key: cord-345299-4k7qymqd
authors: Xiong, Hua-Long; Cao, Jia-Li; Shen, Chen-Guang; Ma, Jian; Qiao, Xiao-Yang; Shi, Tian-Shu; Yang, Yang; Ge, Sheng-Xiang; Zhang, Jun; Zhang, Tian-Ying; Yuan, Quan; Xia, Ning-Shao
title: Several FDA-approved drugs effectively inhibit SARS-CoV-2 infection in vitro
date: 2020-06-05
journal: bioRxiv
DOI: 10.1101/2020.06.05.135996
sha: 
doc_id: 345299
cord_uid: 4k7qymqd

To identify drugs that are potentially used for the treatment of COVID-19, the potency of 1403 FDA-approved drugs were evaluated using a robust pseudovirus assay and the candidates were further confirmed by authentic SARS-CoV-2 assay. Four compounds, Clomiphene (citrate), Vortioxetine, Vortioxetine (hydrobromide) and Asenapine (hydrochloride), showed potent inhibitory effects in both pseudovirus and authentic virus assay. The combination of Clomiphene (citrate), Vortioxetine and Asenapine (hydrochloride) is much more potent than used alone, with IC50 of 0.34 μM.

As of 26 May 2020, the COVID-19 pandemic has claimed more than 342,000 lives, but yet effective drug is not available. It is time-consuming to develop vaccines or specific drugs for a disease caused by a novel defined virus like SARS-CoV-2. Re-purposing of approved drugs may be a faster way to find treatment for COVID-19. Verification of drugs that might suppress SARS-CoV-2 by prediction, including drugs against similar virus and broad-spectrum antiviral agents (BSAAs), is time-saving for drug re-purposing at the expense of missing some potential candidates. Integrative, antiviral drug repurposing methods based on big data analysis or molecular docking and molecular dynamics are timesaving and high throughput. However, drugs identified by virtual screening still need to be verified in vitro and in vivo.

In our previous research, a robust neutralization assay was established based on SARS-CoV-2 S-bearing vesicular stomatitis virus (VSV) pseudovirus and human ACE2expressing BHK21 cells (BHK21-hACE2) 1 . Single-cycle infectious of recombinant VSV-SARS-CoV-2-Sdel18 mimics the entry of SARS-CoV-2. The infection of pseudovirus can be detected by fluorescence 12 hours after infection, enabling the assay time-saving for high-throughput screening 1 . This pseudovirus based assay is suitable for screening drugs that can block the infection of SARS-CoV-2.

In this study, the anti-SARS-Cov-2 potentiality of 1403 FDA approved drugs were quantitatively evaluated by the pseudovirus-based assay. The screening procedure was illustrated in Figure 1A and described in methods. The numbers of GFP-positive cells from drug treated wells were counted and divided by the number of infected cells from the well without treatment of drugs to calculate the relative value of infection rate. The results of two repetitions showed that most of drugs did not inhibit viral infection ( Figure 1B ). Fortyfour drugs with relatively better inhibitory effect were selected for further validation. In the second round of screening, inhibition of VSV-SARS-CoV-2-Sdel18 virus infection and cell cytotoxicity were both detected (Supplementary Figure 1) . Among them, 31 drugs were excluded due to cytotoxicity. 13 drugs were selected for analysis of specificity to VSV-SARS-CoV-2-Sdel18 and verification by authentic SARS-CoV-2 assay.

To verify whether these selected drugs act on spike protein of SARS-CoV-2 on the pseudovirus or the VSV backbone, we evaluated the inhibitory effect of these compounds on VSV-G (The sequence of GFP was inserted into the genome of VSV, so that the infection of VSV could be indicated by green fluorescence.). There were three compounds, including chloroquine diphosphate, ribavirin, and cetylpyridinium (chloride monohydrate), exhibited significant inhibitor effects on VSV-G, whereas no effect was noted for other compounds ( Figure 1C ). including Clomiphene (citrate), Amiodarone (hydrochloride), Vortioxetine, Vortioxetine (hydrobromide) and Asenapine (hydrochloride), were selected and the function of these compounds was confirmed using authentic SARS-CoV-2 assay ( Figure 1D and E). Among them, the inhibitory effects of Clomiphene (citrate) and Vortioxetine were comparable to Chloroquine diphosphate, while Vortioxetine (hydrobromide) and Asenapine (hydrochloride) were slightly less effective. Whereas Amiodarone (hydrochloride) inhibited the infection of pseudovirus efficiently with IC50 around 4.44 μM, but it showed 5 no effect on authentic SARS-CoV-2 virus infection even used at a concentration of 100 μM.

To further evaluate the potential of applying these drugs in prophylaxis and combination therapy, we treated the cell with pseudovirus and different drug combinations.

The drug combinations were added either at the same time of pseudovirus infection or 6 hours pre-infection (Table 1 Vortioxetine is an antidepressant drug that is used to treat major depressive disorder in adults. Vortioxetine was safe and well tolerated, it was approved in 2013 9 . So far, no previous study described its antiviral roles. It is reported that sever COVID-19 patients have a high probability of suffering from mental illness. Recently, another antidepressant drug fluvoxamine is evaluated for the potential to treat COVID-19 by researchers from the Washington University School of Medicine, because the drug may prevent an overreaction 7 of the immune system called cytokine storms, which could result in life-threatening organ failure. The antiviral mechanism of Vortioxetine remains unknow. However, it may bring physical and psychological benefits for COVID-19 patients.

Asenapine is an antipsychotic medicine that is used to treat schizophrenia and bipolar I disorder and has been approved since 2009. Notably, it showed less cytotoxicity in this study comparing to other drugs that could inhibit the infection of SARS-CoV-2.

In summary, our study identified four FDA-approved drugs that have the potential to suppress SARS-CoV-2 infection. The robust assay based on VSV-SARS-CoV-2-Sdel18 pseudovirus screened out the potential drugs with high efficiency, then the inhibitory effect was confirmed by authentic SARS-CoV-2 assay. The inhibitory effect of Vortioxetine and Clomifene is superior and the mechanism of these drugs seems different from Chloroquine.

The combination of Clomifene (citrate), Vortioxetine and Asenapine (hydrochloride) greatly decreases the IC50/IC90 of blocking virus infection. The clinical safety of these compounds has been evaluated and the availability of pharmacological data are expected to enable rapid preclinical and clinical evaluation for treatment of COVID-19. Based on the existing clinical results, it seems that it is difficult for one particular drug alone to significantly benefit COVID-19 patients, and combination therapy is more likely to make the patient recover faster. This work identified novel drugs that suppress the infection of virus and provided more candidates for post-exposure prophylaxis and combination therapies. Notice that no test in vivo has been conducted and the mechanism of these compounds also remains unknown. More researches are required to support the clinical application of these drugs for treatment of COVID-19. 

VSV pseudovirus carrying truncated spike protein of SARS-CoV-2, named VSV-SARS-CoV-2-Sdel18 virus, was packaged as previously described 1 . VSV-G was prepared in similar way 10 

The relative value or inhibition rate of candidate drugs were calculated according to the decrease of GFP positive cell number (for pseudovirus-based assay) or cytopathic effect (for authentic SARS-CoV-2-based assay). The IC50 (the half maximal inhibitory concentration), IC90 (the concentration for the 90% of the maximum inhibition) and CC50

(the 50% cytotoxic concentration) values were calculated with non-linear regression, i.e. log (inhibitor) vs. normalized response -Variable slope or log(agonist) vs. response-Find ECanything using GraphPad Prism 7.00 (GraphPad Software, Inc., San Diego, CA, USA). 106.20 na "Clo" means Clomiphene (citrate), "Vor" means Vortioxetine, "Ase" means Asenapine (hydrochloride) and "CQ" means Chloroquine diphosphate.

"Pre-treatment" means cell was treated with drugs 6 hours before infection, while "Cotreatment" means cells were treated with drugs at the time of infection.

IC50, IC90 and CC50 were calculated using prism software (GraphPad). "na" means the value can't be calculated. MOI=0.1

Robust neutralization assay based on SARS-CoV-2 S-bearing vesicular stomatitis virus (VSV) pseudovirus and ACE2-overexpressed BHK21 cells

Triple combination of interferon beta-1b, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with COVID-19: an open-label, randomised, phase 2 trial. The Lancet %@

FDA-approved selective estrogen receptor modulators inhibit Ebola virus infection

IFITM proteins inhibit entry driven by the MERS-coronavirus spike protein: evidence for cholesterol-independent mechanisms

Clomiphene and Its Isomers Block Ebola Virus Particle Entry and Infection with Similar Potency: Potential Therapeutic Implications

Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry

Coronavirus Cell Entry Occurs through the Endo-/Lysosomal Pathway in a Proteolysis-Dependent Manner

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

The safety and tolerability of vortioxetine: Analysis of data from randomized placebo-controlled trials and open-label extension studies

Generation of VSV pseudotypes using recombinant DeltaG-VSV for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines

Hua-Long Xiong, Tian-Ying Zhang, Quan Yuan and Ning-Shao Xia had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy 

The authors declare that they have no conflicts of interest.
