目的:以新型冠状病毒木瓜样蛋白酶(PLpro)为靶点,利用荧光共振能量转移(FRET)技术,建立用来筛选PLpro抑制剂的高通量筛选(HTS)模型。方法:通过大肠杆菌原核表达系统进行质粒构建、原核表达、分离纯化,获得高活性PLpro(以FRET检测PLpro生物活性),用的底物为两端被荧光基团Edans与淬灭基团Dabcyl修饰的多肽。通过优化实验条件,建立并评价PLpro抑制剂FRET高通量筛选模型,筛选苗头化合物。结果:利用大肠杆菌原核表达系统成功表达并分离出PLpro,其比活力＞3 000 U/mg。经过优化实验,确定模型筛选条件:设定HEPES缓冲液(pH 7.0)中NaCl浓度为50 mmol/L,孵育温度为25 ℃,孵育时间为30 min,PLpro 浓度为18 μmol/L,底物浓度为2 μmol/L。测得Z′因子值为 0.74,说明所建模型可用于高通量筛选。通过对天然产物化合物库进行筛选,发现十七烷一烯银杏酸(GA17∶1)对PLpro具有混合型抑制作用且IC50值为15.5 μmol/L。结论:成功建立PLpro小分子抑制剂高通量筛选模型,初步确定GA17∶1是一种新型抗PLpro的苗头化合物,该筛选方法对于快速筛选新型冠状病毒PLpro的靶向抑制剂具有十分重要的意义。
Objective: This study aimed to screen SARS-CoV-2 papain-like protease(PLpro) inhibitors, a high-throughput screening (HTS) assay based on fluorescence resonance energy transfer (FRET) technology was established. Methods: The recombiant PLpro was expressed in Escherichia coli Rosetta(DE3) cells, and a FRET-based method that used a fluorescently labeled substrate was used to test the biological activity of PLpro. For screening the hit compounds, the FRET HTS assay of PLpro was developed by optimizing experimental conditions, and evaluated by determining the Z′ factor value of the HTS assay. Results: The soluble PLpro was successfully expressed in in E. coli cells and the enzymatic activity of PLpro was more than 3000 U/mg. After optimization, the HTS assay conditions were determined: the concentration of NaCl in HEPES buffer (pH 7.0) was set at 50 mmol/L, the incubation temperature was 25 ℃, the incubation time was 30 min, the concentration of PLpro was 18 μmol/L, and the substrate concentration was 2 μmol/L.The Z′factor value was 0.74, indicating that the model can be used for screening PLpro inhibitors. The screening of natural product compound library revealed that ginkgolic acid C17∶1(GA17∶1) had mixed inhibitory effects on PLpro with an IC50 value of 15.5 μmol/L. Conclusion: A FRET-based HTS assay for PLpro inhibitors was successfully established, and GA17∶1 was initially identified as a novel anti-PLpro hit compounds. This screening assay is essential for rapid screening of inhibitors targeting SARS-CoV-2 PLpro.
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