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Identification of SARS-CoV-2 Main Protease Cleavage Sites in Bovine β-Casein
János András Mótyán1, Tibor Nagy2, Ágota Nagyné Veres1
1Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary.
Abstract:
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease of 2019 (COVID-19) and has persistently caused infections since its emergence in late 2019. The main protease (Mpro) of SARS-CoV-2 plays a crucial role in its life-cycle; thus, it is an important target for drug development. One of the first virus-specific drugs that has been approved for the treatment of COVID-19 patients is Paxlovid, which contains nirmatrelvir, a covalent inhibitor of Mpro. Screening of inhibitor candidates and specificity studies also rely on efficient substrates and activity assays. Casein is one of the most commonly applied universal substrates that can be used to study a wide range of proteases, including SARS-CoV-2 Mpro. Casein is a known substrate for Mpro in vitro, but the specific casein isoform cleaved by Mpro remained unidentified, and the cleavage sites have yet to be determined. This work studied cleavage of α-, β- and κ-isoforms of bovine casein by SARS-CoV-2 Mpro, using in vitro and in silico approaches. The candidate cleavage sites were predicted in silico based on the protein sequences, and the cleavage positions were identified based on mass spectrometric analysis of cleavage fragments. Based on our results, only β-casein contains cleavage sites for Mpro and thus can be used as its substrate in vitro. The newly identified cleavage site sequences further widen the knowledge about the specificity of SARS-CoV-2 Mpro.
Insights
Researchers identified that only beta-casein serves as an effective substrate for SARS-CoV-2 main protease (Mpro). This finding is crucial for developing new COVID-19 antiviral drugs and understanding protease specificity.
Area of Science:
- Biochemistry
- Virology
- Drug Discovery
Background:
- Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19.
- The SARS-CoV-2 main protease (Mpro) is vital for viral replication and a key drug target.
- Paxlovid, an Mpro inhibitor, is an approved COVID-19 treatment.
Purpose of the Study:
- To identify the specific casein isoform cleaved by SARS-CoV-2 Mpro.
- To determine the exact cleavage sites within casein by Mpro.
- To validate casein as a substrate for Mpro activity assays.
Main Methods:
- In vitro enzymatic assays using alpha-, beta-, and kappa-casein isoforms.
- In silico prediction of potential Mpro cleavage sites.
- Mass spectrometry to identify Mpro cleavage fragments and confirm cleavage sites.
Main Results:
- Only beta-casein was identified as a substrate for SARS-CoV-2 Mpro.
- Specific cleavage sites within beta-casein were determined.
- Alpha- and kappa-casein isoforms were not cleaved by Mpro.
Conclusions:
- Beta-casein is a suitable substrate for SARS-CoV-2 Mpro in biochemical assays.
- The identified cleavage sites enhance understanding of Mpro substrate specificity.
- This research aids in the development of novel antiviral strategies and drug screening.
