Yan Gangan, Li Dongsheng, Lin Yuan, Fu Zhenghao, Qi Haiyan, Liu Xiaoping, Zhang Jing, Si Shuyi, Chen Yunyu
Institute for Drug Screening and Evaluation, Wannan Medical College, 241002, Wuhu, China.
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, 100050, Beijing, China.
Cell Biosci. 2021 Dec 5;11(1):199. doi: 10.1186/s13578-021-00720-3.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmissible and has caused a pandemic named coronavirus disease 2019 (COVID-19), which has quickly spread worldwide. Although several therapeutic agents have been evaluated or approved for the treatment of COVID-19 patients, efficacious antiviral agents are still lacking. An attractive therapeutic target for SARS-CoV-2 is the main protease (Mpro), as this highly conserved enzyme plays a key role in viral polyprotein processing and genomic RNA replication. Therefore, the identification of efficacious antiviral agents against SARS-CoV-2 Mpro using a rapid, miniaturized and economical high-throughput screening (HTS) assay is of the highest importance at the present.
In this study, we first combined the fluorescence polarization (FP) technique with biotin-avidin system (BAS) to develop a novel and step-by-step sandwich-like FP screening assay to quickly identify SARS-CoV-2 Mpro inhibitors from a natural product library. Using this screening assay, dieckol, a natural phlorotannin component extracted from a Chinese traditional medicine Ecklonia cava, was identified as a novel competitive inhibitor against SARS-CoV-2 Mpro in vitro with an IC value of 4.5 ± 0.4 µM. Additionally, dieckol exhibited a high affinity with SARS-CoV-2 Mpro using surface plasmon resonance (SPR) analysis and could bind to the catalytic sites of Mpro through hydrogen-bond interactions in the predicted docking model.
This innovative sandwich-like FP screening assay enables the rapid discovery of antiviral agents targeting viral proteases, and dieckol will be an excellent lead compound for generating more potent and selective antiviral agents targeting SARS-CoV-2 Mpro.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）具有高度传染性，已引发了一场名为2019冠状病毒病（COVID-19）的大流行，并迅速在全球传播。尽管已经评估或批准了几种治疗药物用于治疗COVID-19患者，但仍然缺乏有效的抗病毒药物。SARS-CoV-2的一个有吸引力的治疗靶点是主要蛋白酶（Mpro），因为这种高度保守的酶在病毒多聚蛋白加工和基因组RNA复制中起关键作用。因此，目前使用快速、小型化且经济的高通量筛选（HTS）测定法来鉴定针对SARS-CoV-2 Mpro的有效抗病毒药物至关重要。
在本研究中，我们首先将荧光偏振（FP）技术与生物素-抗生物素蛋白系统（BAS）相结合，开发了一种新颖的逐步夹心式FP筛选测定法，以从天然产物库中快速鉴定SARS-CoV-2 Mpro抑制剂。使用这种筛选测定法，从中药海蕴中提取的天然间苯三酚单宁成分二eckol，在体外被鉴定为一种针对SARS-CoV-2 Mpro的新型竞争性抑制剂，IC值为4.5±0.4μM。此外，通过表面等离子体共振（SPR）分析表明二eckol与SARS-CoV-2 Mpro具有高亲和力，并且在预测的对接模型中可以通过氢键相互作用与Mpro的催化位点结合。
这种创新的夹心式FP筛选测定法能够快速发现针对病毒蛋白酶的抗病毒药物，并且二eckol将成为生成更有效和选择性更高的针对SARS-CoV-2 Mpro的抗病毒药物的优秀先导化合物。
