新型三明治样荧光偏振筛选模型在新型冠状病毒主蛋白酶小分子抑制剂筛选中的应用

Identifying SARS-CoV-2 main protease inhibitors by a novel sandwich-like fluorescence polarization screening assay

摘要:

新型冠状病毒主蛋白酶（main protease，Mpro）通过水解多聚蛋白质体（polyprotein）调控病毒基因组RNA复制，且人体不存在其同源蛋白酶，这使Mpro成为抗新型冠状病毒药物开发的理想靶标之一。本研究基于荧光偏振技术（fluorescence polarization，FP）和生物素-亲和素反应（biotin-avidin system，BAS）原理，成功地建立了三明治样荧光偏振筛选模型用于Mpro小分子抑制剂的快速筛选。通过对天然产物化合物库进行高通量筛选，发现了漆树酸（anacardic acid，AA）是Mpro的竞争型抑制剂，1，2，3，4，6-O-五没食子酰葡萄糖（1，2，3，4，6-O-pentagalloylglucose，PGG）是Mpro的混合型抑制剂，且已报道的部分抑制剂是非特异性Mpro小分子抑制剂。文中建立的三明治样荧光偏振筛选模型具有良好的简便性、灵敏性和稳定性，初步证实了漆树酸和PGG是一类新型苗头化合物，建立科学严谨的活性评价体系对于抗新型冠状病毒药物的筛选与发现是至关重要的。

Abstract:

SARS-CoV-2 main protease (Mpro) is responsible for polyprotein cleavage to release non-structural proteins (nsps) for viral genomic RNA replication, and its homologues are absent in human cells. Therefore, Mpro has been regarded as one of the ideal drug targets for the treatment of coronavirus disease 2019 (COVID-19). In this study, we first combined the fluorescence polarization (FP) technique with biotin-avidin system (BAS) to develop a novel sandwich-like FP screening assay for quick discovery of SARS-CoV-2 Mpro inhibitors from a natural product library. With this screening assay, anacardic acid (AA) and 1,2,3,4,6-O-pentagalloylglucose (PGG) were found to be the competitive inhibitor and mixed-type inhibitor targeting Mpro, respectively. Importantly, our results showed that the majority of the reported Mpro inhibitors are promiscuous cysteine inhibitors that are not specific to Mpro. In summary, this novel sandwich-like FP screening assay is simple, sensitive, and robust, which is ideal for large-scale screening. Natural products AA and PGG will be the promising lead compounds for generating more potent antiviral agents targeting Mpro, and the stringent hit validation at the early stage of drug discovery is urgently needed.