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] × 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 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] × 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.
Here we first described a 2019-nCoVr model for research of 2019-nCoV. This model is suitable for work at biosafety level-2. We then identified three clinically approved drugs (CEP, selamectin, and mefloquine hydrochloride) that can inhibit a 2019-nCoVr infection, and suggested that these drugs be considered for further investigation in the treatment of 2019-nCoV infection.
Our finding of CEP as a potential drug for 2019-nCoV is especially instructive. This drug is an anti-inflammatory and anti-neoplastic alkaloid and is approved for leukopenia. It has multiple functions, such as inhibiting the efflux transporter ABCC10 of anti-tumor drugs,inhibiting the entry of human immunodeficiency virus type 1 (HIV-1) by reducing plasma membrane fluidity,[15] and binding to central portion of heat shock protein 90.[16] Importantly, as a naturally occurring plant alkaloid with more than 40 years of clinic use, CEP has low toxicity in animals and has no significant side effects in humans.Given the observed strong inhibition of virus replication and the drug's established role of anti-inflammatory response, we think CEP is a promising candidate for the treatment of 2019-nCoV infection.
Nonetheless, our finding of CEP and mefloquine as anti-2019-nCoVr agents was in agreement with previous studies in other coronaviruses of the genus Betacoronavirus. Two groups reported CEP as a drug candidate for SARS-CoV and HCoV-OC43, respectively.Mefloquine, which is approved for malaria, was found to have anti-viral activity against both MERS-CoV and SARS-CoV.Furthermore, we identified a previously unknown anti-CoV compound, selamectin, which is marketed as a topical broad-spectrum parasiticide in cats and dogs to control fleas, heartworms, hookworms, roundworms, etc. The anti-viral mechanisms of these three drugs are unknown. We speculate that CEP and mefloquine are likely to target host cell pathways while selamectin might be a 2019-nCoVr-specific inhibitor.
The libraries of drugs used in this study contain 2406 compounds in total. Many of them have anti-viral activities against MERS-CoV and SARS-CoV.[19] Clearly, our finding of only three inhibitors of 2019-nCoVr is not a comprehensive answer of all potential inhibitors in our libraries, as our goal is to find drugs that have the most potent anti-viral activities and we did the initial screening of virus inhibition by observing the existence of intact cell monolayers, not by quantitative methods.
In conclusion, this is the first report of a 2019-nCoVr model. We suggest the three drugs (CEP, selamectin, and mefloquine hydrochloride) be considered for further investigation to treat the 2019-nCoV infection. Due to its amenable nature, our 2019-nCoVr model could play a more important role in the development of therapies and vaccines against 2019-nCoV. With high homology to 2019-nCoV, this 2019-nCoVr isolate could be a potential live vaccine candidate. Cultured long before the outbreak of 2019-nCoV, our 2019-nCoVr isolate might play a significant role in the combat against COVID-19. Thus, our model in part reflects the importance of sustained coronavirus surveillance in wildlife.
Reference & Source information: https://journals.lww.com/
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