新冠病毒主蛋白酶小分子抑制剂荧光共振能量转移高通量筛选模型的优化与应用

Discovery of SARS-CoV-2 main protease inhibitors using an optimized FRET-based high-throughput screening assay

摘要:

基于荧光共振能量转移（fluorescence resonance energy transfer，FRET）原理，以新冠病毒主蛋白酶（main protease，Mpro）为靶标，建立并应用Mpro小分子抑制剂FRET高通量筛选模型，以期快速筛选新型Mpro小分子抑制剂。利用大肠杆菌原核表达与分离纯化高活性的Mpro，再以FRET法进行比活力测定。基于FRET原理，以7-甲氧基香豆素-4-乙酸（7-methoxycoumarin-4-acetic acid，MCA）与2，4-二硝基苯酚（2，4-dinitropheno，Dnp）标记的多肽作为Mpro水解底物，通过优化反应缓冲液、Mpro反应浓度、反应温度与时间及DMSO耐受浓度，建立并应用Mpro小分子抑制剂FRET高通量筛选模型进行苗头化合物的筛选。利用大肠杆菌实现了高活性Mpro的原核表达与分离纯化，且比活力不低于40 000 U/mg。通过一系列优化实验，使用0.4 μmol/L Mpro与5 μmol/L底物建立了Z'因子值为0.79的Mpro小分子抑制剂FRET高通量筛选模型，且反应体系中含有的二硫苏糖醇（1，4-dithiothreitol，DTT）是影响FRET筛选模型可靠性的重要因素。通过对天然产物化合物库进行高通量筛选，发现白花丹素与银杏酸在体外对Mpro酶活性具有良好的抑制作用。本研究建立了基于FRET原理的Mpro小分子抑制剂高通量筛选模型，初步证实了白花丹素与银杏酸是一类新型苗头化合物，为抗新型冠状病毒药物先导化合物的筛选与发现奠定了基础。

Abstract:

For rapid discovery of novel SARS-CoV-2 main protease (Mpro) inhibitors, an optimized fluorescence resonance energy transfer (FRET)-based high-throughput screening (HTS) assay was developed. The recombinant Mpro was expressed in Escherichia coli Rosetta (DE3) cells and the specific activity of purified Mpro was assessed by a FERT assay using a fluorescently labeled substrate. Subsequently, the reaction buffer, working concentration of Mpro, incubation temperature and length, and DMSO tolerance were systematically optimized. The Mpro was solubly expressed in E. coli cells and exhibited an expected enzymatic activity (40 000 U/mg) in a FRET assay. Through these systematic optimizations, we selected 0.4 μmol/L Mpro and 5 μmol/L FRET substrate as the optimal working concentrations in this FRET screening assay, and a high Z' factor of 0.79 was achieved. More importantly, the addition of reducing reagent 1,4-dithiothreitol in reaction buffer is necessary to faithfully assess the reliability of the screening assay. Using this assay, plumbagin (PLB) and ginkgolic acid (GA) were identified as potential Mpro inhibitors in vitro from a natural product library. In summary, we developed an optimized FRET-based HTS assay for the discovery of Mpro inhibitors, and PLB and GA could serve as the promissing lead compounds to generate more potent antiviral agents targeting SARS-CoV-2 Mpro.