Repurposing of Clinically Approved Drugs for Treatment of Coronavirus Disease 2019 in a 2019-Novel Coronavirus-Related Coronavirus Model
The global outbreak of coronavirus disease 2019 (COVID-19), caused by the 2019 novel coronavirus (2019-nCoV), has posed an unprecedented challenge to public health worldwide. As of early 2020, the number of confirmed cases and deaths continued to rise, particularly in mainland China, highlighting the urgent need for effective treatments and preventive measures. This study aimed to address this critical issue by repurposing clinically approved drugs for the treatment of COVID-19, using a 2019-nCoV-related coronavirus model.
The primary challenge in developing treatments for 2019-nCoV infections lies in the requirement for high-level biosafety facilities when working with live viruses. This limitation significantly hinders drug screening efforts, especially for institutions without access to such facilities or live 2019-nCoV samples. To overcome this obstacle, the researchers established a 2019-nCoV-related pangolin coronavirus model, GX_P2V/pangolin/2017/Guangxi, as an alternative system for drug screening and research.
The GX_P2V coronavirus model shares significant similarities with 2019-nCoV, particularly in its spike protein, which shows 92.2% amino acid identity with that of the 2019-nCoV isolate Wuhan-hu-1. Both viruses utilize angiotensin-converting enzyme 2 (ACE2) as their cell receptor for infection, a crucial feature that was confirmed through small interfering RNA (siRNA)-mediated silencing of ACE2 expression. When ACE2 expression was knocked down in Vero E6 cells using specific siRNAs, both ACE2 mRNA levels and viral RNA yields were significantly reduced, demonstrating the essential role of ACE2 in GX_P2V infection.
The researchers conducted a comprehensive drug screening using two libraries containing a total of 2406 clinically approved drugs and antiviral compounds. The screening was performed in Vero E6 cells infected with GX_P2V, with each drug tested at a concentration of 10 μmol/L. The primary readout was the inhibition of cytopathic effects (CPE) observed at 72 hours post-infection (p.i.). Through this screening, three drugs emerged as potent inhibitors of GX_P2V infection: cepharanthine (CEP), selamectin, and mefloquine hydrochloride.
Among these candidates, CEP demonstrated the most potent antiviral activity, with a concentration for 50% of maximal effect (EC50) of 0.98 μmol/L and a selectivity index of 39.91. The viral RNA yield in cells treated with 10 μmol/L CEP was dramatically reduced, showing a 15,393-fold decrease compared to untreated cells at 72 h p.i. Furthermore, plaque assays revealed no production of live viruses in media containing 10 μmol/L CEP at 48 h p.i.
To elucidate the mechanism of CEP’s antiviral activity, the researchers conducted time-of-addition experiments. These experiments demonstrated that CEP exerts its antiviral effects at both the viral entry and post-entry stages. In the viral entry assay, where cells were treated with CEP during the first 2 hours of infection, the viral RNA yield was 2.17-fold lower than in untreated cells. In the post-entry assay, where CEP was added after the initial 2 hours of infection, the viral RNA yield was reduced by 1618-fold. When CEP was present throughout the infection (full-time treatment), the viral RNA yield was 12,459-fold lower than in untreated cells.
The other two identified drugs, selamectin and mefloquine hydrochloride, also showed significant antiviral activity, though their mechanisms of action remain to be fully elucidated. Mefloquine, an antimalarial drug, has previously shown activity against other coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Selamectin, a broad-spectrum parasiticide used in veterinary medicine, represents a novel finding in coronavirus research.
The use of the GX_P2V pangolin coronavirus model offers several advantages for 2019-nCoV research. First, its close genetic relationship to 2019-nCoV, particularly in the spike protein, makes it a relevant model for studying viral entry and replication mechanisms. Second, its use of ACE2 as a receptor, similar to 2019-nCoV, allows for the study of receptor-mediated entry and potential therapeutic interventions targeting this pathway. Third, the model’s non-pathogenicity in humans enables research to be conducted at biosafety level 2, significantly lowering the barrier for institutions to participate in COVID-19 research.
The identification of CEP as a potent inhibitor of 2019-nCoV-related coronavirus infection is particularly noteworthy. CEP is a naturally occurring plant alkaloid with a long history of clinical use, primarily for the treatment of leukopenia. Its established safety profile, combined with its demonstrated antiviral activity, makes it a promising candidate for further investigation in the treatment of COVID-19. Additionally, CEP’s known anti-inflammatory properties could potentially address the cytokine storm often associated with severe COVID-19 cases.
The study also highlights the importance of continued coronavirus surveillance in wildlife populations. The GX_P2V coronavirus was isolated from a smuggled pangolin in 2017, well before the COVID-19 outbreak, demonstrating the potential value of such surveillance efforts in identifying and characterizing novel coronaviruses that could pose future threats to human health.
In conclusion, this research provides a valuable model system for 2019-nCoV research and identifies three clinically approved drugs with potent antiviral activity against a 2019-nCoV-related coronavirus. The findings strongly suggest that CEP, in particular, warrants further investigation as a potential treatment for COVID-19. The study also underscores the importance of drug repurposing strategies in responding to emerging infectious diseases and highlights the potential of wildlife coronavirus surveillance in pandemic preparedness.
doi.org/10.1097/CM9.0000000000000797
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