SARS-COV-2 has recently emerged as a new public health threat. Herein, we report that the FDA-approved gold drug, auranofin, inhibits SARS-COV-2 replication in human cells at low micro molar concentration. Treatment of cells with auranofin resulted in a 95% reduction in the viral RNA at 48 hours after infection. Auranofin treatment dramatically reduced the expression of SARS-COV-2-induced cytokines in human cells. These data indicate that auranofin could be a useful drug to limit SARS-CoV-2 infection and associated lung injury due to its anti-viral, anti-inflammatory and anti-ROS properties. Auranofin has a well-known toxicity profile and is considered safe for human use.
We investigated the anti-viral activity of auranofin against SARS-CoV-2 and its effect on virus-induced inflammation in human cells. We infected Huh7 cells with SARS-CoV-2 (USA-WA1/2020) at a multiplicity of infection (MOI) of 1 for 2 hours, followed by the addition of 4 μM of auranofin. DMSO (0.1%) was used as control (the solvent was used to prepare drug stock). Cell culture supernatants and cell lysates were collected at 24 and 48 hours after infection. Virus RNA copies were measured by RT-PCR using two separate primers specific for the viral N1 gene and N2 gene. As depicted, treatment of cells with auranofin resulted in a 70% reduction in the viral RNA in the supernatant compared to the DMSO at 24 hours after infection. At 48 hours, there was an 85% reduction in the viral RNA in the supernatant compared to the DMSO. Similarly, the levels of intracellular viral RNA decreased by 85% at 24 hours and 95% at 48 hours in auranofin-treated cells compared to the DMSO-treated cells. Both set of primers showed nearly identical results. Auranofin showed no toxicity against Huh7 cells at the used concentration at 24 and 48 hours.
To determine the effective concentration of auranofin that inhibits 50% of viral replication (EC50), we treated SARS-COV-2 infected Huh7 cells with serial dilutions of auranofin. Supernatants and cell lysates were collected at 48 hours after infection and viral RNA was quantified by RT-PCR. The data were plotted in graphs using non-linear regression model (GraphPad software). At 48 hours, there was a dose-dependent reduction in viral RNA levels in the auranofin-treated cells.Represents the EC50 values of auranofin treatment against SARS-CoV-2 infected Huh7 cells. Auranofin inhibited virus replication in the infected cells at EC50 of approximately 1.5 μM.
To assess the effect of auranofin on inflammatory response during SARS-COV-2 infection, we measured the levels of key cytokines in auranofin and DMSO-treated cells at 24 and 48 hours after infection. SARS-COV-2 infection induces a strong up-regulation of IL-6, IL-1β, TNFα and NF-kB in Huh7 cells. Treatment with auranofin dramatically reduced the expression of SARS-COV-2-induced cytokines in Huh7 cells. SARS-COV-2 infection resulted in a 200-fold increase in the mRNA expression of IL-6 at 48 hours after infection compared to corresponding mock-infected cells. In contrast, there was only a 2-fold increase in expression of IL-6 in auranofin-treated cells. TNF-α levels increased by 90-fold in the DMSO-treated cells at 48 hours after infection, but this increase was absent in the auranofin-treated cells. Similarly, no increase in the expression of IL-1β and NF-kB was observed in the auranofin-treated cells.
Methods
SARS-COV-2 infection and drug treatment In this study, we used a novel SARS-COV-2 (USA-WA1/2020) isolated from an oropharyngeal swab from a patient in Washington, USA (BEI NR-52281). Virus strain was amplified once in Vero E6 cells and had titers of 5 × 10 plaque-forming units (PFU)/mL. Huh7 cells (human liver cell line) were grown in DMEM (Gibco) supplemented with 5% heat-inactivated fetal bovine serum. Cells were infected with SARS-COV-2 or PBS (Mock) at a multiplicity of infection (MOI) of 1 for 2 hours. Cell were washed twice with PBS and media containing different concentrations of auranofin (Sigma) or DMSO (Sigma) was added to cells. Supernatants and cell lysates were harvested at 24 and 48 hours after infection.
Viral RNA quantification Virus RNA levels were analyzed in the supernatant and cell lysates by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). RNA from cell culture supernatants was extracted using a Viral RNA Mini Kit (Qiagen) and RNA from cell lysates was extracted using a RNeasy Mini Kit (Qiagen) as described previously. qRT-PCR was used to measure viral RNA levels using previously published primers and probes specific for the SARS-COV-2. Forward (5′-GACCCCAAAATC AGCGAAAT-3′), reverse (5′-TCTGGTTACTGCCAGTTGAATCTG-3′), probe, (5′-FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1-3’) targeting the SARS-COV-2 N1 gene and Forward (5′-TTACAAACATTGGCCGCAAA-3′), reverse (5′-GCGCGACATTCCGAAGAA3′), probe, (5′-FAM-ACAATTTGCCCCCAGCGCTTCAG-BHQ1-3’) targeting the SARS-COV-2 N2 gene (Integrated DNA Technologies). Viral RNA copies were determined after comparison with a standard curve produced using serial 10-fold dilutions of SARS-COV-2 RNA.
Cytokine analysis For mRNA analysis of IL-6, IL-1β, TNFα and NF-kB, cDNA was prepared from RNA isolated from the cell lysates using a iScript™ cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA), and qRT-PCR was conducted as described previously. The primer sequences used for qRT-PCR are listed
Reference & Source information: https://www.biorxiv.org/
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