Beijing Advanced Innovation Center for Soft Matter Science and Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China.
Chin Med J (Engl). 2020 May 5;133(9):1051-1056. doi: 10.1097/CM9.0000000000000797.
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.
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.
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).
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.
急需治疗 2019 年新型冠状病毒（2019-nCoV）感染的药物。然而，使用活的 2019-nCoV 进行药物筛选需要高水平的生物安全设施，这对于没有这些设施或 2019-nCoV 的机构来说是一个障碍。本研究旨在利用临床批准的药物治疗 2019-nCoV 相关冠状病毒模型中的冠状病毒疾病 2019（COVID-19）。
描述了一种 2019-nCoV 相关穿山甲冠状病毒 GX_P2V/穿山甲/2017/广西。通过使用小干扰 RNA（siRNA）介导的 ACE2 沉默来研究 GX_P2V 是否使用血管紧张素转换酶 2（ACE2）作为细胞受体。使用穿山甲冠状病毒模型来鉴定治疗 2019-nCoV 感染的药物候选物。筛选了 2406 种临床批准药物的两个文库，以确定它们抑制 GX_P2V 感染对 Vero E6 细胞产生细胞病变效应的能力。进一步研究了潜在药物的抗病毒活性和抗病毒机制。通过定量实时聚合酶链反应（qRT-PCR）和噬菌斑测定分别定量病毒 RNA 和感染性颗粒的产量。
冠状病毒 GX_P2V 的刺突蛋白与 2019-nCoV 分离株 Wuhan-hu-1 的氨基酸序列同源性为 92.2%，与 2019-nCoV 一样，使用 ACE2 作为感染受体。三种药物，包括石蒜碱（CEP）、塞拉菌素和盐酸甲氟喹，在 10 μmol/L 时完全抑制细胞培养中的细胞病变效应。CEP 对 GX_P2V 感染的抑制作用最为有效，其 50%最大效应浓度[EC50]为 0.98 μmol/L。用 10 μmol/L CEP 处理的细胞中病毒 RNA 的产量比没有 CEP 处理的细胞低 15393 倍[6.48±0.02]×10与 1.00±0.12，t=150.38，P<0.001）在感染后 72 小时（p.i.）。在 48 h p.i.时，噬菌斑测定发现含 10 μmol/L CEP 的培养基中没有活病毒的产生。此外，我们发现 CEP 对病毒进入（0.46±0.12，vs.1.00±0.37，t=2.42，P<0.05）和病毒复制[6.18±0.95]×10与 1.00±0.43，t=3.98，P<0.05）均具有有效的抗病毒活性。
我们的穿山甲冠状病毒 GX_P2V 是研究 2019-nCoV 的可行模型。CEP、塞拉菌素和盐酸甲氟喹是治疗 2019-nCoV 感染的潜在药物。我们的研究结果强烈表明 CEP 是一种广谱的泛β冠状病毒抑制剂，值得进一步研究 CEP 治疗 2019-nCoV 感染。
