Repurposing of clinically approved drugs for treatment of coronavirus disease 2019 in a 2019-novel coronavirus-related coronavirus model

Background:Medicines for the treatment of 2019-novel coronavirus(2019-nCoV)infections are urgently needed.However,drug screening using live 2019-nCoV requires high-level biosafety facilities,which imposes an obstacle for those institutions without such facilities or 2019-nCoV.This study aims to repurpose the clinically approved drugs for the treatment of coronavirus disease 2019(COVID-19)in a 2019-nCoV-related coronavirus model.Methods:A 2019-nCoV-related pangolin coronavirus GX_P2V/pangolin/2017/Guangxi was described.Whether GX_P2V uses angiotensin-converting enzyme 2(ACE2)as the cell receptor was investigated by using small interfering RNA(siRNA)-mediated silencing of ACE2.The pangolin coronavirus model was used to identify drug candidates for treating 2019-nCoV infection.Two libraries of 2406 clinically approved drugs were screened for their ability to inhibit cytopathic effects on Vero E6 cells by GX_P2V infection.The anti-viral activities and anti-viral mechanisms of potential drugs were further investigated.Viral yields of RNAs and infectious particles were quantified by quantitative real-time polymerase chain reaction(qRT-PCR)and plaque assay,respectively.Results:The spike protein of coronavirus GX_P2V shares 92.2%amino acid identity with that of 2019-nCoV isolate Wuhanhu-1,and uses ACE2 as the receptor for infection just like 2019-nCoV.Three drugs,including cepharanthine(CEP),selamectin,and mefloquine hydrochloride,exhibited complete inhibition of cytopathic effects in cell culture at 10μmol/L.CEP demonstrated the most potent inhibition of GX_P2V infection,with a concentration for 50%of maximal effect[EC50]of 0.98μmol/L.The viral RNA yield in cells treated with 10μmol/L CEP was 15,393-fold lower than in cells without CEP treatment([6.48±0.02]×10-4vs.1.00±0.12,t=150.38,P<0.001)at 72 h post-infection(p.i.).Plaque assays found no production of live viruses in media containing 10μmol/L CEP at 48 h p.i.Furthermore,we found CEP had potent anti-viral activities against both viral entry(0.46±0.12,vs.1.00±0.37,t=2.42,P<0.05)and viral replication([6.18±0.95]×10-4vs.1.00±0.43,t=3.98,P<0.05).Conclusions:Our pangolin coronavirus GX_P2V is a workable model for 2019-nCoV research.CEP,selamectin,and mefloquine hydrochloride are potential drugs for treating 2019-nCoV infection.Our results strongly suggest that CEP is a wide-spectrum inhibitor of pan-betacoronavirus,and further study of CEP for treatment of 2019-nCoV infection is warranted.