Broad Anti-coronavirus Activity of Food and Drug Administration-Approved Drugs against SARS-CoV-2 In Vitro and SARS-CoV In Vivo

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China at the end of 2019 and has rapidly caused a pandemic, with over 20 million recorded COVID-19 cases in August 2020 (https://covid19.who.int/). There are no FDA-approved antivirals or vaccines for any coronavirus, including SARS-CoV-2. Current treatments for COVID-19 are limited to supportive therapies and off-label use of FDA-approved drugs. Rapid development and human testing of potential antivirals is urgently needed. Numerous drugs are already approved for human use, and subsequently, there is a good understanding of their safety profiles and potential side effects, making them easier to fast-track to clinical studies in COVID-19 patients. Here, we present data on the antiviral activity of 20 FDA-approved drugs against SARS-CoV-2 that also inhibit SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). We found that 17 of these inhibit SARS-CoV-2 at non-cytotoxic concentrations. We directly followed up seven of these to demonstrate that all are capable of inhibiting infectious SARS-CoV-2 production. Moreover, we evaluated two of these, chloroquine and chlorpromazine, in vivo using a mouse-adapted SARS-CoV model and found that both drugs protect mice from clinical disease.IMPORTANCE There are no FDA-approved antivirals for any coronavirus, including SARS-CoV-2. Numerous drugs are already approved for human use that may have antiviral activity and therefore could potentially be rapidly repurposed as antivirals. Here, we present data assessing the antiviral activity of 20 FDA-approved drugs against SARS-CoV-2 that also inhibit SARS-CoV and MERS-CoV in vitro We found that 17 of these inhibit SARS-CoV-2, suggesting that they may have pan-anti-coronaviral activity. We directly followed up seven of these and found that they all inhibit infectious-SARS-CoV-2 production. Moreover, we evaluated chloroquine and chlorpromazine in vivo using mouse-adapted SARS-CoV. We found that neither drug inhibited viral replication in the lungs, but both protected against clinical disease.

Keywords: FDA-approved drugs; SARS-CoV-2; antiviral therapeutics; coronavirus; drug repurposing; nCoV-2019; pandemic.

FIG 1

Percentage inhibition and percentage cytotoxicity graphs from drug screens starting at 50 μM using an 8-point, 1:2 dilution series. Results are from one representative drug screen of three showing percentage inhibition and cytotoxicity for each of the tested drugs. Triplicate wells of cells were pretreated with the indicated drug for 2 h prior to infection with SARS-CoV-2 at an MOI of 0.01. Cells were incubated for 72 h prior to CellTiter-Glo assays to assess cytopathic effect. Data are percent inhibition of relative cell viability for drug-treated cells versus vehicle control. The values are means, with error bars displaying standard deviation between the triplicate wells. Seven graphs are boxed in red to highlight drugs that become the subject of follow-up study.

FIG 2

Hydroxychloroquine and chloroquine inhibit production of SARS-CoV-2 N and RdRp mRNA. Vero E6 cells were pretreated with hydroxychloroquine sulfate (A, C, and E) or chloroquine phosphate (B, D, and F) at the indicated concentration (or 0.1% water as a vehicle control) for 2 h prior to infection with SARS-CoV-2 at an MOI of 0.1. Cells were collected in TRIzol 24 hpi. RNA was extracted from the TRIzol sample, and qRT-PCR was performed for viral RdRp (A and B) or N (C and D) mRNA using WHO primers. RNA levels were normalized with 18S RNA, and fold change for drug-treated cells relative to vehicle control was calculated (dotted lines indicate a fold change of 1, which is no change over control). Data are from 3 independent infections performed on triplicate wells; the fold change was calculated in each independent experiment, and the mean fold change is plotted, with error bars displaying standard deviations. Along with TRIzol samples for RNA, supernatant was collected from cells and used for TCID₅₀ assays to determine infectious-virus production following treatment with HCQ (E) or CQ (F). Data are from 3 independent infections performed on triplicate wells, with the TCID₅₀ per milliliter being averaged across all wells. Error bars show standard deviations. (G) Cells were treated with 50 μM HCQ or 0.1% water as a control. Drug was added either at 2 h prior to infection, at the time of infection, or at 2 h after infection with SARS-CoV-2 at an MOI of 0.1. After 24 h infection, supernatant was collected and used for TCID₅₀ assays to determine infectious-virus production. Data are from 3 independent infections performed on triplicate wells, with the values being averaged across all wells. Error bars show standard deviations. (H) SARS-CoV spike pseudoviruses (PV) were used for infection of BSC1 cells. The cells were treated with 10 μM CQ or CPZ for 1 h prior to infection with PV for 3 h. The PV carry BlaM, and cells were loaded with CCF2 to monitor cleavage and shift in fluorescence output for evidence of S-mediated entry into cells. Data are normalized to those for PV alone and are from 3 independent experiments, with error bars representing standard deviations. In all cases, t tests were performed for vehicle control versus drug-treated samples, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

FIG 3

Antiviral activity of additional FDA-approved compounds against SARS-CoV-2. Other drugs that showed antiviral activity in our initial CellTiter-Glo screening were tested for inhibition of productive virus infection. Cells were treated with the indicated concentrations of amodiaquine dihydrochloride dihydrate (A), amodiaquine hydrochloride (B), chlorpromazine (C), imatinib (D), and mefloquine (E) for 2 h prior to infection with SARS-CoV-2 at an MOI of 0.1 for 24 h. Supernatant was collected and used for TCID₅₀ assays to quantify infectious virus production. Data are from a representative experiment of four performed on triplicate wells. Data are means, with error bars indicating standard deviations. In all cases, t tests were performed for vehicle control versus drug-treated samples, *, P < 0.05.

FIG 4

Antiviral activity of selected FDA-approved drugs in A549-hACE2 cells infected with SARS-CoV-2. A549-hACE2 cells were pretreated with hydroxychloroquine (HCQ), chloroquine (CQ), amodiaquine dihydrochloride dihydrate (AmD), amodiaquine hydrochloride (AmH), chlorpromazine (CPZ), imatinib (Imat), or mefloquine (Mefl) at the indicated concentrations for 2 h prior to infection with SARS-CoV-2 at MOI 0.1. At 48 hpi, supernatant was collected for titer determination by TCID₅₀ assay, and cells were collected in TRIzol for RNA extraction and qRT-PCR. qRT-PCR was carried out for the RdRp gene (A) and the N gene (B), and data are presented as fold change relative to vehicle control (0.1% H₂O for HCQ and CQ and 0.1% DMSO for all other drugs), with RNA levels being normalized to GAPDH. Data are from a representative experiment of three performed in triplicate. Error bars indicate standard deviations. (C) Titers of virus produced from drug-treated and vehicle control cells, presented as TCID₅₀/ml. Data are from three independent experiments performed in triplicate. Error bars indicate standard deviations. t tests were performed for vehicle control versus drug-treated samples, ****, P < 0.001.

FIG 5

CQ is protective against SARS-CoV (MA15) disease in vivo but does not inhibit viral replication. Mice were treated with CQ 1 day prior to infection with SARS-CoV (MA15) and dosed across the 4-day infection time course. Water was used as the vehicle control, and PBS was used as a control for uninfected mice. (A) Weight loss of mice treated with CQ at two different doses (0.8 mg and 1.6 mg) over the 4-day infection. Data are presented as relative weight loss compared to the mouse weight on day 0. In each treatment group there were 5 mice, and the data are means and standard deviations. (B) At day 4, mice were euthanized, and lung sections were used for H&E staining. (C) In addition to collecting lungs for section staining, there was also collection to determine titer of virus by plaque assay.

FIG 6

CQ is protective against SARS-CoV (MA15) disease in vivo but does not inhibit viral replication. As for Fig. 5, mice were treated with CPZ 1 day prior to infection with SARS-CoV (MA15) and through the 4-day infection time course. Water was used as the vehicle control for both drugs, and PBS was used as a control for uninfected mice. (A) Weight loss of mice treated with CPZ at three different doses (20 μg, 100 μg, and 200 μg) over the 4-day infection. Data are presented as relative weight loss compared to the mouse weight on day 0. In each treatment group, there were 5 mice, and data are means and standard deviations. (B) At day 4, mice were euthanized, and lung sections were used for H&E staining. (C) In addition to collecting lungs for section staining, there was also collection to determine titer of virus by plaque assay.