Identification of Plitidepsin as Potent Inhibitor of SARS-CoV-2-Induced Cytopathic Effect After a Drug Repurposing Screen

Abstract

There is an urgent need to identify therapeutics for the treatment of Coronavirus disease 2019 (COVID-19). Although different antivirals are given for the clinical management of SARS-CoV-2 infection, their efficacy is still under evaluation. Here, we have screened existing drugs approved for human use in a variety of diseases, to compare how they counteract SARS-CoV-2-induced cytopathic effect and viral replication in vitro. Among the potential 72 antivirals tested herein that were previously proposed to inhibit SARS-CoV-2 infection, only 18 % had an IC₅₀ below 25 µM or 10² IU/ml. These included plitidepsin, novel cathepsin inhibitors, nelfinavir mesylate hydrate, interferon 2-alpha, interferon-gamma, fenofibrate, camostat along the well-known remdesivir and chloroquine derivatives. Plitidepsin was the only clinically approved drug displaying nanomolar efficacy. Four of these families, including novel cathepsin inhibitors, blocked viral entry in a cell-type specific manner. Since the most effective antivirals usually combine therapies that tackle the virus at different steps of infection, we also assessed several drug combinations. Although no particular synergy was found, inhibitory combinations did not reduce their antiviral activity. Thus, these combinations could decrease the potential emergence of resistant viruses. Antivirals prioritized herein identify novel compounds and their mode of action, while independently replicating the activity of a reduced proportion of drugs which are mostly approved for clinical use. Combinations of these drugs should be tested in animal models to inform the design of fast track clinical trials.

Keywords: SARS-CoV-2; antivirals; plitidepsin; synergy; viral entry.

FIGURE 1

Antiviral activity of entry inhibitors against SARS-CoV-2. (A) Antiviral activity of hydroxychloroquine and azithromycin. Cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of hydroxychloroquine, azithromycin, and their combination. Drugs were used at a concentration ranging from 0.0512 nM to 100 µM. When combined, each drug was added at the same concentration. Non-linear fit to a variable response curve from one representative experiment with two replicates is shown (red lines), excluding data from drug concentrations with associated toxicity. The particular IC₅₀ value of this graph is indicated. Cytotoxic effect on Vero E6 cells exposed to increasing concentrations of drugs in the absence of virus is also shown (grey lines). (B) Cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of amantadine, a clathrin-mediated endocytosis inhibitor, E-64d, a pan-cathepsin inhibitor acting downstream once viruses are internalized in endosomes, NB-DNJ, an inhibitor of ganglioside biosynthesis and methyl-β-cyclodextrin, a cholesterol-depleting agent. All drugs were used at a concentration ranging from 0.0512 nM to 100 µM aside from methyl-β-cyclodextrin, which was used 10 times more concentrated. Non-linear fit to a variable response curve from one experiment with two replicates is shown (red lines). Cytotoxic effect on Vero E6 cells exposed to increasing concentrations of drugs in the absence of virus is also shown (grey lines). (C) Cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of camostat, a TMPRSS2 inhibitor, and ATT, an alpha-1 antyitrypsin, a broad cellular protease inhibitor, as described in (A). (D) Effect of entry inhibitors on luciferase expression of reporter lentiviruses pseudotyped with SARS-CoV-2 Spike in ACE2 expressing HEK-293T cells. Values are normalized to luciferase expression by mock-treated cells set at 100%. Mean and s.e.m. from two experiments with one to three replicates. Cells were exposed to fixed amounts of SARS-CoV-2 Spike lentiviruses in the presence of a non-toxic constant concentration of the drugs tested on Vero E6. Significant statistical deviations from 100% were assessed with a one sample t test. (E) Comparison of entry inhibitors blocking viral endocytosis, such as chloroquine, with inhibitors blocking serine protease TMPRSS2 expressed on the cellular membrane, such as camostat, on different cell lines. ACE2 expressing HEK-293T cells transfected or not with TMPRSS2 were exposed to SARS-CoV-2 Spike lentiviruses as described in (B). Values are normalized to luciferase expression by mock-treated cells set at 100%. Mean and s.e.m. from at least two representative experiments with two replicates. Statistical deviations from 100% were assessed with a one sample t test.

FIGURE 2

Antiviral activity of post-entry inhibitors. (A) Cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of Remdesivir. Drug was used at a concentration ranging from 0.0512 nM to 100 μM. Non-linear fit to a variable response curve from one representative experiment with two replicates is shown (red lines), excluding data from drug concentrations with associated toxicity. The particular IC₅₀ value of this graph is indicated. Cytotoxic effect on Vero E6 cells exposed to increasing concentrations of drugs in the absence of virus is also shown (grey lines). (B). Cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of remdesivir and its combination with hydroxychloroquine, as detailed in (A). Drugs in combination were used at a concentration ranging from 0.0512 nM to 100 μM (left panel). (C). Cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of protease inhibitors against HIV-1. Nelfinavir mesylate hydrate was the only drug with activity. Inhibitors were used at a concentration ranging from 0.0512 nM to 100 μM. The particular IC₅₀ value of this graph is indicated (D). Cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of plitidepsin and its combinations with hydroxychloroquine and remdesivir. When combined, each drug was added at the same concentration. Drugs were used at a concentration ranging from 0.5 nM to 10 μM. The particular IC₅₀ value of these graphs is indicated.

FIGURE 3

Antiviral activity of inhibitors with unknown mechanism of action. (A). Cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of Itraconazole. Drug was used at a concentration ranging from 0.0512 nM to 100 µ. Non-linear fit to a variable response curve from one representative experiment with two replicates is shown (red lines), excluding data from drug concentrations with associated toxicity. The particular IC₅₀ value of this graph is indicated. Cytotoxic effect on Vero E6 cells exposed to increasing concentrations of drugs in the absence of virus is also shown (grey lines). (B). Cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of Fenofibrate, as detailed in (A). (C). Effect of fenofibrate on the entry of luciferase expressing lentiviruses pseudotyped with SARS-CoV-2 Spike in ACE2-expressing HEK-293T cells. Values are normalized to luciferase expression by mock-treated cells set at 100%. Mean and s.e.m. from two experiments with two replicates. Statistical deviations from 100% were assessed with a one sample t test. (D). Cytopathic effect on Vero E6 cells exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of MDL 28170, as detailed in (A). (E). Comparison of MDL 28170 activity with entry inhibitors blocking viral endocytosis, such as chloroquine and E-64d, and inhibitors blocking serine protease TMPRSS2, such as camostat. ACE2 expressing HEK-293T cells transfected or not with TMPRSS2 were exposed to SARS-CoV-2 Spike lentiviruses in the presence of these compounds. Values are normalized to luciferase expression by mock-treated cells set at 100%. Mean and s.e.m. from at least two experiments with two replicates. Statistical deviations from 100% were assessed with a one sample t test.

FIGURE 4

Decreased release of SARS-CoV-2 in the presence of inhibitors with antiviral activity. (A). Viral release to the supernatant in the presence of the indicated compounds added at increasing concentrations 3 days post-infection of Vero E6 cells. SARS-CoV-2 nucleoprotein was detected with an ELISA at concentrations were drugs were nontoxic. Mean and s.e.m. from two experiments. (B). Viral release to the supernatant in the presence of the indicated interferons as described in A. Mean and s.e.m. from one experiment.