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.
QI Haiyan
,
YAN Gangan
,
YAN Haohao
,
LIU Zhicheng
,
LIU Xiaoli
,
LIU Xiaoping
,
CHEN Yunyu
. Development and application of a high-throughput screening assay based on fluorescence resonance energy transfer for SARS-CoV-2 papain-like protease inhibitors[J]. Journal of Baotou Medical College, 2024
, 40(9)
: 74
-80
.
DOI: 10.16833/j.cnki.jbmc.2024.09.014
[1] 温亚亚,宋丽,汪巧菊,等. 新冠肺炎疫苗的研究现状及面临的挑战[J].生物技术通报,2022,38(7):136-145.
[2] Wu CR, Zheng MZ, Yang YY, et al. Furin: a potential therapeutic target for COVID-19[J]. iScience,2020,23(10):101642.
[3] Shang J, Wan YS, Luo CM, et al. Cell entry mechanisms of SARS-CoV-2[J]. Proc Natl Acad Sci USA,2020,117(21):11727-11734.
[4] Anirudhan V, Lee H, Cheng H, et al. Targeting SARS-CoV-2 viral proteases as a therapeutic strategy to treat COVID-19[J]. J Med Virol, 2021,93(5):2722-2734.
[5] Rimanshee A, Shweta K, Bharati P, et al. Structural insights into SARS-CoV-2 proteins[J]. J Mol Biol, 2021,433(2):166725.
[6] Donghyuk S, Rukmini M, Diana G, et al. Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity[J]. Nature, 2020,587(7835):657-662.
[7] 杜倩倩,张智慧,廉政,等. 新型冠状病毒PLpro蛋白酶结构与功能的生物信息学分析[J].病毒学报,2021,37(1):43-51.
[8] Brendan TF, Ian AD, Jackelyn M, et al. Characterization and noncovalent inhibition of the deubiquitinase and deISGylase activity of SARS-CoV-2 papain-like protease[J]. ACS Infect Dis, 2020,6(8):2099-2109.
[9] 闫干干,李淼,戚海燕,等. 新型冠状病毒木瓜样蛋白酶在大肠杆菌中的可溶表达与酶活性测定[J].中国现代应用药学,2022,39(1):5-11.
[10] Fu ZY, Huang B, Tang JL, et al. The complex structure of GRL0617 and SARS-CoV-2 PLpro reveals a hot spot for antiviral drug discovery[J]. Nat Commun, 2021,12(1):488.
[11] 戚海燕,闫干干,付正豪,等. 新型冠状病毒主蛋白酶抑制剂的筛选方法研究进展[J].生命的化学,2021,41(2):207-214.
[12] 李志伟,李本杰,刘志兵,等. 基于FRET原理构建AtLHT1转运底物筛选探针[J].华南农业大学学报,2022,43(1):77-84.
[13] Ronen B, Barbara AH, Ruben MM, et al. Ginkgolic acid inhibits fusion of enveloped viruses[J].Sci Rep,2020,10(1):4746.
[14] Vanbeek TA. Chemical analysis of Ginkgo biloba leaves and extracts[J]. J Chromatogr A,2002,967(1):21-55.
[15] Ibrahim MA,Ramadan HH,Mohammed RN. Evidence that Ginkgo Biloba could use in the influenza and coronavirus COVID-19 infections[J].J Basic Clin Physiol Pharmacol, 2021,32(3):131-143.
[16] 闫干干,李东升,戚海燕,等. 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[J].生物工程学报,2022,38(6):2236-2249.
