Development and application of a high-throughput screening assay based on fluorescence resonance energy transfer for SARS-CoV-2 papain-like protease inhibitors
Development and application of a high-throughput screening assay based on fluorescence resonance energy transfer for SARS-CoV-2 papain-like protease inhibitors
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 es-tablished.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 de-termining 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 suc-cessfully 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.
关键词
新型冠状病毒/木瓜样蛋白酶抑制剂/荧光共振能量转移法/高通量筛选/十七烷一烯银杏酸
Key words
SARS-CoV-2/papain-like protease inhibitor/fluorescence resonance energy transfer/high-throughput screening/ginkgolic acid C17:1
维普
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