key: cord-0707803-fo07nxvi
authors: Yang, Cheng-Wei; Peng, Tzu-Ting; Hsu, Hsing-Yu; Lee, Yue-Zhi; Wu, Szu-Huei; Lin, Wen-Hsing; Ke, Yi-Yu; Hsu, Tsu-An; Yeh, Teng-Kuang; Huang, Wen-Zheng; Lin, Jiunn-Horng; Sytwu, Huey-Kang; Chen, Chiung-Tong; Lee, Shiow-Ju
title: Repurposing old drugs as antiviral agents for coronaviruses
date: 2020-05-23
journal: Biomed J
DOI: 10.1016/j.bj.2020.05.003
sha: 24fd099a1b168c63655e2957425441a3648a67ee
doc_id: 707803
cord_uid: fo07nxvi

BACKGROUND: New therapeutic options to address the ongoing COVID-19 pandemic are urgently needed. One possible strategy is the repurposing of existing drugs approved for other indications as antiviral agents for SARS-CoV-2. Due to the commercial unavailability of SARS-CoV-2 drugs for treating COVID-19, we screened approximately 250 existing drugs or pharmacologically active compounds for their inhibitory activities against feline infectious peritonitis coronavirus (FIPV) and human coronavirus OC43 (HCoV-OC43), a human coronavirus in the same genus (Betacoronavirus) as SARS-CoV-2. METHODS: FIPV was proliferated in feline Fcwf-4 cells and HCoV-OC43 in human HCT-8 cells. Viral proliferation was assayed by visualization of cytopathic effects on the infected Fcwf-4 cells and immunofluorescent assay for detection of the nucleocapsid proteins of HCoV-OC43 in the HCT-8 cells. The concentrations (EC(50)) of each drug necessary to diminish viral activity to 50% of that for the untreated controls were determined. The viabilities of Fcwf-4 and HCT-8 cells were measured by crystal violet staining and MTS/PMS assay, respectively. RESULTS: Fifteen out of the 252 drugs or pharmacologically active compounds screened were found to be active against both FIPV and HCoV-OC43, with EC(50) values ranging from 11 nM to 75 μM. They are all old drugs as follows, anisomycin, antimycin A, atovaquone, chloroquine, conivaptan, emetine, gemcitabine, homoharringtonine, niclosamide, nitazoxanide, oligomycin, salinomycin, tilorone, valinomycin, vismodegib. CONCLUSION: All of the old drugs identified as having activity against FIPV and HCoV-OC43 have seen clinical use in their respective indications and are associated with known dosing schedules and adverse effect or toxicity profiles in humans. Those, when later confirmed to have an anti-viral effect on SARS-CoV-2, should be considered for immediate uses in COVID-19 patients.

Graphic abstract: FIPV: feline infectious peritonitis coronavirus; HCoV-OC43: human coronavirus; SARS-CoV-2: severe acute respiratory syndrome associated coronavirus-2.

New therapeutic options to address the ongoing COVID-19 pandemic are urgently needed. One possible strategy is the repurposing of existing drugs approved for other indications as antiviral agents for SARS-CoV-2. Due to the commercial unavailability of SARS-CoV-2 drugs for treating COVID-19, we screened approximately 250 existing drugs or pharmacologically active compounds for their inhibitory activities against feline infectious peritonitis coronavirus (FIPV) and human coronavirus OC43 (HCoV-OC43), a human coronavirus in the same genus (Betacoronavirus) as SARS-CoV-2.

FIPV was proliferated in feline Fcwf-4 cells and HCoV-OC43 in human HCT-8

cells. Viral proliferation was assayed by visualization of cytopathic effects on the infected Fcwf-4 cells and immunofluorescent assay for detection of the nucleocapsid proteins of HCoV-OC43 in the HCT-8 cells. The concentrations (EC 50 ) of each drug necessary to diminish viral activity to 50% of that for the untreated controls were determined. The viabilities of Fcwf-4 and HCT-8 cells were measured by crystal violet staining and MTS/PMS assay, respectively.

Fifteen out of the 252 drugs or pharmacologically active compounds screened were found to be active against both FIPV and HCoV-OC43, with EC 50 values ranging from 11 nM to 75 µM. They are all old drugs as follows, anisomycin, antimycin A, atovaquone, chloroquine, conivaptan, emetine, gemcitabine, homoharringtonine, niclosamide, nitazoxanide, oligomycin, salinomycin, tilorone, valinomycin, vismodegib.

All of the old drugs identified as having activity against FIPV and HCoV-OC43 have seen clinical use in their respective indications and are associated with known dosing schedules and adverse effect or toxicity profiles in humans. Those, when later confirmed to have an anti-viral effect on SARS-CoV-2, should be considered for immediate uses in COVID-19 patients.

COVID-19, the disease caused by severe acute respiratory syndrome associated coronavirus-2 (SARS-CoV-2), has infected over 2,970,000 people and killed more than 206,000 in 213 countries since the first outbreak in WuHan, China [1] was reported in December 2019. To control this disease, effective treatments, including SARS-CoV-2 inhibitors, are being actively pursued [1] . One potentially efficient approach is the repurposing of drugs previously approved to treat other diseases, since they are immediately available for use in clinical trials with COVID-19 patients and have known safety profiles.

Coronavirus (CoV) has a large genome and its mutation rate is higher than that of other RNA viruses [2, 3] . Animal CoVs cause persistent enzootic infections that inevitably infect new host species and become zoonotic, as happened for SARS-CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. Due to the diversity and relentlessness of these viruses, their individual mitigation is difficult [4] ; and therefore broad-spectrum inhibitors of emerging and endemic CoVs are needed, especially for those prone to be periodically cycling in and out of humans and livestock. Therefore, the small molecule inhibitors, either old drugs or new chemical entities, were actively pursued and developed aiming to prevent or cure the SARS-CoV and MERS-CoV infections [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] . Previous work established the clinical efficacy of pegylated interferon-α-2a, ribavirin, and lopinavir/ritonavir for the treatment of the MERS-CoV [15, 16] . Therefore, the strategy of repurposing existing drugs to treat patients infected with novel or zoonotic CoV has been validated and merits consideration in the contest of tackling the SARS-CoV-2 and COVID-19 pandemic.

Two representative CoVs were selected for existing drugs to be tested against. Feline infectious peritonitis coronavirus (FIPV) belongs to the genus Alphacoronavirus and causes enteritis in domestic and wild cats. Approximately 5-15% of infected cats develop feline infectious peritonitis, which is usually fatal [17, 18] . The pathogenesis, epidemiology, and pulmonary lesions associated with FIPV are similar to those associated with human SARS [19] . Human coronavirus OC43 (HCoV-OC43) belongs to the same viral genus (Betacoronavirus) as SARS-CoV and SARS-CoV-2, infects humans and cattle, and is one of the viruses responsible for the common cold [20, 21] .

Approximately ~250 old drugs or pharmacologically active compounds were assessed for their inhibitory activity of FIPV [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] , of which 23 were found to exhibit some anti-FIPV activity and advanced to testing against HCoV-OC43. In total, 15 exerted an inhibitory effect on both FIPV and HCoV-OC43 and they turned out are old drugs. These included chloroquine, which was recently demonstrated to be capable of inhibiting SARS-CoV-2 in vitro, and exhibited some efficacy in the clinical treatment of COVID-19 patients [22] . Moreover, GS-441524, the active metabolite of remdesivir [23, 24] which is currently being investigated in clinical trials for severe cases of COVID-19 [24, 25] , also exerted potent inhibitory activities against both FIPV and HCoV-OC43 herein.

Felis catus whole fetus-4 (Fcwf-4) cells (ATCC ® CRL-2787) were maintained in Dulbecco's modified Eagle's medium (DMEM, Hyclone Laboratories, Logan, UT, USA) containing 10% fetal bovine serum (FBS) with 1% penicillin/streptomycin at 37 o C with 5% CO 2 . The serotype Ⅱ FIPV Taiwan isolate NTU156 strain, a kind gift from National Taiwan University, was propagated and titrated in Fcwf-4 cells [26] . Confluent Fcwf-4 cells were seeded in 96-well plates and treated with various concentrations of testing compounds of up to 100 µM at 37°C under an atmosphere of 5% CO 2 for 48 h. Sixteen h post inoculation, cells were infected with FIPV NTU156 strain at 300 TCID 50 per well and incubated at 37 o C. After 1 h, the supernatant was discarded and a series of 7 concentrations at different dilution of testing compounds in DMEM containing 2% FBS added. Plates were incubated at 37 o C under an atmosphere of 5% CO 2 for additional 48 h; then, the cells were fixed with 10% formalin and stained with 0.1% crystal violet. The cytopathic effect (CPE) of the virus was assumed to correlate with the intensity of the crystal violet staining and measured visually for determination of the 50% effective concentrations (EC 50 ). Cell cytotoxicity was also measured by crystal violet staining. The 50% cytotoxicity concentration (CC 50 ) was calculated according a Reed and Muench method [27] .

HCT-8 colon epithelial cells (ATCC ® CCL-244™) were grown as monolayers in a growth medium consisting of DMEM and 10% FBS, (Biological Industries, Cromwell, CT, USA). HCoV-OC43 (ATCC ® VR1558™) was grown and propagated in HCT-8 ells cultured with DMEM and 2% FBS. EC 50 was measured using an indirect immunofluorescent assay (IFA). HCT-8 cells were deposited in 96-well plates, pre-treated with solutions of the compounds to be tested for 30 minutes, and then infected with HCoV-OC43 at a multiplicity of infection (MOI) of 0.05, and incubated 

About 250 drugs were collected and screened for their inhibitory activity against FIPV; this activity was ascertained by visual observation of their cytopathic effects. Of these 252, 23 were also tested for their inhibition of HCoV-OC43 by an IFA against HCoV-OC43 nucleocapsid protein ( Table 1) . Fifteen of these, all old drugs, exhibited inhibitory activity against HCoV-OC43 and exhibited -EC 50 values ranging from 11 nM to 75 µM for FIPV and 62 nM to 48 µM for HCoV-OC43 (Table 1) . Representative results from the cytopathy (FIPV) and IFA assays (HCoV-OC43) are depicted in Figures 1 and 2 [30] and showed in vitro activity against SARS-CoV [11] .

Atovaquone, a hydroxy-1,4-naphthoquinone with antipneumocystic activity [31] , exhibited EC 50 values of 4.78 µM against FIPV and 6.78 µM against HCoV-OC43. Conivaptan, a non-peptide inhibitor of the receptor vasopressin and originally approved for hyponatremia [32] , exhibited EC 50 values of 16.9 µM against FIPV and 12.2 µM against HCoV-OC43. Atovaquone is predicted to inhibit SARS-CoV-2 through targeting of the viral RNA-dependent RNA polymerase or 3C like protease [33] .

Chloroquine is being intensively studied in clinical for treating COVID-19 and in preclinical for efficacies against SARS-CoV-2 both in vitro and in vivo [22] , but its EC 50 values against FIPV (27.9 µM) and HCoV-OC43 (27.4 µM) were higher than of the compounds mentioned above. Nonetheless, this identified EC 50 of ~27 µM is comparable or better than the clinically used effective dosages of chloroquine, 200 ~ 1000 mg in qd or bid [34] [35] [36] . In addition, we also tested the nucleotide analogue GS-441524, the active metabolite of remdesivir [23, 24] , and it exhibited an EC 50 of 3.5 µM against FIPV, compared to an EC 50 of 6.77 µM against HCoV-OC43;

GS-441524 has been reported to exhibit good in vivo efficacy against FIPV in cats [23] and against SARS-CoV in vitro [37] , but it is not an FDA approved drug yet.

Remdesivir (GS-5734), the prodrug of GS-441524, has also shown efficacy for the treatment of COVID-19 patients [24, 25] . Moreover, this identified EC 50 (3 ~7 µM) of GS-441524 is also equivalent or better than the clinically used effective dosages of remdesivir, 100~200 mg [24, 25] .Thus the concentrations tested in this particular study are of clinical significance.

Other drugs which have already been repurposed in the context of COVID-19 include ivermectin, a type of avermectin used to treat many types of parasite infestations [38] , and the combination of hydroxychloroquine and the antibiotic azithromycin [39] . Therefore, all of these above-mentioned drugs are therefore proposed as potential treatments for COVID-19.In conclusion, several existing drugs were identified as having good inhibitory activities against FIPV and HCoV-OC43, and the doses at which they are currently used for their respective disease indications 

The authors declare no conflict of interest. 

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The authors gratefully acknowledge Dr. Kung-Yee Liang, National Health Research Institutes, for his full support and supervision on this work. This work was funded by the National Health Research Institutes, Taiwan, R.O.C. and the Ministry of Science