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Updated: Aug 21, 2026
A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation
Published on: January 9, 2019
Characterization of SARS-CoV main protease and identification of biologically active small molecule inhibitors using
Richard Y Kao1, Amanda P C To, Louisa W Y Ng
1Department of Microbiology, The University of Hong Kong, Pokfulam, Hong Kong, China. rytkao@hkucc.hku.hk
Abstract:
Severe acute respiratory syndrome associated coronavirus main protease (SARS-CoV Mpro) has been proposed as a prime target for anti-SARS drug development. We have cloned and overexpressed the SARS-CoV Mpro in Escherichia coli, and purified the recombinant Mpro to homogeneity. The kinetic parameters of the recombinant SARS-CoV Mpro were characterized by high performance liquid chromatography-based assay and continuous fluorescence-based assay. Two novel small molecule inhibitors of the SARS-CoV Mpro were identified by high-throughput screening using an internally quenched fluorogenic substrate. The identified inhibitors have Ki values at low microM range with comparable anti-SARS-CoV activity in cell-based assays.
Insights
Researchers identified two novel small molecule inhibitors targeting the SARS-CoV Mpro, a key enzyme in severe acute respiratory syndrome coronavirus replication. These inhibitors show potential for developing new anti-SARS therapies.
Area of Science:
- Biochemistry
- Virology
- Drug Discovery
Background:
- Severe acute respiratory syndrome coronavirus main protease (SARS-CoV Mpro) is a critical target for antiviral drug development.
- Understanding Mpro's kinetics and identifying inhibitors are crucial for combating SARS-CoV.
Purpose of the Study:
- To clone, overexpress, and purify SARS-CoV Mpro.
- To characterize the kinetic parameters of the recombinant enzyme.
- To identify novel small molecule inhibitors of SARS-CoV Mpro.
Main Methods:
- Cloning and overexpression of SARS-CoV Mpro in Escherichia coli.
- Purification of recombinant Mpro to homogeneity.
- Kinetic parameter characterization using HPLC and fluorescence-based assays.
- High-throughput screening for inhibitors using a fluorogenic substrate.
Main Results:
- Recombinant SARS-CoV Mpro was successfully purified.
- Kinetic parameters of the enzyme were determined.
- Two novel small molecule inhibitors with low microM Ki values were identified.
- Inhibitors demonstrated comparable anti-SARS-CoV activity in cell-based assays.
Conclusions:
- The study successfully produced and characterized SARS-CoV Mpro.
- Novel small molecule inhibitors targeting SARS-CoV Mpro were discovered.
- These inhibitors hold promise for the development of effective anti-SARS-CoV drugs.
