Mótyán János András, Nagy Tibor, Nagyné Veres Ágota, Golda Mária, Mahdi Mohamed, Tőzsér József
Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary.
Department of Applied Chemistry, Faculty of Sciences and Technology, University of Debrecen, 4032 Debrecen, Hungary.
Int J Mol Sci. 2025 Jun 18;26(12):5829. doi: 10.3390/ijms26125829.
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.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）是2019冠状病毒病（COVID-19）的病原体，自2019年末出现以来持续引发感染。SARS-CoV-2的主要蛋白酶（Mpro）在其生命周期中起关键作用；因此，它是药物研发的重要靶点。已获批用于治疗COVID-19患者的首批病毒特异性药物之一是帕罗韦德，其含有Mpro的共价抑制剂奈玛特韦。抑制剂候选物的筛选和特异性研究也依赖于有效的底物和活性测定。酪蛋白是最常用的通用底物之一，可用于研究包括SARS-CoV-2 Mpro在内的多种蛋白酶。酪蛋白在体外是已知的Mpro底物，但Mpro切割的具体酪蛋白异构体仍未确定，切割位点也有待确定。这项工作使用体外和计算机模拟方法研究了SARS-CoV-2 Mpro对牛α-、β-和κ-酪蛋白异构体的切割。基于蛋白质序列在计算机模拟中预测候选切割位点，并基于切割片段的质谱分析确定切割位置。根据我们的结果，只有β-酪蛋白含有Mpro的切割位点，因此可在体外用作其底物。新确定的切割位点序列进一步拓宽了对SARS-CoV-2 Mpro特异性的认识。
