Identification of SARS-CoV-2 entry inhibitors among already approved drugs

Abstract

To discover effective drugs for COVID-19 treatment amongst already clinically approved drugs, we developed a high throughput screening assay for SARS-CoV-2 virus entry inhibitors using SARS2-S pseudotyped virus. An approved drug library of 1800 small molecular drugs was screened for SARS2 entry inhibitors and 15 active drugs were identified as specific SARS2-S pseudovirus entry inhibitors. Antiviral tests using native SARS-CoV-2 virus in Vero E6 cells confirmed that 7 of these drugs (clemastine, amiodarone, trimeprazine, bosutinib, toremifene, flupenthixol, and azelastine) significantly inhibited SARS2 replication, reducing supernatant viral RNA load with a promising level of activity. Three of the drugs were classified as histamine receptor antagonists with clemastine showing the strongest anti-SARS2 activity (EC₅₀ = 0.95 ± 0.83 µM). Our work suggests that these 7 drugs could enter into further in vivo studies and clinical investigations for COVID-19 treatment.

Keywords: COVID-19; SARS-CoV-2; approved drug library; clemastine; high throughput screening assay; histamine receptor antagonists; virus entry inhibitors.

Fig. 1. Flow chart of the HTS screening and validation procedure.

A library of 1800 approved drugs was screened to determine the effects on blocking SARS2-S pseudovirus infection activity (@20 µM). A set of 154 drugs was selected as the primary hits and further screened for confirmation and to exclude nonspecific entry inhibition activity. Fifteen drugs were selected as specific SARS-CoV-2 entry inhibitor candidates. Experiments using a native clinical SARS-CoV-2 strain confirmed that these seven drugs were active viral entry inhibitors against SARS-CoV-2 infection in vitro.

Fig. 2. Results of primary high throughput screening.

Data from the primary screening experiments using the library of 1800 approved drugs were plotted as the cell viability (%) vs SARS2-S pseudovirus infectivity (%) in Huh-7 cells. Cell viability was calculated as a percentage of the OD@570 readout by the MTT method for each well against that of the control well with no drug. Infectivity was calculated in the same way using a luciferase activity readout. Compounds that met different primary criteria were plotted in color (Red: 20% ≥viability, viability/infectivity ≥20; Blue: 50% ≥viability ≥20%, viability/infectivity ≥10; Green: viability ≥50%, viability/infectivity ≥5).

Fig. 3. Antiviral activities of 15 drugs selected by the pseudovirus assay against the native SARS2 virus.

Fifteen drugs selected by the pseudovirus assay were tested for inhibitory activity against native SARS-CoV-2 virus replication in Vero E6 cells at 24 h post infection at a final concentration of 10 µM. Culture supernatant was collected, and the viral genome RNA load was quantified by qRT-PCR methodology as described in “Methods”. Data are shown as the viral RNA load decrease (in log10 scale) vs the DMSO control. Remdesivir was used as a positive control at 10 µM as well. Data are shown as the means ± SEM of two independent experiments.

Fig. 4. Activity against native SARS-CoV-2 virus replication in vitro.

Antiviral activity of the seven selected drugs against native SARS-CoV-2 replication. Vero E6 cells were treated with drugs at the indicated concentrations and infected with SARS-CoV-2 virus at an MOI = 0.01. Culture supernatants were collected at 24 h post infection, and viral RNA was quantified by qRT-PCR with a TaqMan probe targeting the RBD region of the S gene (circle, solid line). Data are shown as the viral genome load vs the DMSO control (mean ± SEM, n = 6). Cell viability after drug treatment was assayed by the MTT method (square, dashed line); data are shown as percentages of the DMSO control (mean ± SEM, n = 2).