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A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation
Published on: January 9, 2019
Design of wide-spectrum inhibitors targeting coronavirus main proteases
Haitao Yang1, Weiqing Xie, Xiaoyu Xue
1Tsinghua-IBP Joint Research Group for Structural Biology, Tsinghua University, Beijing, China.
Abstract:
The genus Coronavirus contains about 25 species of coronaviruses (CoVs), which are important pathogens causing highly prevalent diseases and often severe or fatal in humans and animals. No licensed specific drugs are available to prevent their infection. Different host receptors for cellular entry, poorly conserved structural proteins (antigens), and the high mutation and recombination rates of CoVs pose a significant problem in the development of wide-spectrum anti-CoV drugs and vaccines. CoV main proteases (M(pro)s), which are key enzymes in viral gene expression and replication, were revealed to share a highly conservative substrate-recognition pocket by comparison of four crystal structures and a homology model representing all three genetic clusters of the genus Coronavirus. This conclusion was further supported by enzyme activity assays. Mechanism-based irreversible inhibitors were designed, based on this conserved structural region, and a uniform inhibition mechanism was elucidated from the structures of Mpro-inhibitor complexes from severe acute respiratory syndrome-CoV and porcine transmissible gastroenteritis virus. A structure-assisted optimization program has yielded compounds with fast in vitro inactivation of multiple CoV M(pro)s, potent antiviral activity, and extremely low cellular toxicity in cell-based assays. Further modification could rapidly lead to the discovery of a single agent with clinical potential against existing and possible future emerging CoV-related diseases.
Insights
Developing broad-spectrum antiviral drugs is challenging due to coronavirus (CoV) diversity. Researchers identified a conserved active site in CoV main proteases (Mpro), leading to novel inhibitors effective against multiple CoV strains.
Area of Science:
- Virology
- Drug Discovery
- Structural Biology
Background:
- Coronaviruses (CoVs) are significant pathogens causing severe diseases in humans and animals.
- Existing treatments lack broad-spectrum efficacy due to CoV genetic diversity and rapid mutation rates.
- No specific antiviral drugs are currently licensed for CoV infections.
Purpose of the Study:
- To identify a conserved target for developing broad-spectrum antiviral agents against coronaviruses.
- To design and evaluate novel inhibitors targeting the conserved CoV main protease (Mpro).
Main Methods:
- Comparative analysis of crystal structures and homology modeling of CoV main proteases (Mpro).
- Enzyme activity assays to confirm protease function and inhibition.
- Structure-based design and optimization of mechanism-based irreversible inhibitors.
- In vitro and cell-based assays to assess antiviral activity and cytotoxicity.
Main Results:
- A highly conserved substrate-recognition pocket was identified across different CoV Mpro enzymes.
- Mechanism-based irreversible inhibitors targeting this conserved pocket demonstrated potent in vitro inactivation of multiple CoV Mpros.
- Optimized compounds exhibited significant antiviral activity with low cellular toxicity.
Conclusions:
- The conserved Mpro active site represents a promising target for broad-spectrum coronavirus drug development.
- Structure-assisted drug design yielded effective inhibitors with potential for clinical application against current and emerging CoV diseases.
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