key: cord-325420-e9fjo7tl
authors: Xiao, Xia; Wang, Conghui; Chang, De; Wang, Ying; Dong, Xiaojing; Jiao, Tao; Zhao, Zhendong; Ren, Lili; Dela Cruz, Charles S; Sharma, Lokesh; Lei, Xiaobo; Wang, Jianwei
title: Identification of potent and safe antiviral therapeutic candidates against SARS-CoV-2
date: 2020-07-06
journal: bioRxiv
DOI: 10.1101/2020.07.06.188953
sha: 
doc_id: 325420
cord_uid: e9fjo7tl

COVID-19 pandemic has infected millions of people with mortality exceeding 300,000. There is an urgent need to find therapeutic agents that can help clear the virus to prevent the severe disease and death. Identifying effective and safer drugs can provide with more options to treat the COVID-19 infections either alone or in combination. Here we performed a high throughput screen of approximately 1700 US FDA approved compounds to identify novel therapeutic agents that can effectively inhibit replication of coronaviruses including SARS-CoV-2. Our two-step screen first used a human coronavirus strain OC43 to identify compounds with anti-coronaviral activities. The effective compounds were then screened for their effectiveness in inhibiting SARS-CoV-2. These screens have identified 24 anti-SARS-CoV-2 drugs including previously reported compounds such as hydroxychloroquine, amlodipine, arbidol hydrochloride, tilorone 2HCl, dronedarone hydrochloride, and merfloquine hydrochloride. Five of the newly identified drugs had a safety index (cytotoxic/effective concentration) of >600, indicating wide therapeutic window compared to hydroxychloroquine which had safety index of 22 in similar experiments. Mechanistically, five of the effective compounds were found to block SARS-CoV-2 S protein-mediated cell fusion. These FDA approved compounds can provide much needed therapeutic options that we urgently need in the midst of the pandemic.

of the screened compounds. The SI for the hydroxychloroquine was 22 in our study, indicating 116 increased safety of newly identified drugs compared to the hydroxychloroquine. agent of COVID-19. Positive compounds from the initial screen were tested for their antiviral 120 efficacy against SARS-CoV-2 in Vero cells. SARS-CoV-2 replicates within the Vero cells and 121 causes cytopathic effects in these cells in absence of any antiviral treatment. We generated the 122 dose response inhibition curves along with the cytotoxicity curves for these compounds in 123 presence of SARS-CoV-2 (Fig. 3) . Our data show that 24 of these compounds show significant 124 efficacy in inhibiting SARS-CoV-2 replication with sub micromolar IC50 for many of these 125 drugs such as nilotinib, clofazimine and raloxifene. The effects also confirmed by 126 immunofluorescence assay (data not shown). These compounds also belong to a wide variety of 127 classes including cardiac glycosides, anti-malarial drug hydroxychloroquine, cyclooxygenase-2 128 inhibitors and ion channel blockers, among others. The IC50, CC50 and SI of these compounds 129 is shown in Table 2 . 130

Five candidate drugs inhibit cell fusion. Finally, in order to clarify the mechanism by which 131

The current pandemic of COVID-19 is the third major outbreak in this century and the largest 141 needed. In this study, we have identified many FDA approved therapies that are highly effective 153 against coronaviruses, including 24 of the agents that are effective against SARS-CoV-2. This 154 screening confirms previous reports demonstrating anti-SARS-CoV-2 activity of 155 hydroxychroquine, amlodipine besylate, arbidol hydrochloride, tilorone 2HCl, dronedarone 156 hydrochloride, tetrandrine, merfloquine, and thioridazine hydrochloride 8-10, 23 , while identifying 157 additional 14 drugs. The underlying mechanisms of viral replication inhibition by these drugs is 158 not clear. It is highly unlikely that these compounds will have similar antiviral mechanisms given 159 the vast structural and pharmacological diversities of the effective antiviral compounds in our drugs can affect various steps in the viral life cycle including attachment, entry, replication, 162 assembly and budding of viral progeny. Five drugs may inhibit S-mediated cell fusion as 163 indicated by our data (Fig. 4) . Further studies are required to understand the precise mechanisms 164 of each of the effective compounds found in this study. 165

Toxicity is one of the limiting factors in the therapeutic application of many drugs despite their 166 known antiviral activities. Many of these drugs had SI of > 600, showing promise of their 167 usefulness at safe doses. For comparison, the SI of hydroxychloroquine was found to be 22 in 168 our study while SI of amlodipine besylate was found to be ~3, demonstrating much lower safety 169 profile of this drug. Similarly, other drugs that are known to have low selective index such as 170 digoxin for their approved use, also show lower SI in our screen. Five of the drugs with SI 171 of >600 include tyrosine kinase inhibitor nilotinib, antibiotics such as clofazimine and actidione, 172 selective estrogen receptor modulator such as raloxifene and non-steroidal anti-inflammatory 173 drug celecoxib. 174

etacoronaviruses have raised great public health threats to human beings, as most known HCoVs 175 including all the three virulent HCoVs (SARS-CoV, MERS-CoV and SARS-CoV-2) and two 176 seasonal HCoVs (OC43 and HKU1) belong to this species 3-7, 19, 24 . It is of great value to identify 177 antivirals against a broad spectrum of HCoVs, particularly the Betacoronaviruses, to tackle such 178 threats by pharmaceutical interventions. To this end, we first screened the compounds which 179 showed apparent activity of anti-OC43, the most prevalent HCoV circulates worldwide 25 . We 180 then narrowed down the candidates by the screening on SARS-CoV-2, resulting in the 9 provides a foundation for subsequent anti-HCoVs drug screening of broad spectrum. However, 183 further tests are warranted to verify their efficacies. 184

In summary, our screen identified 14 previously unknown FDA approved compounds that are 185 effective in inhibiting SARS-CoV-2 beside confirming the antiviral properties of ten previously 186 reported compounds, validating our approach. This screen identified five new compounds that 187 are highly efficacious in inhibiting the viral replication of SARS-CoV-2 with SI >600. Further 188 studies are needed to confirm the in vivo efficacy of these drugs in humans and COVID-19 189 relevant mouse models such as those with human ACE2 transgene 26 . The positively identified drugs from this screen were used to perform dose response curves 220 against OC43 on LLC-MK2 and against SARS-CoV-2 using Vero cells as described below. Immunofluorescence: Cells were fixed with 4% paraformaldehyde for 20 min at room 238 temperature, and permeabilized with 0.5% Triton X-100 for 10 min. Cells were then blocked 239 with 5% BSA and stained with primary antibodies, followed by staining with an Alexa Fluor 488 240 secondary antibodies. Nuclei were counterstained with DAPI. gene. The reference standard was tenfold diluted from 1×10 9 copies to 1×10 4 copies. PCR 248 amplification procedure was 50℃, 15min, 95℃, 3min; 95℃, 15s, 60℃, 45s+Plate Read, 50 249 cycles. The amplification process, fluorescence signal detection, data storage and analysis were 12 all completed by fluorescence quantitative PCR and its own software (Bio-Rad CFX Manager). 251

The copies of virus were calculated according to the standard curve. The inhibition ratio was 252 obtained by dividing the number of copies of the virus in the vehicle control group. The data 253 were nonlinear fitting by graphpad 7.0 software to calculate IC 50 of each drug. 254

Cell-cell fusion assay: Cell-cell fusion assays were performed as described previously (19). 255

Briefly, HEK-293T cells were co-transfected with SARS-CoV-2-S glycoprotein and eGFP. At 256 24 h post transfection, cells were digested with trypsin (0.25%) and overlaid on a 50% confluent 257 monolayer of 293T-ACE2 cells at a ratio of 1:1 which were treated with candidate drugs for 1h. 258

After 24h incubation, images of syncytia were captured with Operetta (PerkinElmer, 259

Massachusetts, USA). 

Could chloroquine /hydroxychloroquine be 318 harmful in Coronavirus Disease 2019 (COVID-19) treatment? Clinical infectious