Development of a Fluorescence-Based, High-Throughput SARS-CoV-2 3CL(pro) Reporter Assay.
Development of a Fluorescence-Based, High-Throughput SARS-CoV-2 3CL(pro) Reporter Assay.
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DOI：
10.1128/jvi.01265-20
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发表时间：
2020-10-27
影响因子：
5.4
通讯作者：
Heaton NS
中科院分区：
文献类型：
--
作者：
Froggatt HM;Heaton BE;Heaton NS
COVID-19大流行已导致全球超过70万人死亡，日常生活发生无数变化。沿着疫苗的开发，鉴定治疗感染患者的有效抗病毒药物是最重要的。然而，快速的药物发现需要有效的方法来鉴定可以抑制病毒的新化合物。在这项工作中，我们提出了一种方法，用于确定抑制剂的SARS冠状病毒2的主要蛋白酶，3CLpro。这种基于荧光素酶的检测允许在生物安全水平2（BSL 2）的人类细胞培养物中进行抗病毒药物筛选，具有高通量兼容的样品处理和分析。这种检测方法可能有助于识别新型抗病毒药物，以控制COVID-19大流行。在2019年底，一种人类冠状病毒，现在被称为严重急性呼吸道综合征冠状病毒2（SARS-CoV-2），出现了，可能来自人畜共患病水库。这种病毒导致COVID-19，感染了数百万人，并导致地球仪数十万人死亡。虽然控制并最终阻止大流行的最佳干预措施是预防性疫苗，但抗病毒治疗对于限制已感染者的发病率和死亡率也很重要。目前，只有一种FDA批准的抗SARS-CoV-2抗病毒药物Remdesivir可用，不幸的是，其疗效似乎有限。因此，鉴定新的和有效的抗病毒药物是最重要的。为了促进快速药物发现，需要灵活、灵敏和高通量的筛选方法。关于药物靶点，大多数注意力集中在病毒RNA依赖性RNA聚合酶或主要病毒蛋白酶3CLpro上。3CLpro是抗病毒治疗的一个有吸引力的靶标，因为它对于加工新翻译的病毒蛋白是必不可少的，并且没有蛋白酶活性就不能完成病毒生命周期。在这项工作中，我们报告了一种新的测定，以确定3CLpro的抑制剂。我们的报告基因是基于一种绿色荧光蛋白（GFP）衍生的蛋白质，该蛋白质仅在被3CLpro切割后才发出荧光。这种实验优化的报告基因检测允许在生物安全水平2（BSL 2）的人类细胞培养物中使用高通量兼容方案进行抗病毒药物筛选。使用这种筛选方法与现有的药物库相结合，可能会导致快速识别新的抗病毒药物，以抑制SARS-CoV-2的复制和传播。重要性COVID-19大流行已导致全球超过70万人死亡，日常生活发生无数变化。沿着疫苗的开发，鉴定治疗感染患者的有效抗病毒药物是最重要的。然而，快速的药物发现需要有效的方法来鉴定可以抑制病毒的新化合物。在这项工作中，我们提出了一种方法，用于确定抑制剂的SARS冠状病毒2的主要蛋白酶，3CLpro。这种基于荧光素酶的检测允许在生物安全水平2（BSL 2）的人类细胞培养物中进行抗病毒药物筛选，具有高通量兼容的样品处理和分析。这种检测方法可能有助于识别新型抗病毒药物，以控制COVID-19大流行。
The COVID-19 pandemic has already led to more than 700,000 deaths and innumerable changes to daily life worldwide. Along with development of a vaccine, identification of effective antivirals to treat infected patients is of the highest importance. However, rapid drug discovery requires efficient methods to identify novel compounds that can inhibit the virus. In this work, we present a method for identifying inhibitors of the SARS-CoV-2 main protease, 3CLpro. This reporter-based assay allows for antiviral drug screening in human cell culture at biosafety level 2 (BSL2) with high-throughput compatible sample processing and analysis. This assay may help identify novel antivirals to control the COVID-19 pandemic. In late 2019, a human coronavirus, now known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged, likely from a zoonotic reservoir. This virus causes COVID-19, has infected millions of people, and has led to hundreds of thousands of deaths across the globe. While the best interventions to control and ultimately stop the pandemic are prophylactic vaccines, antiviral therapeutics are important to limit morbidity and mortality in those already infected. At this time, only one FDA-approved anti-SARS-CoV-2 antiviral drug, remdesivir, is available, and unfortunately, its efficacy appears to be limited. Thus, the identification of new and efficacious antivirals is of the highest importance. In order to facilitate rapid drug discovery, flexible, sensitive, and high-throughput screening methods are required. With respect to drug targets, most attention is focused on either the viral RNA-dependent RNA polymerase or the main viral protease, 3CLpro. 3CLpro is an attractive target for antiviral therapeutics, as it is essential for processing newly translated viral proteins and the viral life cycle cannot be completed without protease activity. In this work, we report a new assay to identify inhibitors of 3CLpro. Our reporter is based on a green fluorescent protein (GFP)-derived protein that fluoresces only after cleavage by 3CLpro. This experimentally optimized reporter assay allows for antiviral drug screening in human cell culture at biosafety level 2 (BSL2) with high-throughput compatible protocols. Using this screening approach in combination with existing drug libraries may lead to the rapid identification of novel antivirals to suppress SARS-CoV-2 replication and spread. IMPORTANCE The COVID-19 pandemic has already led to more than 700,000 deaths and innumerable changes to daily life worldwide. Along with development of a vaccine, identification of effective antivirals to treat infected patients is of the highest importance. However, rapid drug discovery requires efficient methods to identify novel compounds that can inhibit the virus. In this work, we present a method for identifying inhibitors of the SARS-CoV-2 main protease, 3CLpro. This reporter-based assay allows for antiviral drug screening in human cell culture at biosafety level 2 (BSL2) with high-throughput compatible sample processing and analysis. This assay may help identify novel antivirals to control the COVID-19 pandemic.