Glycyl-tRNA Synthetase as a Target for Antiviral Drug Screening Against Influenza Virus

Figures

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  Figure 1. Changes in Aminoacyl-tRNA synthetase expression post Influenza A Virus Infection. The changes in GlyRS (A), FARSA (C), and HisRS (E) expression were measured at 24, 48, and 72 h post-viral infection by Western blot. The experiment was repeated three times, and the results presented here are from one representative experiment. The results from the three repeated experiments were quantified using ImageJ in (B,D,F). Compared and analyzed using a t-test. **: p < 0.01, ****: p < 0.0001.

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  Figure 2. Generation of aaRSKD cells and the role of GARS in influenza A virus infection. Stable cell lines were established by infecting A549 cells with packaged lentivirus, and the knockdown efficiency of GARS (A), FARSA (B), and HisRS (C) in puromycin-selected A549 cells was assessed through immunoblot analysis. In A549 cells, Western blot analysis was conducted 48 h post-transient transfection with HA-GARS (D), HA-FARSA (E), HA-HisRS (F), and HA-vector, utilizing HA antibodies for detection. (G) GARSKD, GARSOE (transient transfection of HA-GARS), or negative control A549 cells were infected with the PR8 virus at a MOI of 1. (H) FARSAKD, FARSAOE (transient transfection of HA-FARSA), or negative control A549 cells were infected with the PR8 virus at a MOI of 1. (I) HisRSKD, HisRSOE (transient transfection of HA-HisRS), or negative control A549 cells were infected with the PR8 virus at a MOI of 1. The virus titers were measured using the hemagglutination method at 24, 48, and 72 h post-viral infection. Data are presented as the mean of three independent experiments, with error bars representing the standard error of the mean (SEM). ns: not significant, *: p < 0.05, **: p < 0.01.

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  Figure 3. A549 cells were infected with A/Guangdong-MaonanSWL1536/2019(H1N1)pdm09 virus (A) at an MOI of 1, A/Victoria/4897/2022(H1N1) (B) at an MOI of 0.5, A/Kansas/14/2017(H3N2)-like virus (C) at an MOI of 0.5, and B/Colorado/06/2017(BV) (D) at an MOI of 0.1. Virus titers were measured using the hemagglutination method at 24, 48, and 72 h post-infection. Data are presented as the mean of three independent experiments, with error bars representing the standard error of the mean (SEM). ns: not significant, *: p < 0.05, **: p < 0.01, ****: p < 0.0001.

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  Figure 4. Preliminary screening of antiviral drugs through GlyRS. (A) Flow chart of antiviral drug screening through GlyRS. Drug screening strategy includes thermal shift assay, bio-layer interferometry, enzyme inhibition assay, and viral titration assay. (B) Out of 5090 compounds, 40 exhibited a Tm shift > 5.0 °C, indicating positive hits. Black points within the black dashed lines represent negative results, while gray and colored points outside the gray dashed lines indicate positive hits.

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  Figure 5. The identification of GlyRS inhibitors. Binding sensorgrams for the interaction of Iodoquiol (A), Chloramine-T (B), Lansoprazole (C), Nisoldipine (D), Velpatasvir (E), Mometasone furoate (F), Carnosic acid (G), Scutellarein (H), N-Acetyl-L-alanine (I), and Lycobetaine (J) with immobilized GlyRS. The five curves are generated from 20, 10, 5, 2.5, and 1.25 μM molecules from top to bottom. The BLI affinity of 10 molecules to GlyRS was presented as mean ± SD, as shown in Table 1. The experiment was repeated three times, and the result shown in the image is a representative outcome of one of these repetitions. (K) The impact of 10 small molecules on GlyRS enzyme activity. The IC50 values of Lycobetaine (L) or Scutellarein (M) were measured based on the ATP consumption assay and presented as mean ± SD.

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  Figure 6. Binding modes and molecular dynamics simulations of inhibitors with GlyRS. The binding pocket and site of Lycobetaine (A) or Scutellarein (B) and the GlyRS protein and its interaction with surrounding amino acids. The two-dimensional plot was generated using Schrödinger software (Release 2019-2, Schrödinger LLC, New York, NY, USA, 2019). The curve of RMSD values over time during the molecular dynamics simulation of the Lycobetaine (C) or Scutellarein (D) and GlyRS complex. The MD simulation (RMSF analysis) of GlyRS and Lycobetaine (E) or Scutellarein (F) complexes for 100 ns.

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  Figure 7. GlyRS inhibitors block virus infection. The CCK-8 assay revealed the CC50 value of Lycobetaine (A) or Scutellarein (B) on A549 cells. Data were presented as mean ± SD. The vertical markers indicate the positions of the drugs at 10 μM or 20 μM. After inhibitor treatment for 24h and during viral infection, A/PuertoRico/8/34 (H1N1) (C) and A/Guangdong-MaonanSWL1536/2019 (H1N1) pdm09 virus (D) at a MOI of 1, A/Victoria/4897/2022(H1N1) (E) at a MOI of 0.5, and B/Colorado/06/2017(BV) (F) at a MOI of 0.1 were used to infect A549 cells. The virus titers were measured using the hemagglutination method at 24 h post-viral infection. *: p < 0.05, **: p < 0.01, ***: p < 0.001.

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  Figure 8. Lycobetaine induces conformational changes in GlyRS and inhibits virus infection by GlyRS. In the limited proteolysis analysis, the GlyRS proteins (in the presence and absence of Lycobetaine (A) or Scutellarein (B)) were incubated with trypsin at different concentrations for 1 h at 37 °C before the reactions were quenched, and the products were separated by SDS-PAGE. (C) After inhibitor treatment for 24h and PR8 infection at a MOI of 1 on GARSOE A549 cells, with the inhibitor present throughout the entire treatment. The virus titers were measured using the hemagglutination method at 24 h post-viral infection. *: p < 0.05. (D) After inhibitor treatment for 24 h and PR8 infection at a MOI of 1 on GARSKD A549 cells, with the inhibitor present throughout the entire treatment. The virus titers were measured using the hemagglutination method at 24 h post-viral infection. *: p < 0.05, **: p < 0.01. ns: none significance.