[Discovery of SARS-CoV-2 main protease inhibitors using an optimized FRET-based high-throughput screening assay].
Sheng Wu Gong Cheng Xue Bao
; 38(6): 2236-2249, 2022 Jun 25.
Article
in Chinese
| MEDLINE | ID: covidwho-1912217
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.
Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
High-Throughput Screening Assays
/
COVID-19
Type of study:
Systematic review/Meta Analysis
Limits:
Humans
Language:
Chinese
Journal:
Sheng Wu Gong Cheng Xue Bao
Journal subject:
Biotechnology
Year:
2022
Document Type:
Article
Affiliation country:
J.cjb.210657
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