Journal: Nature Communications

Article Title: Tubeimosides are pan-coronavirus and filovirus inhibitors that can block their fusion protein binding to Niemann-Pick C1

doi: 10.1038/s41467-023-44504-4

Figure Lengend Snippet: A A total of 19 compounds from a library containing 974 plant-sourced small molecules were tested at 1 μM for antiviral activity to EBOV, HIV-1, and VSV using HIV-1 Luc-reporter pseudoviruses. EBOV-GP (red)- and VSV-G (blue)-mediated infection were conducted in Vero-E6 cells, and HIV-1 Env (green)-mediated infection was done in TZM-bI cells. Five compounds specifically targeting EBOV are circled. Viral infection was shown as intracellular luciferase activity. RLU, relative light unit. B The chemical structures of six compounds are shown. C The anti-EBOV activity of Toremifene citrate (Tor), Tamoxifen citrate (Tam), and Tubeimosides (Tubs) I/II/III were determined at indicated concentrations using HIV-1 Luc-pseudoviruses, EBOV trVLPs, and HIV-1 GFP-pseudoviruses. Levels of viral infections were calculated as relative values, with the DMSO treatment set as 100. The percentage of inhibition was calculated by deduction of the relative viral infection value from 100. Error bars indicate SEMs ( n = 3 biologically independent experiments). One-way ANOVA was applied. ** P < 0.01, *** P < 0.001, ****<0.0001. D Vero-E6 cells were infected with HIV-1 GFP-pseudoviruses expressing EBOV-GP and treated with Tor, Tam, and Tubs I/II/III at indicated concentrations. Infected cells were imaged by EVOS FL Auto Imaging System (scale bar, 200 μm). E The anti-EBOV activity of Tubs I/II/III was titrated in Vero-E6, SNB-19, and Huh-7 cells using HIV-1 Luc-pseudoviruses. The anti-EBOV activity of U18666A (U18) was also titrated in Huh-7 cells similarly. The percentage of inhibition was calculated similarly as aforementioned. F The anti-EBOV activity of Tam, Tor, and Tubs I/II/III was titrated in HEK293T cells using EBOV trVLPs. The percentage of inhibition was calculated similarly as aforementioned. G A summary of IC 50 values on viral infection. H The cytotoxic effect of Tubs I/II/III was titrated in Vero-E6 cells using CellTiter-Glo® Luminescent Cell Viability Assay. Their CC 50 values are shown.

Article Snippet: Goat anti-human ACE2 affinity purified IgG (Cat No. AF933) (1:5000) was from R&D Systems; rabbit anti-human NPC1 (Cat No. ab134113) (1:5000) was from Abcam; rabbit anti-SARS-CoV-spike protein (Cat No. NB100-56047) (1:3000) was from Novus Biologicals; rabbit anti-SARS-CoV-2 spike RBD protein (Cat No. 40592-T62) (1:3000), rabbit anti-Zaire EBOV-GP (Cat No. 40442-T48) (1:5000) were from Sino Biological; mouse monoclonal anti-VSV-G (Cat No. Cat#A02180) (1:2000) was from Abbkine; rat monoclonal anti-FLAG (Cat No. F3165) (1:10,000), horseradish Peroxidase (HRP)-conjugated anti-FLAG (Cat No. A8592) (1:10,000), HRP-conjugated anti-HA (Cat No. H6533) (1:10,000), and HRP-conjugated anti-β-Actin (Cat No. A3854) (1:5000)were from Sigma Aldrich ; HRP-conjugated AffiniPure donkey anti-goat IgG (H + L) (Cat. No. 705-035-003), HRP-conjugated goat anti-mouse IgG (Cat No. 115-035-003) (1:5000) and anti-rabbit IgG (Cat No. 111-035-003) (1:5000), and APC-conjugated polyclonal anti-goat antibody (Cat No. 705-136-147) (1:500) were from Jackson ImmunoResearch.

Techniques: Activity Assay, Infection, Luciferase, Inhibition, Expressing, Imaging, Cell Viability Assay

Journal: Nature Communications

Article Title: Tubeimosides are pan-coronavirus and filovirus inhibitors that can block their fusion protein binding to Niemann-Pick C1

doi: 10.1038/s41467-023-44504-4

Figure Lengend Snippet: A Docking of indicated compounds to NPC1-C and EBOV-GP complex (PDB: 5F1B) was analyzed via Webina. B SNB-19 cells were treated overnight with indicated compounds. After being fixed, cells were stained with filipin (50 µg/mL) and visualized by confocal microscopy (scale bar, 40 μm). Experiments were repeated 3 times independently, and representative results are shown. C Vero-E6 cells were treated with Tubs I/II/III at 0.5 μM and infected with HIV-1 Luc-pseudovirus expressing MARV-GP. Viral infection was determined as previously. D Vero-E6 cells were treated with indicated compounds and infected with HIV-1 Luc-pseudovirus expressing EBOV-GP. Tor, Tam, and Tubs I/II/III were used at 2.5 μM, 5.0 μM, or 0.5 μM, respectively. DMSO was used as a control (Ctr). E Vero-E6 cells were pretreated for 1 h with EIPA and Tubs I/II/III and spinoculated with GFP-labeled EBOV-VLPs expressing EBOV-GP or VSV-G. After removal of VLPs and culture for another 3 h, GFP-positive cells were quantified by flow cytometry. EIPA [5-(N-ethyl-N-isopropyl) amiloride] and Tubs I/II/III were used at 25 μM, or 0.5 μM, respectively. F CTS-B or CTS-L substrates were incubated with cell lysate from A549 cells treated with Tubs I/II/III, and their activity was determined. E-64-d ethyl ester (EST) was used at 10 μM and Tubs I/II/III were used at indicated concentrations. G A549 cells were treated with NH 4 Cl at 100 µM and Tubs I/II/III at 0.5 μM. After being stained with LysoTracker Red, cells were visualized by fluorescence microscope (EVOS FL Auto Imaging System) (scale bar, 200 μm). Experiments were repeated 3 times independently, and representative results are shown. Error bars in C and D indicate SEMs ( n = 3 biologically independent experiments). One-way ANOVA was applied. *** P < 0.001, **** P < 0.0001; n.s. not significant.

Article Snippet: Goat anti-human ACE2 affinity purified IgG (Cat No. AF933) (1:5000) was from R&D Systems; rabbit anti-human NPC1 (Cat No. ab134113) (1:5000) was from Abcam; rabbit anti-SARS-CoV-spike protein (Cat No. NB100-56047) (1:3000) was from Novus Biologicals; rabbit anti-SARS-CoV-2 spike RBD protein (Cat No. 40592-T62) (1:3000), rabbit anti-Zaire EBOV-GP (Cat No. 40442-T48) (1:5000) were from Sino Biological; mouse monoclonal anti-VSV-G (Cat No. Cat#A02180) (1:2000) was from Abbkine; rat monoclonal anti-FLAG (Cat No. F3165) (1:10,000), horseradish Peroxidase (HRP)-conjugated anti-FLAG (Cat No. A8592) (1:10,000), HRP-conjugated anti-HA (Cat No. H6533) (1:10,000), and HRP-conjugated anti-β-Actin (Cat No. A3854) (1:5000)were from Sigma Aldrich ; HRP-conjugated AffiniPure donkey anti-goat IgG (H + L) (Cat. No. 705-035-003), HRP-conjugated goat anti-mouse IgG (Cat No. 115-035-003) (1:5000) and anti-rabbit IgG (Cat No. 111-035-003) (1:5000), and APC-conjugated polyclonal anti-goat antibody (Cat No. 705-136-147) (1:500) were from Jackson ImmunoResearch.

Techniques: Staining, Confocal Microscopy, Infection, Expressing, Labeling, Flow Cytometry, Incubation, Activity Assay, Fluorescence, Microscopy, Imaging

Journal: Nature Communications

Article Title: Tubeimosides are pan-coronavirus and filovirus inhibitors that can block their fusion protein binding to Niemann-Pick C1

doi: 10.1038/s41467-023-44504-4

Figure Lengend Snippet: A Huh-7 cells were treated with SARS2-VLPs and infected with HIV-1 Luc-pseudovirus expressing EBOV-GP. Alternatively, Huh-7-A-T cells were treated with EBOV-VLPs and infected with HIV-1 Luc-pseudovirus expressing SARS2-S. Viral infection is presented as relative values, with the infection in the absence of VLPs set as 100. B Huh-7-A-T cells were infected with HIV-1 Luc-pseudoviruses expressing VSV-G or SARS2-S and treated with indicated compounds at 1 µM. DMSO was used as a control (Ctr). The percentage of inhibition was calculated similarly as aforementioned. C The anti-SARS2 activity of Tubs I/II/III were measured in Huh-7-A-T cells using HIV-1 pseudovirus expressing SARS2-S, and their IC 50 values are indicated. D Huh-7-A-T cells were treated with 1 μM Tubs I/II/III at indicated time points and infected with HIV-1 Luc-pseudovirus expressing SARS2-S. DMSO was used as a control (Ctr). Viral inhibition was determined as previously, E NPC1 was expressed with EBOV-GP, VSV-G, or SARS2-S in HEK293T cells. NPC1 was immunoprecipitated (IP), and proteins in cell lysate (input) and pulldown samples were analyzed by WB. F NPC1 and deletion mutants 1-620 or 1-377 were expressed with EBOV-GP or SARS2-S in HEK293T cells. These NPC1 proteins were immunoprecipitated and proteins were detected by WB. G HIV-1 Luc-pseudoviruses expressing EBOV-GP or SARS2-S were purified by ultracentrifugation. After treatment with thermolysin (TL) at 200 µg/mL, proteins were analyzed by WB. H Recombinant NPC1 proteins were purified by immunoprecipitation from HEK293T cells transfected with a NPC1-expression vector. NPC1 was incubated with purified HIV-1 pseudoviruses expressing EBOV-GP or SARS2-S pre-treated with TL. Proteins associated with NPC1 were pulled down and analyzed by WB. I The anti-SARS2 activity of U18666A (U18) was measured in Huh-7-A-T cells using HIV-1 Luc-pseudovirus expressing SARS2-S. J Purified recombinant NPC1 proteins were incubated with indicated compounds, followed by incubation with HIV-1 pseudoviruses expressing EBOV-GP or SARS2-S after cleavage by TL. Proteins associated with NPC1 were pulled down and analyzed by WB. Experiments in E – J were repeated 3 times independently, and representative results are shown.

Article Snippet: Goat anti-human ACE2 affinity purified IgG (Cat No. AF933) (1:5000) was from R&D Systems; rabbit anti-human NPC1 (Cat No. ab134113) (1:5000) was from Abcam; rabbit anti-SARS-CoV-spike protein (Cat No. NB100-56047) (1:3000) was from Novus Biologicals; rabbit anti-SARS-CoV-2 spike RBD protein (Cat No. 40592-T62) (1:3000), rabbit anti-Zaire EBOV-GP (Cat No. 40442-T48) (1:5000) were from Sino Biological; mouse monoclonal anti-VSV-G (Cat No. Cat#A02180) (1:2000) was from Abbkine; rat monoclonal anti-FLAG (Cat No. F3165) (1:10,000), horseradish Peroxidase (HRP)-conjugated anti-FLAG (Cat No. A8592) (1:10,000), HRP-conjugated anti-HA (Cat No. H6533) (1:10,000), and HRP-conjugated anti-β-Actin (Cat No. A3854) (1:5000)were from Sigma Aldrich ; HRP-conjugated AffiniPure donkey anti-goat IgG (H + L) (Cat. No. 705-035-003), HRP-conjugated goat anti-mouse IgG (Cat No. 115-035-003) (1:5000) and anti-rabbit IgG (Cat No. 111-035-003) (1:5000), and APC-conjugated polyclonal anti-goat antibody (Cat No. 705-136-147) (1:500) were from Jackson ImmunoResearch.

Techniques: Infection, Expressing, Inhibition, Activity Assay, Immunoprecipitation, Purification, Recombinant, Transfection, Plasmid Preparation, Incubation

Journal: Nature Communications

Article Title: Tubeimosides are pan-coronavirus and filovirus inhibitors that can block their fusion protein binding to Niemann-Pick C1

doi: 10.1038/s41467-023-44504-4

Figure Lengend Snippet: A NPC1 was knocked out in indicated cells (see Supplementary Figs. – ). At least one KO clone was obtained from each cell line. The expression of NPC1 and ACE2 in these cells was determined by WB. The ACE2 expression on the cell surface was also determined in A549 WT (−) and WT-A (+) cells by flow cytometry after cells were gated by higher forward scatter (FSC) and side scatter (SSC). Experiments were repeated 3 times independently, and representative results are shown. B Indicated CHO WT and NPC1 -KO cells were infected with HIV-1 Luc-pseudoviruses expressing VSV-G, EBOV-GP, or SARS2-S, and viral infection was determined. C Human NPC1 and its three mutants P691S, L656F, and D786N were expressed in CHO NPC1 -KO cells expressing ACE2 (CHO KO-A). Cells were infected with HIV-1 Luc-pseudoviruses expressing EBOV-GP or SARS2-S, and viral infection was determined. D Indicated A549 cells were infected with HIV-1 Luc-pseudoviruses expressing EBOV-GP or VSV-G, and viral infection was determined. E Indicated A549 cells were transfected with an ACE2- or a TMPRSS2-expression vector and infected with HIV-1 Luc-pseudovirus expressing SARS2-S. Viral infection was determined. F Indicated Caco2 and Vero-E6 cells were infected with HIV-1 Luc-pseudoviruses expressing EBOV-GP, VSV-G, or SARS2-S, and viral infection was determined. G Indicated cell lines were infected with SARS2 authentic viruses. After 24 or 48 h, viral RNA copy numbers were determined by real-time PCR. H HPAE II cells were transfected with indicated siRNAs and infected with SARS2 authentic viruses. NPC1 expression in these cells was detected by WB and viral infection was detected by real-time PCR after 24 or 48 h of infection. I The IC 50 of Tubs I/II/III for authentic SARS2 were measured in Vero-E6 cells. Viral titers were determined by plaque assay after 24 h of infection. PFU, plaque forming units. Error bars in C , E , and F indicate SEMs ( n = 4 biologically independent experiments). One-way ANOVA was applied. *** P < 0.001, **** P < 0.0001.

Article Snippet: Goat anti-human ACE2 affinity purified IgG (Cat No. AF933) (1:5000) was from R&D Systems; rabbit anti-human NPC1 (Cat No. ab134113) (1:5000) was from Abcam; rabbit anti-SARS-CoV-spike protein (Cat No. NB100-56047) (1:3000) was from Novus Biologicals; rabbit anti-SARS-CoV-2 spike RBD protein (Cat No. 40592-T62) (1:3000), rabbit anti-Zaire EBOV-GP (Cat No. 40442-T48) (1:5000) were from Sino Biological; mouse monoclonal anti-VSV-G (Cat No. Cat#A02180) (1:2000) was from Abbkine; rat monoclonal anti-FLAG (Cat No. F3165) (1:10,000), horseradish Peroxidase (HRP)-conjugated anti-FLAG (Cat No. A8592) (1:10,000), HRP-conjugated anti-HA (Cat No. H6533) (1:10,000), and HRP-conjugated anti-β-Actin (Cat No. A3854) (1:5000)were from Sigma Aldrich ; HRP-conjugated AffiniPure donkey anti-goat IgG (H + L) (Cat. No. 705-035-003), HRP-conjugated goat anti-mouse IgG (Cat No. 115-035-003) (1:5000) and anti-rabbit IgG (Cat No. 111-035-003) (1:5000), and APC-conjugated polyclonal anti-goat antibody (Cat No. 705-136-147) (1:500) were from Jackson ImmunoResearch.

Techniques: Expressing, Flow Cytometry, Infection, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Plaque Assay