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

Abstract

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 Wuhan-hu-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 10 μmol/L. CEP demonstrated the most potent inhibition of GX_P2V infection, with a concentration for 50% of maximal effect [EC50] of 0.98 μmol/L. The viral RNA yield in cells treated with 10 μmol/L CEP was 15,393-fold lower than in cells without CEP treatment ([6.48 ± 0.02] × 10vs. 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 10 μmol/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] × 10vs. 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.

Figure 1

Pangolin coronavirus GX_P2V has two residue changes among the five key residues for receptor binding when compared to SARS-CoV, but likely still utilizes ACE2 as the cell receptor. (A) Alignment of receptor binding domains of 2019-nCoV-related CoVs and SARS-CoV. Dots represent residues that are identical to those in SARS-CoV and hyphens represent gaps. Five key residues for binding between SARS-CoV RBD and ACE2 protein are indicated as underlined capital letters. (B) The ACE2 expressions and (C) viral RNA yields were significantly reduced in ACE2-specific siRNA treated cells (^∗P < 0.01). Vero cells were transfected with 0.8, 4, and 20 nmol/L ACE2-specific siRNAs or NSC siRNA by using the RNAiMax transfection reagent. At 48 h p.t., the cells were infected with pangolin CoV at an MOI of approximately 10. At 24 h p.i., CoV-infected cells were lysed in lysis buffer, and the RNA levels of ACE2, CoV, and β-actin were determined by qRT-PCR. SARS-CoV: Severe acute respiratory syndrome-coronavirus; ACE2: Angiotensin-converting enzyme 2; 2019-nCoV: 2019 Novel coronavirus; CoV: Coronavirus; RBD: Receptor binding domain; siRNAs: Small interfering RNA; NSC: Non-specific control; p.t.: Post-transfection; MOI: Multiplicity of infection; p.i.: Post-infection; qRT-PCR: Quantitative real-time polymerase chain reaction; mRNA: Messenger RNA; SiNC: siRNA of negative control; SiACE2: siRNA of ACE2; nM = nmol/L.

Figure 2

Screening of clinically approved drugs for their anti-viral activities against 2019-nCoV by observing CPE inhibition and relative quantification of viral RNA yields. (A) Vero E6 cells (5000 cells/well) were infected with pangolin CoV (MOI = 0.01) and were incubated in media containing different chemical drugs with a final concentration of 10 μmol/L. At 72 h p.i., the CPE was observed by using phase microscopy. (B) Infected cells with cepharanthine treatment had no obvious CPE and were further analyzed by detecting viral RNA level. ^∗P < 0.001. 2019n-CoV: 2019 Novel coronavirus; CPE: Cytopathic effect; CoV: Coronavirus; MOI: Multiplicity of infection; p.i.: Post-infection; mRNA: Messenger RNA.

Figure 3

Anti-viral activities of cepharanthine against CoV in vitro. (A) Vero E6 cells were infected with CoV at an MOI of 0.05 in the treatment of different doses of cepharanthine for 48 h. The viral yield in the cell was then quantified by qRT-PCR and normalized by β-actin level. Cytotoxicity of these drugs to Vero E6 cells was measured by CellTiter-Blue assay. The left and right Y-axis of the graphs represent mean percentage of inhibition of virus yield and cytotoxicity of cepharanthine, respectively. The experiments were done in triplicates. (B) Time-of-addition experiment of cepharanthine. The detailed steps for time-of-addition experiment of cepharanthine were described in the method section, and virus and β-actin mRNA levels in the infected cell were quantified by qRT-PCR at 48 h p.i. ^∗P < 0.05 (C) Cepharanthine inhibited infectious virus production in the supernatant. The virus titers of supernatant of control cells (left) and cepharanthine treated cells (right) in the “full time” experiment were determined by plaque assay. CoV: Coronavirus; MOI: Multiplicity of infection; qRT-PCR: Quantitative real-time polymerase chain reaction; p.i.: Post-infection. EC₅₀: Concentration for 50% of maximal effect; CC₅₀: Cytotoxicity concentration 50%; SI: Selectivity index; mRNA: Messenger RNA; μM: μmol/L.