Discovery and Preliminary Characterization of Translational Modulators that Impair the Binding of eIF6 to 60S Ribosomal Subunits

Abstract

Eukaryotic initiation factor 6 (eIF6) is necessary for the nucleolar biogenesis of 60S ribosomes. However, most of eIF6 resides in the cytoplasm, where it acts as an initiation factor. eIF6 is necessary for maximal protein synthesis downstream of growth factor stimulation. eIF6 is an antiassociation factor that binds 60S subunits, in turn preventing premature 40S joining and thus the formation of inactive 80S subunits. It is widely thought that eIF6 antiassociation activity is critical for its function. Here, we exploited and improved our assay for eIF6 binding to ribosomes (iRIA) in order to screen for modulators of eIF6 binding to the 60S. Three compounds, eIFsixty-1 (clofazimine), eIFsixty-4, and eIFsixty-6 were identified and characterized. All three inhibit the binding of eIF6 to the 60S in the micromolar range. eIFsixty-4 robustly inhibits cell growth, whereas eIFsixty-1 and eIFsixty-6 might have dose- and cell-specific effects. Puromycin labeling shows that eIF6ixty-4 is a strong global translational inhibitor, whereas the other two are mild modulators. Polysome profiling and RT-qPCR show that all three inhibitors reduce the specific translation of well-known eIF6 targets. In contrast, none of them affect the nucleolar localization of eIF6. These data provide proof of principle that the generation of eIF6 translational modulators is feasible.

A Quick Guide to Small-Molecule Inhibitors of Eukaryotic Protein Synthesis

“…More recently, targeted screening identified a group of compounds that interfere with the 60S subunit binding of initiation factor eIF6 (figure, i.8). These compounds are eIFsixty 1 (clofazimine), eIFsixty 4, and eIFsixty 6 (eIFsixty 4 exhibits the most pronounced effect on the translation and cell growth) [142]. eIF6 is involved most ly in the preparation of newly synthesized ribosomes for the first round of translation after their export from the nucleus, but it may also take part in the regular translation cycle [143].…”

Arginine-rich C9ORF72 ALS Proteins Stall Ribosomes in a Manner Distinct From a Canonical Ribosome-Associated Quality Control Substrate

“…eIF6 is a translation initiation factor that binds 60S ribosomal subunits which prevents them premature joining with 40S subunits. Reduction of eIF6 levels causes a decrease in the amount of available 60S subunits and impairs protein synthesis (55). We therefore hypothesized that eIF6 knockout reduces ribosome load on mRNA which, according to the collision-based model, should mitigate arrest on stall sequences.…”

Inhibition of eIF6 Activity Reduces Hepatocellular Carcinoma Growth: An In Vivo and In Vitro Study

“…Recently, we identified three inhibitors of eIF6 binding to the 60S subunit. All three reduce the translation of lipogenic enzymes [ 28 , 30 , 34 ]. The inhibitors will be referred to as eIFsixty-1, eIFsixty-4 and eIFsixty-6.…”

“…The molecular action of eIF6 on reinitiation is exerted through its regulated binding to 60S ribosomal subunits [ 22 ]. In this context, we have identified three compounds, eIFsixty-1, eIFsixty-4, and eIFsixty-6 that inhibit the binding of eIF6 to 60S and the translation of lipogenic enzymes [ 30 ]. However, the extent by which eIF6 genetic or pharmacological inhibition impairs the formation and growth of HCC is still not fully addressed.…”