Translation in yeast cell lysates was performed in the presence of DMSO, 40 m QL47, or 40 m compound 14. small-molecule inhibitor that can be utilized to probe the eukaryotic translation machinery and that can be further developed as a new therapeutic agent. transcribed reporter DV subgenomic RNA bearing the virus’s seven nonstructural genes (1 h and 24 h post-treatment. Analysis of the neosynthesized proteins was performed as in transcribed reporter RNA that had the characteristics of cellular mRNA and harbored a cap structure and a poly(A) tail (Fig. S2). Interestingly, we observed that this effect on host protein synthesis is acute and lost when cells are treated with QL47 for 24 h (Fig. 1and translation reactions conducted in the presence of QL47 or harringtonine. Likewise, we detected limited impact by WP1130, a promiscuous deubiquitinase inhibitor that potently promotes protein degradation in cell-based assays (23), on the abundance of the subgenomic viral polyprotein in the translation system (Fig. 2translations performed in rabbit reticulocyte lysates for 90 min at 30 C in the presence of precharged FluoroTectTM lysine tRNA and DMSO, 40 m QL47, or 30 g/ml CHX. An transcribed reporter RNA bearing the EMCV IRES and a luciferase (transcribed reporter DV subgenomic RNA (40) was added, and lysates were incubated in the presence of DMSO or 40 m of the indicated small molecules for 90 min at 30 C. The luciferase signal was measured, and data are presented as means S.D. of two technical replicates. One representative experiment is shown from two independent experiments. transcribed reporter DV subgenomic RNA in rabbit reticulocyte lysates was performed for 90 min at 30 C in the presence of DMSO, 30 g/ml CHX, or 40 m of QL47 and the indicated analogs. The luciferase signal was measured, and data are presented as means S.D. of two technical replicates. One representative experiment is shown from three independent experiments. To further demonstrate that this inhibition of translation is pertinent to QL47’s cellular activity, we took advantage of our previously reported SAR studies (6, 9) and tested the activity of QL47 analogs in this system. Consistent with our hypothesis, we found a good correlation between their reported activities and their activities in the translation TCS ERK 11e (VX-11e) assay (Fig. 2using a reconstituted cell-free synthesis system (25). QL47 does, however, inhibit translation in yeast cell lysates, demonstrating that this small molecule specifically affects eukaryotic translation (Fig. 3cells carrying the pUA66-plasmid that constitutively expresses GFP (24) were treated with DMSO, 250 g/ml G418, or 50 m QL47. The intracellular GFP fluorescence signal was then measured continuously for 14 h at 37 C. The signal obtained from growth medium was subtracted, and data are presented as means S.D. of 12 experimental replicates. One representative experiment is shown from two independent experiments. translation assays performed Enpep in rabbit reticulocyte lysates, yeast cell lysates, or a reconstituted cell-free synthesis system (PURExpress?). Translation in rabbit reticulocyte lysates was performed in the presence of DMSO, 30 g/ml CHX, 40 m QL47, or 40 m compound 14. An transcribed reporter DV subgenomic RNA was used as a template, and the luciferase signal was measured after 90-min incubation at 30 C. Data are presented as means normalized to DMSO S.D. of four experimental replicates. Translation in yeast cell lysates was performed in the presence of DMSO, 40 m QL47, or 40 m compound 14. An transcribed vesicular stomatitis virus (VSV) RNA bearing a luciferase reporter gene (44) was used as a template, and the luciferase signal was measured after 2-h incubation at 25 C. Data are presented as means normalized to DMSO S.D. of three experimental replicates. Translation in a reconstituted cell-free synthesis system (PURExpress?) was performed in the presence of DMSO, 250 g/ml G418, 100 m QL47, or 100 m compound 14. A plasmid expressing GFP under control of a T7 promoter was used as a template. After 1-h incubation at 37 C, the total protein content was analyzed by Western blotting. The reporter protein was detected using a GFP TCS ERK 11e (VX-11e) antibody, and its abundance was normalized to the loading control (histidine tag). Data are presented as means normalized to DMSO S.D. of two technical replicates. One representative experiment is shown from four (rabbit reticulocyte lysates) or two (yeast cell lysates and cell-free synthesis system) independent experiments. indicate that the differences between experimental samples and the DMSO-treated control samples are statistically significant when compared using unpaired test: ***, 0.001; nonsignificant ( 0.05. QL47 inhibits an early step in the translation process We next sought to examine the mechanism by which QL47 inhibits protein synthesis by analyzing active translation complexes in mammalian cells.Y. probe the eukaryotic translation machinery and that can be further developed as a new therapeutic agent. transcribed reporter DV subgenomic RNA bearing the virus’s seven nonstructural genes (1 h and 24 h post-treatment. Analysis of the neosynthesized proteins was performed as in transcribed reporter RNA that had the characteristics of cellular mRNA and harbored a cap structure and a poly(A) tail (Fig. S2). Interestingly, we observed that this effect on host protein synthesis is acute and lost when cells are treated with QL47 for 24 h (Fig. 1and translation reactions conducted in the presence of QL47 or harringtonine. Likewise, we detected limited impact by WP1130, a promiscuous deubiquitinase inhibitor that potently promotes protein degradation in cell-based assays (23), on the abundance of the subgenomic viral polyprotein in the translation system (Fig. 2translations performed in rabbit reticulocyte lysates for 90 min at 30 C in the presence of precharged FluoroTectTM lysine tRNA and DMSO, 40 m QL47, or 30 g/ml CHX. An transcribed reporter RNA bearing the EMCV IRES and a luciferase (transcribed reporter DV subgenomic RNA (40) was added, and lysates were incubated in the presence of DMSO or 40 m of the indicated small molecules for 90 min at 30 C. The luciferase signal was measured, and data are presented as means S.D. of two technical replicates. One representative experiment is shown from two independent experiments. transcribed reporter DV subgenomic RNA in rabbit reticulocyte lysates was performed for 90 min at 30 C in the presence of DMSO, 30 g/ml CHX, or 40 m of QL47 and the indicated analogs. The luciferase signal was measured, and data are presented as means S.D. of two technical replicates. One representative experiment is shown from three independent experiments. To further demonstrate that this inhibition of translation is pertinent to QL47’s cellular activity, we took advantage of our previously reported SAR studies (6, 9) and tested the activity of QL47 analogs in this system. Consistent with our hypothesis, we found a good correlation between their reported activities and their activities in the translation assay (Fig. 2using a reconstituted cell-free synthesis system (25). QL47 does, however, inhibit translation in yeast cell lysates, demonstrating that this TCS ERK 11e (VX-11e) small molecule specifically affects eukaryotic translation (Fig. 3cells carrying the pUA66-plasmid that constitutively expresses GFP (24) were treated with DMSO, 250 g/ml G418, or 50 m QL47. The intracellular GFP fluorescence signal was then measured continuously for 14 h at 37 C. The signal obtained from growth medium was subtracted, and data are presented as means S.D. of 12 experimental replicates. One representative experiment is shown from two independent experiments. translation assays performed in rabbit reticulocyte lysates, yeast cell lysates, or a reconstituted cell-free synthesis system (PURExpress?). Translation in rabbit reticulocyte lysates was performed in the presence of DMSO, 30 g/ml CHX, 40 m QL47, or 40 m compound 14. An transcribed reporter DV subgenomic RNA was used as a template, and the luciferase signal was measured after 90-min incubation at 30 C. Data are presented as means normalized to DMSO S.D. of four experimental replicates. Translation in yeast cell lysates was performed in the presence of DMSO, 40 m QL47, or 40 m compound 14. An transcribed vesicular stomatitis virus (VSV) RNA bearing a luciferase reporter gene (44) was used as a template, and the luciferase signal was measured after 2-h incubation at 25 C. Data are presented as means normalized to DMSO S.D. of three experimental replicates. Translation in a reconstituted cell-free synthesis system (PURExpress?) was performed in the presence of DMSO, 250 g/ml G418, 100 m QL47, or 100 m compound 14. A plasmid expressing GFP under control of a T7 promoter was used as a.