Coronaviruses (CoVs) act as cross-species viruses and have the potential to spread rapidly into new host species and cause epidemic diseases. Despite the severe public health threat of severe acute respiratory syndrome coronavirus and Middle East respiratory syndrome CoV (MERS-CoV), there are currently no drugs available for their treatment; therefore, broad-spectrum inhibitors of emerging and endemic CoVs are urgently needed. To search for effective inhibitory agents, we performed high-throughput screening (HTS) of a 2,000-compound library of approved drugs and pharmacologically active compounds using the established genetically engineered human CoV OC43 (HCoV-OC43) strain expressing Renilla luciferase (rOC43-ns2Del-Rluc) and validated the inhibitors using multiple genetically distinct CoVs in vitro We screened 56 hits from the HTS data and validated 36 compounds in vitro using wild-type HCoV-OC43. Furthermore, we identified seven compounds (lycorine, emetine, monensin sodium, mycophenolate mofetil, mycophenolic acid, phenazopyridine, and pyrvinium pamoate) as broad-spectrum inhibitors according to their strong inhibition of replication by four CoVs in vitro at low-micromolar concentrations. Additionally, we found that emetine blocked MERS-CoV entry according to pseudovirus entry assays and that lycorine protected BALB/c mice against HCoV-OC43-induced lethality by decreasing viral load in the central nervous system. This represents the first demonstration of in vivo real-time bioluminescence imaging to monitor the effect of lycorine on the spread and distribution of HCoV-OC43 in a mouse model. These results offer critical information supporting the development of an effective therapeutic strategy against CoV infection.IMPORTANCE Currently, there is no approved therapy to treat coronavirus infection; therefore, broad-spectrum inhibitors of emerging and endemic CoVs are needed. Based on our high-throughput screening assay using a compound library, we identified seven compounds with broad-spectrum efficacy against the replication of four CoVs in vitro Additionally, one compound (lycorine) was found to protect BALB/c mice against HCoV-OC43-induced lethality by decreasing viral load in the central nervous system. This inhibitor might offer promising therapeutic possibilities for combatting novel CoV infections in the future.

中文翻译：

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高通量筛选和鉴定冠状病毒的有效广谱抑制剂。

冠状病毒（CoV）充当跨物种病毒，并有可能迅速传播到新的宿主物种中并引起流行病。尽管严重急性呼吸系统综合症冠状病毒和中东呼吸系统综合症冠状病毒（MERS-CoV）严重威胁公共卫生，但目前尚无可用于治疗的药物。因此，迫切需要广谱的新兴和地方性冠状病毒抑制剂。为了寻找有效的抑制剂，我们对2种药物进行了高通量筛选（HTS）使用已建立的表达雷尼利亚荧光素酶的基因工程人类CoV OC43（HCoV-OC43）菌株的000种批准的药物和药理活性化合物的化合物文库，并使用多种遗传上不同的CoV在体外验证了抑制剂我们筛选了56个基因从HTS数据中提取，并使用野生型HCoV-OC43在体外验证了36种化合物。此外，根据它们在低微摩尔浓度下对四种CoV的体外复制的强烈抑制作用，我们确定了七种化合物（茄子碱，依替丁，莫能菌素钠，霉酚酸酯，霉酚酸酯，吩唑并吡啶和棕榈酸丙酮酯）为广谱抑制剂。另外，我们发现依替丁根据伪病毒进入检测阻止了MERS-CoV的进入，而蛋氨酸通过降低中枢神经系统的病毒载量来保护BALB / c小鼠免受HCoV-OC43诱导的致死性。这代表了体内实时生物发光成像的首次演示，该成像可监测lycorine对小鼠模型中HCoV-OC43的扩散和分布的影响。这些结果提供了关键信息，支持了针对CoV感染的有效治疗策略的开发。重要提示当前，尚无批准的用于治疗冠状病毒感染的疗法。因此，需要广谱的新兴和地方性冠状病毒抑制剂。根据我们使用化合物库进行的高通量筛选分析，我们鉴定了7种具有广谱功效的化合物，可在体外复制4株CoV。此外，还发现一种化合物（lycorine）可通过降低中枢神经系统的病毒载量来保护BALB / c小鼠免受HCoV-OC43诱导的致死性。这种抑制剂可能为将来对抗新型CoV感染提供有希望的治疗可能性。