Repurposing of clinically approved drugs for treatment of coronavirus disease 2019 in a 2019-novel coronavirus-related coronavirus model
Repurposing of clinically approved drugs for treatment of coronavirus disease 2019 in a 2019-novel coronavirus-related coronavirus model
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DOI：
10.1097/cm9.0000000000000797
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发表时间：
2020-05-05
影响因子：
6.1
通讯作者：
Tong, Yi-Gang
中科院分区：
文献类型：
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作者：
Fan, Hua-Hao;Wang, Li-Qin;Tong, Yi-Gang
背景：目前迫切需要治疗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，这对那些没有此类设施或2019-nCoV的机构构成了障碍。本研究旨在在2019-nCoV相关冠状病毒模型中重新使用临床批准的药物治疗2019冠状病毒病（COVID-19）。方法：描述了一株2019-nCoV相关的穿山甲冠状病毒GX_P2V/pangolin/2017/Guangxi。通过使用小干扰RNA（siRNA）介导的ACE 2沉默来研究GX_P2V是否使用血管紧张素转换酶2（ACE 2）作为细胞受体。穿山甲冠状病毒模型用于鉴定治疗2019-nCoV感染的候选药物。筛选了两个2406种临床批准药物的文库，以确定其抑制GX_P2V感染对Vero E6细胞的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。分别通过定量实时聚合酶链反应（qRT-PCR）和空斑试验定量RNA和感染性颗粒的病毒产量。结果如下：冠状病毒GX_P2V的刺突蛋白与2019-nCoV分离株Wuhan-hu-1的刺突蛋白具有92.2%的氨基酸同源性，并与2019-nCoV一样使用ACE 2作为感染受体。三种药物，包括千金藤素（CEP），赛拉菌素，盐酸甲氟喹，在10 μ mol/L的细胞培养中表现出完全抑制细胞病变效应。CEP对GX_P2V感染的抑制作用最强，半数最大效应浓度[EC 50]为0.98 μ mol/L。在感染后72 h（p.i.），经10 μ mol/L CEP处理的细胞中病毒RNA产量比未经CEP处理的细胞低15，393倍（[6.48 +/- 0.02] x 10（-4）vs. 1.00 +/- 0.12，t = 150.38，P < 0.001）。空斑试验发现在含有10 μ mol/L CEP的培养基中在感染后48 h没有活病毒产生。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] x 10（-4）vs.1.00 +/- 0.43，t = 3.98，P < 0.05）都具有有效的抗病毒活性。结论：我们的穿山甲冠状病毒GX_P2V是2019-nCoV研究的可行模型。CEP、赛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是泛β冠状病毒的广谱抑制剂，有必要进一步研究CEP治疗2019-nCoV感染。
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 mu mol/L. CEP demonstrated the most potent inhibition of GX_P2V infection, with a concentration for 50% of maximal effect [EC50] of 0.98 mu mol/L. The viral RNA yield in cells treated with 10 mu mol/L CEP was 15,393-fold lower than in cells without CEP treatment ([6.48 +/- 0.02] x 10(-4)vs. 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 mu mol/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] x 10(-4)vs. 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.