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Updated: Oct 3, 2025
Quantitative Structure-Activity Relationship, Activity Prediction, and Molecular Dynamics of Non-nucleotide Reverse Transcriptase Inhibitors
Published on: May 9, 2025
Non-covalent SARS-CoV-2 Mpro inhibitors developed from in silico screen hits
Giacomo G Rossetti1,2, Marianna A Ossorio1, Stephan Rempel2
1Department of Molecular Biology, University of Geneva, 1205, Geneva, Switzerland.
Abstract:
Mpro, the main protease of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is essential for the viral life cycle. Accordingly, several groups have performed in silico screens to identify Mpro inhibitors that might be used to treat SARS-CoV-2 infections. We selected more than five hundred compounds from the top-ranking hits of two very large in silico screens for on-demand synthesis. We then examined whether these compounds could bind to Mpro and inhibit its protease activity. Two interesting chemotypes were identified, which were further evaluated by characterizing an additional five hundred synthesis on-demand analogues. The compounds of the first chemotype denatured Mpro and were considered not useful for further development. The compounds of the second chemotype bound to and enhanced the melting temperature of Mpro. The most active compound from this chemotype inhibited Mpro in vitro with an IC50 value of 1 μM and suppressed replication of the SARS-CoV-2 virus in tissue culture cells. Its mode of binding to Mpro was determined by X-ray crystallography, revealing that it is a non-covalent inhibitor. We propose that the inhibitors described here could form the basis for medicinal chemistry efforts that could lead to the development of clinically relevant inhibitors.
Insights
Researchers identified novel SARS-CoV-2 Mpro inhibitors from in silico screens. One compound non-covalently binds and inhibits the main protease, suppressing viral replication in cell culture, offering a basis for new COVID-19 therapeutics.
Area of Science:
- Virology
- Biochemistry
- Medicinal Chemistry
Background:
- The main protease (Mpro) of SARS-CoV-2 is crucial for viral replication.
- In silico screens have been employed to discover potential Mpro inhibitors for treating COVID-19.
Purpose of the Study:
- To synthesize and evaluate compounds identified from in silico screens for Mpro inhibition.
- To characterize novel Mpro inhibitors and assess their potential for therapeutic development.
Main Methods:
- On-demand synthesis of over 500 compounds from in silico screens.
- Biochemical assays to assess Mpro binding and protease activity.
- X-ray crystallography to determine inhibitor binding mode.
- Cell-based assays to evaluate antiviral activity against SARS-CoV-2.
Main Results:
- Two distinct chemotypes of Mpro inhibitors were identified.
- Compounds of the second chemotype bound to Mpro, stabilizing it and inhibiting its activity.
- The most potent compound demonstrated an IC50 of 1 μM and suppressed SARS-CoV-2 replication in vitro.
- X-ray crystallography revealed a non-covalent binding mode for the lead inhibitor.
Conclusions:
- Novel non-covalent Mpro inhibitors were discovered and characterized.
- These compounds show promise as a foundation for developing clinically relevant COVID-19 therapeutics.
- Further medicinal chemistry optimization could lead to effective antiviral drugs.
