Inhibitors of eIF1A-ribosome interaction unveil uORF-dependent regulation of translation initiation and antitumor and antiviral effects

Figure 1. Discovery of eIF1A-RPS10 inhibitors (1Ais) using a high-throughput drug screen (HTS) in-vitro. (A) A scheme describing eIF1A and RPS10 split-Renilla constructs used for bacterial expression and in vitro HTS. (B) A flow chart describing the various steps of the screening process for eIF1A-RPS10 interaction inhibitors. (C) The effect of 1Ais on scanning. HEK293T cells were transfected with the described constructs that differ in their 5'UTR (the 111 nt was used as a reference, and the 427 nt is the eIF1A-sensitive IRF7) and exposed to 1Ais. Transfection efficiencies were normalized by a co-transfected GFP reporter gene. Levels of expression from reference and IRF7 5'UTR normalized to GFP were measured after overnight incubation. All of the results are relative to DMSO. Error bars represent SEM, n ≥ 4 independent biological replicates, Student t test comparing to DMSO control was performed, *P < 0.05. (D) The effect of 1Ais on translation initiation fidelity. HEK293T cells were transfected with the constructs that differ in their starting codon. The luciferase protein in these constructs is exclusively initiated from these starting codons. 6 h after transfection, cells were exposed…

Figure 2. Biochemical characterization of 1Ais interaction with eIF1A and RPS10 and the 48S complex. (A, B) Recombinant eIF1A and Rps10 were each expressed in E. coli, purified and fluorescently labeled. Then, each of these proteins was incubated with increasing amounts of 1Ais. The binding data are presented as a heat map showing the binding of the tested 1Ais with eIF1A (A) and RPS10 (B). These data are also presented as binding curves in Appendix Figs. S2A and 3A. Binding plots of 1Ai-3638, 1Ai-8214 and 1Ai-4927 with purified and fluorescently labeled eIF1A WT or eIF1A without 30 N-terminal or C-terminal amino acids and Rps10, are shown at the bottom. All fluorescence results were normalized to 0 1Ai concentration (DMSO). The IC50 concentrations were calculated using the AAT Bioquest online tool and shown in Appendix Fig. S3C. (C) Rabbit Reticulocyte lysates (RRL) were pre-incubated with 1Ais, and then 48S complexes were formed by the addition of GMP-PNP and in vitro synthesized luciferase mRNA. The 48S complexes were then separated on a sucrose gradient (8–32%), and the OD at 254 nm was measured. Each gradient was separated into top, 48S, and bottom fractions according to the 48S peak…

Figure 3. 1Ais disrupt eIF1A interaction with RPS10/ribosome in cells. (A–C) HEK293 cells were grown on a glass slide, exposed to the selected 1Ai for 3 h, fixed and subjected to Proximity ligation assay (PLA). Cells were reacted with the indicated matching antibody pairs, control pairs and eIF1A and RPS10 pair (A) and the DMSO and 1Ai-treated cells with the eIF1A and RPS10 pair (B). The samples were treated according to PLA protocol and stained with DAPI to visualize cellular orientation and numbers. PLA dots (red), nuclei (blue) were visualized using confocal microscopy. Each picture is a merge of ~15 pictures covering all cell volume. (C) Quantification of PLA results for eIF1A and RPS10 antibodies and 1Ais treatment, n = 4 independent biological replicates, error bars represent SEM, ** denotes P < 0.01 according to Student t test. Source data are available online for this figure.

Figure 4. 1Ais impairment of translation initiation is related to AUG context and regulation by uORFs. (A) HEK293 cells were treated with DMSO or 1Ais for 3 h and subjected to polysome profiling by fractionating cell lysates by centrifugation on a sucrose density gradient (10–50%) followed by OD measurement (254 nm) of each fraction. The quantified polysomes to monosomes ratio is shown on the right. The bars represent the mean, n = 2 independent biological replicates. (B–I) The effect of translation inhibition by 1Ais on global translation using Ribo-seq. HEK293T cells were treated with DMSO or 1Ai-3638 and 1Ai-8214 for 3 h then cycloheximide addition, followed by preparation of ribosome footprinting libraries, deep sequencing and analysis according to standard RF protocol. In parallel the same samples were also subjected to RNA-seq for total mRNA level measurements. (B) Relative mRNA abundance after 1Ais or DMSO treatment. The bars represent the mean, n = 2–3 independent biological replicates. (C) Metagene analysis. The p-site in each ribosome footprint was located and the ribosomal occupancy of each area on the mRNA was calculated and normalized to mRNA total counts. Data is normalized…

Figure 5. 1Ai-3638 and 1Ai-8214 decrease ovarian tumor growth in xenograft mouse model. (A) Gene and pathways enrichment analysis of 1Ai-3638 and 1Ai-8214 downregulated genes by the Gene Analytics tool. (B) OVCAR8 ovarian carcinoma cell line was incubated with increasing concentrations of 1Ais for 72 h, and then cell viability was assessed. Viability (100%) is normalized to 0 concentration (DMSO), n = 3 independent biological replicates, and error bars represent SD. (C) 1Ais toxicity assessment. Mice weight was measured at different time points after 1Ai-3638 and 1Ai-8214 subcutaneous injection for 3 weeks (assessed every 3–4 days, 3 mice per concentration, 21 mice in total) (D) OVCAR8 cell-derived xenografts weight after 1Ais treatment (10 mice per group). OVCAR8 cells were injected into the ovaries of immunocompromised female mice. Out of ten mice in each group (1Ai-3638,1Ai-8214 and DMSO) nine were included as they presented an ovarian tumor. 1Ais were injected into the mice’s peritoneal cavity five times a week for 4 weeks. Finally, mice were sacrificed, and tumor weight was measured. Results are presented as boxplots in which the center = median, box ends = interquartile range (IQR),…

Figure 6. 1Ai-4927 decreases SARS-COV-2 infection by selective inhibition of a context-specific regulatory CUG element. (A) 1Ais enhances cell viability after SARS-COV-2 infection. Vero6 cells were incubated with increasing concentrations of 1Ais and infected by the SARS-COV-2 virus. After 72 h, cell viability was measured by counting live cells. The 1Ais that presented a significant effect are presented in Bar plots. 100% represent uninfected cells and the black line cells infected with SARS-COV2 but treated with DMSO. Error bars represent SD of the mean, *P < 0.05, **P < 0.01, n = 3 independent biological replicates. All 1Ai viability plots are shown in Appendix Fig. S4A. (B) Vero6 cells infected by SARS-COV-2 at different multiplicities of infection and treated by 1Ais as described in Fig. 6A. Error bars represent the mean ± SD, n = 2 independent biological replicates. (C) mRNA sample was taken from Vero6 cells rescued by 1Ai-4927 (A) and tested by q-PCR for viral mRNA quantity. (D) The effect of 1Ais on NSP1-mediated inhibition of translation. HEK293T Cells were transfected with NSP1 and a GFP reporter. Cells were incubated overnight with 1Ais, n = 6 for DMSO and no NSP1 and n = 2 for…