Broad-Spectrum Host-Based Antivirals Targeting the Interferon and Lipogenesis Pathways as Potential Treatment Options for the Pandemic Coronavirus Disease 2019 (COVID-19)

Abstract

The ongoing Coronavirus Disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) signals an urgent need for an expansion in treatment options. In this study, we investigated the anti-SARS-CoV-2 activities of 22 antiviral agents with known broad-spectrum antiviral activities against coronaviruses and/or other viruses. They were first evaluated in our primary screening in VeroE6 cells and then the most potent anti-SARS-CoV-2 antiviral agents were further evaluated using viral antigen expression, viral load reduction, and plaque reduction assays. In addition to remdesivir, lopinavir, and chloroquine, our primary screening additionally identified types I and II recombinant interferons, 25-hydroxycholesterol, and AM580 as the most potent anti-SARS-CoV-2 agents among the 22 antiviral agents. Betaferon (interferon-β1b) exhibited the most potent anti-SARS-CoV-2 activity in viral antigen expression, viral load reduction, and plaque reduction assays among the recombinant interferons. The lipogenesis modulators 25-hydroxycholesterol and AM580 exhibited EC₅₀ at low micromolar levels and selectivity indices of >10.0. Combinational use of these host-based antiviral agents with virus-based antivirals to target different processes of the SARS-CoV-2 replication cycle should be evaluated in animal models and/or clinical trials.

Keywords: 25-hydroxycholesterol; AM580; COVID-19; coronavirus; interferon; treatment.

Figure 1

Primary screening of 22 antiviral agents with broad-spectrum antiviral activities against coronaviruses and/or other viruses. VeroE6 cells were infected with SARS-CoV-2 (multiplicity of infection = 0.001) and treated with the fixed concentration of 10,000 IU/mL for each IFN or 20 µM for each of the other antiviral agents. The cell culture supernatants were collected at 72 h post-inoculation for viral load quantitation by quantitative reverse transcription-polymerase chain reaction. The experiments were performed in triplicate. The cut-off value of ≥90% inhibition (i.e., ≥1 log10 copies/mL reduction when compared with the DMSO control) was used to select the antiviral agents for further evaluation (magenta dots = recombinant interferons, blue dots = lipogenesis modulators, and green dots = antiviral agents recently reported to be active against SARS-CoV-2). Abbreviation: 25-HC, 25-hydroxycholesterol; DMSO, dimethyl sulfoxide.

Figure 2

SARS-CoV-2 nucleocapsid (N) antigen expression assay. Immunofluorescence staining of SARS-CoV-2-N antigens (labelled with in-house rabbit antiserum against SARS-CoV-2-N in green) and cell nuclei (labelled with 4′,6-diamidino-2-phenylindole in blue). Fixation and staining was performed after each recombinant IFN (3000 IU/mL) or antiviral agent (20 µM) was used to treat the SARS-CoV-2-infected compound (multiplicity of infection = 0.1) VeroE6 cells for 24 h. Scale bar = 100 µm.

Figure 3

SARS-CoV-2 viral load reduction assay. VeroE6 cells were infected with SARS-CoV-2 (multiplicity of infection = 0.01) and treated with different concentrations of the selected antiviral agents as indicated. The culture supernatants of the SARS-CoV-2-infected cells were harvested at 48 h post-inoculation for quantitative reverse transcription-polymerase chain reaction analysis to determine the viral RNA load. * indicates p < 0.05 and ** indicates p < 0.01. The results are presented as mean ± standard deviations. The experiments were performed in triplicate and repeated twice for confirmation. Abbreviation: 25-HC, 25-hydroxycholesterol.

Figure 4

SARS-CoV-2 plaque reduction assay. Fifty plaque-forming units of SARS-CoV-2 were added to each well of VeroE6 cell monolayers with or without the addition of the indicated antiviral agents and the plates were then incubated for 1 h at 37 °C in 5% CO2 before removal of unbound viral particles by aspiration of the media and washing once with DMEM. Monolayers were overlaid with media containing 1% low melting agarose in DMEM and different concentrations of the antiviral agents, inverted and incubated for another 72 h. The wells were then fixed with 10% formaldehyde overnight. After removal of the agarose plugs, the monolayers were stained with 0.7% crystal violet and the plaques counted. The experiments were performed in triplicate and repeated twice for confirmation. Abbreviation: 25-HC, 25-hydroxycholesterol.

Figure 5

Time-of-drug-addition assay for 25-HC and AM580. (A) Schematic representation of the experimental design of time-of-drug-addition assay. The grey blocks indicate the duration of virus adsorption and the black blocks represent the incubation periods between the cells and individual compounds. (B) The viral loads in the culture supernatants normalized by DMSO at the different phases of the assay were shown. The experiments were performed in triplicate and replicated twice. The results are shown as mean ± standard deviations. Abbreviation: 25-HC, 25-hydroxycholesterol.