Inhibitors of protein synthesis identified by a high throughput multiplexed translation screen

Abstract

The use of small molecule inhibitors of cellular processes is a powerful approach to understanding gene function that complements the genetic approach. We have designed a high throughput screen to identify new inhibitors of eukaryotic protein synthesis. We used a bicistronic mRNA reporter to multiplex our assay and simultaneously screen for inhibitors of cap-dependent initiation, internal initiation and translation elongation/termination. Functional screening of >90 000 compounds in an in vitro translation reaction identified 36 inhibitors, 14 of which are known inhibitors of translation and 18 of which are nucleic acid-binding ligands. Our results indicate that intercalators constitute a large class of protein synthesis inhibitors. Four non-intercalating compounds were identified, three of which block elongation and one of which inhibits initiation. The novel inhibitor of initiation affects 5' end-mediated initiation, as well as translation initiated from picornaviral IRESs, but does not significantly affect internal initiation from the hepatitis C virus 5'-untranslated region. This compound should be useful for delineating differences in mechanism of initiation among IRESs.

Reversing chemoresistance by small molecule inhibition of the translation initiation complex eIF4F

“…In vitro transcriptions and translations of bicistronic mRNA reporters were performed as previously described (28). Firefly (FF) and renilla (Ren) luciferase (luc) activity (RLU) was measured using a Berthold Lumat LB 9507 luminometer.…”

Inhibition of Protein Synthesis Alters Protein Degradation through Activation of Protein Kinase B (AKT)

“…To determine whether the cycloheximide-induced AKT phosphorylation results from inhibition of protein synthesis, we treated HEK-293FT cells with NSC119889, another protein synthesis inhibitor that acts by preventing mRNA-ribosome interaction and inhibiting 5Ј-mediated/cap-dependent initiation of protein synthesis (18). A marked increase in AKT phosphorylation at Ser 473 was also observed (Fig.…”

Inhibition of translation by cytotrienin A—a member of the ansamycin family

“…During the course of a high-throughput screen to identify translation inhibitors (Novac et al 2004), Cyt A (Fig. 1A) was identified as a ''hit'' that inhibited both cap-dependent (Firefly [FF] luciferase) and hepatitis C virus (HCV)-driven (Renilla [Ren] luciferase) translation in Krebs-2 extracts (Fig.…”

“…In vitro translations were performed as previously reported (Novac et al 2004). Translations were performed using a capped bicistronic mRNA reporter FF/HCV/ Ren transcribed from pSP/(CAG) 33 /FF/ HCV/Ren.pA 51 , in which firefly (FF) luciferase protein is produced by capdependent translation and Renilla (Ren) luciferase protein is generated by Hepatitis C virus (HCV) IRES-mediated initiation.…”