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: Tubeimosides I (Cas No. 102040-03-9), Tubeimosides II (Cas No. 115810-12-3), and Tubeimosides III (Cas No. 115810-13-4) were purchased from Chengdu Biopurity Phytochemicals Ltd, China; U18666A (Cat No. 50205-0551) was from Cell Signaling Technology; Cathepsin B substrate (Cat No. sc-215529) and Cathepsin L substrate (Cat No. sc-3136) were from Santa Cruz Biotechnology; Filipin III (Cat No. GC12048) and CA074 (Cat No. GC15917) were from GLPBIO; EIPA (Cat No. HY-101840) and EST (E-64-d) (Cat No. HY-100229), and cholesterol (Cat No. HY-N0322) were from MedChemExpress; N-Dodecyl-β-D-maltoside (DDM) (Cat No. D4641), recombinant soluble ACE2 (Cat No. SAE0064), thermolysin from Bacillus thermoproteolyticus (Cat No. P1512), and anti-protease inhibitor cocktail (Cat No. P8340) were from Sigma Aldrich; Lyso-Tracker Red (Cat No. C1046), phosphoramidon (Metalloproteinase inhibitor) (Cat No. SG2024), and acetate buffer pH5.2 (Cat No. ST351) were from Beyotime Biotechnology; Lipofectamine 3000 (Cat No. L3000015) and Blasticidin S HCl (R21001) were from ThermoFisher; Puromycin (Cat No. ant-pr-1) was from InvivoGen; polyethyleneimine (Cat No. 23966-1) was from Polysciences, Inc; recombinant NPC1-C protein (Cat No. 16499-H32H) and recombinant SARS-CoV-2 spike RBD protein (Cat. No. 40592-V08H) were from SinoBiological.

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 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: Tubeimosides I (Cas No. 102040-03-9), Tubeimosides II (Cas No. 115810-12-3), and Tubeimosides III (Cas No. 115810-13-4) were purchased from Chengdu Biopurity Phytochemicals Ltd, China; U18666A (Cat No. 50205-0551) was from Cell Signaling Technology; Cathepsin B substrate (Cat No. sc-215529) and Cathepsin L substrate (Cat No. sc-3136) were from Santa Cruz Biotechnology; Filipin III (Cat No. GC12048) and CA074 (Cat No. GC15917) were from GLPBIO; EIPA (Cat No. HY-101840) and EST (E-64-d) (Cat No. HY-100229), and cholesterol (Cat No. HY-N0322) were from MedChemExpress; N-Dodecyl-β-D-maltoside (DDM) (Cat No. D4641), recombinant soluble ACE2 (Cat No. SAE0064), thermolysin from Bacillus thermoproteolyticus (Cat No. P1512), and anti-protease inhibitor cocktail (Cat No. P8340) were from Sigma Aldrich; Lyso-Tracker Red (Cat No. C1046), phosphoramidon (Metalloproteinase inhibitor) (Cat No. SG2024), and acetate buffer pH5.2 (Cat No. ST351) were from Beyotime Biotechnology; Lipofectamine 3000 (Cat No. L3000015) and Blasticidin S HCl (R21001) were from ThermoFisher; Puromycin (Cat No. ant-pr-1) was from InvivoGen; polyethyleneimine (Cat No. 23966-1) was from Polysciences, Inc; recombinant NPC1-C protein (Cat No. 16499-H32H) and recombinant SARS-CoV-2 spike RBD protein (Cat. No. 40592-V08H) were from SinoBiological.

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