200 μL 0.05% trypsin-EDTA. Cells are pooled with previously collected supernatants and spun for 2 min at 2,000 rpm and 4℃. The cell pellet is ished with 2 mL cold PBS, followed by another spin. After the second spin, the cell pellet is resuspended in 100 μL annexin V binding buffer and propidium iodide added to a final concentration of 5 μg/mL. After the addition of 5 μL annexin V-fluorescein isothiocyanate, samples are incubated for 15 min in the dark at room temperature and diluted. Fluorescence-activated cell sorter (FACS) analyses are performed using a FACScan instrument[1].

4E2RCat is an inhibitor of eIF4E-eIF4G interaction with an IC50 of 13.5 μM.

4E2RCat prevents the interaction between eIF4E (the cap-binding protein) and eIF4G (a large scaffolding protein), inhibiting cap-dependent translation. It significantly decreases human coronavirus 229E (HCoV-229E) replication, reducing the percentage of infected cells and intra- and extracellular infectious virus titers. 4E2RCat inhibits cap-dependent translation in a dose-dependent manner. 4E2RCat inhibits cap-dependent FF translation but not EMCV IRES-driven Ren translation. 4E2RCat inhibits coronavirus replication in a dose- and time-dependent manner[1].

4E2RCat inhibits protein synthesis in vivo and it is not a consequence of increased cell death[1].

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