Glycopeptide Antibiotics Potently Inhibit Cathepsin L in the Late Endosome/Lysosome and Block the Entry of Ebola Virus, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV)

Abstract

Ebola virus infection can cause severe hemorrhagic fever with a high mortality in humans. The outbreaks of Ebola viruses in 2014 represented the most serious Ebola epidemics in history and greatly threatened public health worldwide. The development of additional effective anti-Ebola therapeutic agents is therefore quite urgent. In this study, via high throughput screening of Food and Drug Administration-approved drugs, we identified that teicoplanin, a glycopeptide antibiotic, potently prevents the entry of Ebola envelope pseudotyped viruses into the cytoplasm. Furthermore, teicoplanin also has an inhibitory effect on transcription- and replication-competent virus-like particles, with an IC50 as low as 330 nm Comparative analysis further demonstrated that teicoplanin is able to block the entry of Middle East respiratory syndrome (MERS) and severe acute respiratory syndrome (SARS) envelope pseudotyped viruses as well. Teicoplanin derivatives such as dalbavancin, oritavancin, and telavancin can also inhibit the entry of Ebola, MERS, and SARS viruses. Mechanistic studies showed that teicoplanin blocks Ebola virus entry by specifically inhibiting the activity of cathepsin L, opening a novel avenue for the development of additional glycopeptides as potential inhibitors of cathepsin L-dependent viruses. Notably, given that teicoplanin has routinely been used in the clinic with low toxicity, our work provides a promising prospect for the prophylaxis and treatment of Ebola, MERS, and SARS virus infection.

Keywords: Ebola virus; MERS-CoV; SARS-CoV; antibiotics; glycopeptide; glycoprotein; lysosome; virus entry.

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Figures

FIGURE 1.

Teicoplanin specifically inhibits the entry of Ebola viruses.A, teicoplanin and amiodarone specifically inhibit the entry of Ebola viruses. HEK293T cells were seeded in a 96-well plate, and 24 h later, the cells were incubated with various reagents at 37 °C for 1 h. The cells were then infected with HIV-luc/Zaire EBOV-GP (2014) pseudotyped viruses. After washing and incubation with fresh medium for 48 h, the intracellular luciferase activity was measured.B, chemical structure of teicoplanin.C, HEK293T cells were incubated with teicoplanin at various concentrations at 37 °C for 1 h. The intracellular luciferase activity was measured at 48 h post-infection. The IC50 was calculated using GraphPad Prism software. D, HEK293T cells were incubated with 500 μm teicoplanin at 37 °C for 48 h. Then the cell viability was determined.E, HEK293T cells were infected with HIV-luc/Zaire EBOV-GP (2014) pseudotyped viruses and incubated with 50 μm teicoplanin at 0, 2, 4, and 8 h post-infection. The cells were then incubated for 48 h after which the intracellular luciferase activity was tested. The HIV-luc/VSV-G pseudotyped viruses were used as the controls for specificity. F, HEK293T cells were seeded in a 6-well plate (2 × 105 per well). After 24 h, the cells were incubated with teicoplanin at various concentrations at 37 °C for 1 h and then infected with p24-normalized (100 ng) HIV-luc/Zaire EBOV-GP (2014) or HIV-luc/VSV-G pseudotyped viruses per well. After 6 h, the cells were washed with PBS twice and incubated with 0.25% trypsin at 37 °C for 2 min to remove the viruses that adhered to the cell surfaces. The cells were then collected and lysed, and the intracellular amount of HIV-1 p24 was measured by ELISA. The results are representatives of at least three independent experiments. Thebars show the mean values ± S.D. (error bars). The p value was determined by a Student's t test. ***,p < 0.001.

FIGURE 2.

Ebola virus entry into different cell types is repressed by teicoplanin.A, primary human umbilical vein endothelial cells were seeded in a 96-well plate. After 24 h, the cells were incubated with teicoplanin at various concentrations at 37 °C for 1 h. Subsequently, the cells were infected with HIV-luc/Zaire EBOV-GP (2014) pseudotyped viruses. After washing and incubation with fresh medium for 48 h, the intracellular luciferase activity was measured. B, antiviral entry assay of teicoplanin on the A549 cells was conducted in a similar way. C, antiviral entry assay of teicoplanin on the HeLa cells was conducted in a similar procedure. D, THP-1 cells were incubated with teicoplanin at various concentrations at 37 °C for 1 h. Subsequently, the cells were infected with HIV-luc/Zaire EBOV-GP (2014) pseudotyped viruses. After 12 h, the supernatants were removed by centrifugation at 300 × g for 10 min, and the cells were suspended and cultured with RPMI 1640 medium at 37 °C for 48 h. The intracellular luciferase activity was then tested. The results are representative of at least three independent experiments. The barsshow the mean values ± S.D. (error bars). The p value was determined by a Student'st test. **, p < 0.01; ***, p < 0.001.

FIGURE 3.

Target of teicoplanin is located within the host cells.A, p24-normalized (50 ng) HIV-luc/Zaire EBOV-GP (2014) pseudotyped viruses were incubated with 50 μm teicoplanin in a 96-well plate at 37 °C for 12 h. Then the compound virus mixtures were transferred into a Microcon 30-kDa centrifugal filter device (Millipore) and centrifuged (7000 × g) at 4 °C for 15 min. Subsequently, fresh medium was added twice onto the filter device to wash the compound virus mixtures. Then 0.5 ml of DMEM was used to suspend the compound virus mixtures, and the reversed filter device was centrifuged (500 ×g) at 4 °C for 5 min. The solution was collected and used to infect HEK293T cells after HIV-1 p24 normalization. After 48 h, the intracellular luciferase activity was measured. B, HEK293T cells were incubated with teicoplanin at various concentrations for 12 h. The cells were then infected with HIV-luc/Zaire EBOV-GP (2014) pseudotyped viruses. After washing and incubation with fresh medium for 48 h, the intracellular luciferase activity was measured. The assay during treatment was the same as the antiviral entry assay. The results are representative of at least three independent experiments. The bars show the mean values ± S.D. (error bars). Thep value was determined by a Student'st test. n.s., not significant; **, p < 0.01; ***,p < 0.001.

FIGURE 4.

Teicoplanin does not block the cell receptor.A, schematic representation of the entry of Ebola viruses and SARS-CoVs. B, HEK293T cells were incubated with teicoplanin at various concentrations at 37 °C for 1 h. The cells were then infected with HIV-luc/SARS-CoV-S pseudotyped viruses. After washing and incubation with fresh medium for 48 h, the intracellular luciferase activity was measured. C, HEK293T cells were seeded in a 96-well plate. After 24 h, the cells were transfected with 200 nm siRNAs against NPC1 and ACE2, respectively. After 48 h, the cells were infected with HIV-luc/Zaire EBOV-GP (2014) or HIV-luc/SARS-CoV-S pseudotyped viruses. The results are representative of at least three independent experiments. The bars show the mean values ± S.D. (error bars). Thep value was determined by a Student'st test. n.s., not significant; *, p < 0.05; **,p < 0.01; ***, p < 0.001.

FIGURE 5.

Teicoplanin has no effect on HOPS complexes.A, schematic representation of the entry of Ebola viruses, SARS-CoVs, and VSVs. The host factors thatare essential for the entry of Ebola viruses are illustrated.B, HEK293T cells were transfected with 200 nm siRNAs per well. After 48 h, the cells were infected with HIV-luc/Zaire EBOV-GP (2014), HIV-luc/SARS-CoV-S, or HIV-luc/VSV pseudotyped viruses. After washing and incubation with fresh medium for 48 h, the intracellular luciferase activity was measured. C, HEK293T cells were plated in a glass bottom dish. After 24 h, groups of cells were incubated with 50 μm teicoplanin or control for 12 h. Another group of cells was transfected with siRNAs against VPS39 and VPS41 or the si-control and incubated with these siRNAs at a final concentration of 200 nm for 48 h. Then the cells were incubated with dextran-Alexa Flour 568 (Mr 10,000) for 2 h in 1% serum-containing DMEM. Subsequently, the cells were subjected to immunofluorescence analysis. Images were obtained using Zeiss LSM780 confocal microscopy with Zeiss ZFN software.Scale bar, 10 μm. D, co-localization of dextran and LAMP1 was analyzed from 20 fields (≥5 cells per field) per independent experiment. Pearson's overlap coefficients were used to determine the levels of the co-localization of dextran and LAMP1. Eand F, HEK293T cells were incubated with teicoplanin at the various indicated concentrations at 37 °C for 1 h. The cells were then infected with HIV-luc/Zaire EBOV-GP (2014) or HIV-luc/VSV-G pseudotyped viruses, respectively. Subsequently, the cells were incubated with 0.25% trypsin at 37 °C for 2 min to remove the viruses that adhered to the cell surfaces at 0.5, 1, and 2 h post-infection. The intracellular amount of HIV-1 p24 was then measured by ELISA. The results are representative of at least three independent experiments. The bars show the mean values ± S.D. (error bars). Thep value was determined by a Student'st test. n.s., not significant; ***, p < 0.001.

FIGURE 6.

Teicoplanin directly inhibits the activity of cathepsin L.A, schematic representation of the cathepsin L enzymatic inhibition assay. B, HEK293T cells were incubated with teicoplanin or Z-Phe-Tyr(t-Bu)-diazomethyl ketones and then incubated with 50 μm (Z-Phe-Arg)2-R110 at 37 °C for 90 min. The level of fluorescence was determined using a fluorometer with excitation at 488 nm and emission at 510 nm. C, HEK293T cells were incubated with teicoplanin at various concentrations at 37 °C for 48 h. The cell viability was then determined using the CellTiter® 96 Aqueous One Solution Cell Proliferation Assay. D, HEK293T cells were incubated with teicoplanin or Z-Phe-Tyr(t-Bu)-diazomethyl ketones at 37 °C for 1 h. The cells were then infected with HIV-luc/Zaire EBOV-GP (2014) or HIV-luc/VSV-G pseudotyped viruses. After incubation for 48 h, the intracellular luciferase activity was measured. E, recombinant human cathepsin L (rhCTSL), teicoplanin, or Z-Phe-Tyr(t-Bu)-diazomethyl ketones were incubated 37 °C for 4 h. The rhCTSL-compound mixtures were incubated with (Z-Phe-Arg)2-R110 at 37 °C for 4 h. The fluorescence was then tested using a fluorometer with excitation at 488 nm and emission at 510 nm. The results are representative of at least three independent experiments. Thebars show the mean values ± S.D. (error bars). The p value was determined by a Student's t test.n.s., not significant, *,p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 7.

Entry of Ebola trVLPs is repressed by glycopeptide antibiotics with the exception of vancomycin.A, schematic representation of the EbolatrVLPs entry assay. B, for the pre-transfection experiment, HEK293T cells were pre-transfected with 12.5 ng of pCAGGS-NP, 12.5 ng of pCAGGS-VP35, 7.5 ng of pCAGGS-VP30, 100 ng of pCAGGS-L, 25 ng of pCAGGS-T7, and 25 ng of pCAGGS-Tim1 plasmids. After 24 h, the medium was discarded, and the cells were incubated with teicoplanin at various concentrations at 37 °C for 1 h. After washing and incubation with fresh medium for 48 h, the intracellular luciferase activity was measured. The IC50 was calculated using GraphPad Prism software. C, for the non-pre-transfection experiment, HEK293T cells were incubated with teicoplanin at various concentrations at 37 °C for 1 h without pre-transfection of the above plasmids. The methods used for conducting Ebola trVLPs infection and measuring the intracellular luciferase activity were the same as those described in B. D and E, IC50 values of dalbavancin on the entry of Ebola trVLPs were determined under pre-transfection and non-pre-transfection conditions, respectively.F and G, IC50 values of oritavancin on the entry of Ebola trVLPs were calculated using pre-transfection or non-pre-transfection conditions, respectively. H andI, IC50 values of telavancin on the entry of Ebola trVLPs were determined under pre-transfection and non-pre-transfection conditions, respectively.J and K, effect of vancomycin on the entry of Ebola trVLPs was determined using pre-transfection or non-pre-transfection conditions, respectively. The results are representative of at least three independent experiments. The bars show the mean values ± S.D. (error bars). Thep value was determined by a Student'st test. n.s., not significant.

FIGURE 8.

Entry of MERS-CoVs and SARS-CoVs is inhibited by glycopeptide antibiotics with the exception of vancomycin.A and E, HEK293T cells were incubated with teicoplanin at various concentrations at 37 °C for 1 h. The cells were infected with HIV-luc/MERS-CoV-S or HIV-luc/SARS-CoV-S pseudotyped viruses. After washing and incubation with fresh medium for 48 h, the intracellular luciferase activity was measured. The IC50 was calculated using GraphPad Prism software. B andF, IC50 values of dalbavancin on the entry of HIV-luc/MERS-CoV-S and HIV-luc/SARS-CoV-S pseudotyped viruses were determined. C andG, IC50 values of oritavancin on the entry of HIV-luc/MERS-CoV-S and HIV-luc/SARS-CoV-S pseudotyped viruses were calculated. D andH, IC50 values of telavancin on the entry of HIV-luc/MERS-CoV-S and HIV-luc/SARS-CoV-S pseudotyped viruses were determined. I andJ, The effect of vancomycin on the entry of HIV-luc/MERS-CoV-S and HIV-luc/SARS-CoV-S pseudotyped viruses was determined. The results are representative of at least three independent experiments. The bars show the mean values ± S.D. (error bars). Thep value was determined by a Student'st test. n.s., not significant.

FIGURE 9.

Chemical structures of glycopeptide antibiotics. Teicoplanin, dalbavancin, oritavancin, and telavancin, which inhibit the entry of Ebola trVLPs, MERS-CoVs, and SARS-CoVs, contain key hydrophobic groups. However, vancomycin, which does not exert antiviral activity, does not contain these groups.