Fig 1.
DPUDs inhibit IAV and SARS-CoV-2 infections in tissue culture cells.
(A) Heatmaps of DPUD (10 μM) screens against IAV X-31 (H3N2), WSN (H1N1), and SARS-CoV-2 (D614G). (B) Molecular structures of DPU and ‘hit’ compounds (DPUD-1, -2, -16, -20, and -23) that blocked IAV and SARS-CoV-2 infections by 95–100%. (C) High-content microscopy images of IAV- and SARS-CoV-2-infected cells. Nuclei were stained with Hoechst (magenta), and the viral NP/N proteins (green) were detected by IIF. Scare bars, 50 μm. (D) Western blots showing inhibition of IAV (X-31 and WSN strains) and SARS-CoV-2 (D614G) infections by DPUDs (10 μM) in A549 and Vero-E6 cells, respectively. IAV HA and SARS-CoV-2 Spike protein were detected using anti-HA antibody and anti-S antibody, respectively. GAPDH served as a loading control. (E) Results of viral plaque assay from the supernatants of A549 cells infected with IAV WSN strain. (F) Relative expression of SARS-CoV-2 N and E genes, detected by RT-PCR, in the supernatants of infected cells. (G) High-content microscopy images SARS-CoV-2-infected cells. Nuclei were stained with Hoechst (grey), and the viral Spike protein (cyan) was detected by IIF. All data are represented as mean ± SD. The P-value was determined using one-way ANOVA with multiple comparisons w.r.t. DMSO. ns: P >0.05, *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001.
Fig 2.
DPUDs exhibit high selectivity indices against IAV and SARS-CoV-2 infection.
(A-C) Graphs showing concentration-dependent effect of DPUDs against IAV (X-31 and WSN) and SARS-CoV-2 (D614G) infections in A549 and Vero-E6 cells, respectively. The half-maximal inhibitory concentration (IC50) values corresponding to each compound are shown. (D-E) Cytotoxicity assessment DPUDs in A549 and Vero-E6 cells. The cells were treated with different concentrations of DPUDs for 24 h, following which, LDH cytotoxicity assay was performed. The 50% cytotoxic concentration (CC50) values corresponding to each compound are shown. (F) Selectivity indices of the compounds for IAV X-31 and WSN, and SARS-CoV-2 (D614G) infections.
Fig 3.
(A) Schematic diagram showing the multi-step IAV entry process. (B) Effect of DPUDs (10 μM) on IAV binding to PM, endocytosis, HA acidification in late endosomes, capsid uncoating, and vRNP nuclear import. (C) Schematic diagram showing the steps of PM bypass IAV infection assay. (D) Restoration of IAV infection by induced fusion of virus particles at PM in presence of DPUDs (10 μM). (E) IAV endocytosis is blocked in A549 cells pre-treated with DPUD-1 (10 μM). Cells were pre-treated with DPUD-1 (10 μM) for 2 h, followed by removal of the compound. After pre-treatment, IAV endocytosis assay was performed at 0, 1, 2, 6, and 12 h. The P-value was determined using unpaired t test w.r.t. DMSO. ns: P >0.05, *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001. (F) Images from live cell microscopy, monitoring IAV entry. IAV (X-31) particles were labelled with SP-DiOC18(3) (green) and were allowed to enter A549 cells expressing Rab5-RFP (red) in presence of DMSO or DPUD-1 or DPUD-2 (10 μM) for 25–30 min, following which, images were acquired. Yellow signal indicates colocalization of the virus (green) and Rab5-positive early endosome (red). Cell boundary is shown with dotted line. Scare bars, 10 μm. The P-value was determined using one-way ANOVA with multiple comparisons w.r.t. DMSO. ns: P >0.05, *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001.
Fig 4.
DPUDs target endocytosis and facilitate intracellular Cl- transport.
(A) Effect of DPUDs (10 μM) on the cellular uptake of EGF, Tfn, CTxB, and Dex. (B) Confocal images of A549 cells treated with DPUD-1 (10 μM) or DMSO for 1 h. IIF was performed to detect early and late endosomes using antibodies against EEA1 (cyan) and LAMP1 (red), respectively. Phalloidin-AF647 and Hoechst were used to stain actin filaments (yellow) and nuclei (magenta), respectively. Scale bars, 20 μm. (C) Quantification of EEA1 fluorescence intensities in A549 cells treated with DPUDs (10 μM) for 1 h. One hundred cells were counted for each compound. (D) Treatment with DPUDs (10 μM) during entry phase (2 h) blocked SFV infection in BHK-21 cells. (E) Quantification of vacuolar acidification, probed by an acid-sensing dye LysoTracker Red DND-99. A549 cells were pre-treated with DPUDs (10 μM) for 1 h, followed by incubation with the dye for 1 h in presence of the compounds. Vacuolar acidification was measured by quantifying the intensity of LysoTracker Red DND-99. (F) DPUD-1 facilitates Cl- transport across lipid membrane. Large unilamellar vesicles (LUVs) containing Cl—sensitive lucigenin were generated. In presence of chloride ions, DMSO/DPU (10 μM)/DPUD-1 (10 μM) was added and the fluorescent intensity over time was measured. (G) Lucigenin fluorescence intensity in LUVs over time upon DMSO/DPU (10 μM)/DPUD-1 (10 μM) addition in absence of chloride ions. (H) Calculated pKa values of DPU and DPUDs. (I) Effect of DPUDs (10 μM) in intracellular Cl- accumulation. A genetically-encoded YFP Cl- sensor (mClY) (green) and mCherry-tagged LAMP1 (mCherry-LAMP1) (red) were expressed in A549 cells, and the cells were treated with DPUDs (10 μM) for 1 h. Cell boundary is shown with dotted line. Dark regions in the zoomed images indicate Cl- accumulation. Scale bars, 10 μm. The P-value was determined using one-way ANOVA with multiple comparisons w.r.t. DMSO. ns: P >0.05, *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001.
Fig 5.
DPUD-1 and -23 protect mice against SARS-CoV-2 (MA10) infection.
(A) Schematic representation of the in vivo experimental design. (B) Graph showing % body weights of uninfected mice, and SARS-CoV-2 (MA10)-infected mice treated with DPUD-1 (2 mg/kg/day) or -2 (2 mg/kg/day) or molnupiravir (50 mg/kg/day). (C) Survival curves of mice up to 6 days post-infection. (D-E) Quantification of lung viral titres by RT-PCR targeting SARS-CoV-2 RdRp and N genes, and by viral plaque assay. (F) Microscopy images of lung sections, stained with hematoxylin and eosin. (G) Statistical analysis of lung pathology scores. The P-value was determined using one-way ANOVA with multiple comparisons w.r.t. DMSO. ns: P >0.05, *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001.