The N-terminal peptide of the main protease of SARS-CoV-2, targeting dimer interface, inhibits its proteolytic activity
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The main protease (Mpro) of SARS-CoV-2 cleaves 11 sites ofviral polypeptide chains and generates essential non-structuralproteins for viral replication. Mpro is an important drug targetagainst COVID-19. In this study, we developed a real-time fluorometricturn-on assay system to evaluate Mpro proteolyticactivity for a substrate peptide between NSP4 and NSP5. Itproduced reproducible and reliable results suitable for HTSinhibitor assays. Thus far, most inhibitors against Mpro targetthe active site for substrate binding. Mpro exists as a dimer,which is essential for its activity. We investigated the potentialof the Mpro dimer interface to act as a drug target. The dimerinterface is formed of domain II and domain III of each protomer,in which N-terminal ten amino acids of the domain Iare bound in the middle as a sandwich. The N-terminal partprovides approximately 39% of the dimer interface between twoprotomers. In the real-time fluorometric turn-on assay system,peptides of the N-terminal ten amino acids, N10, can inhibitthe Mpro activity. The dimer interface could be a prospectivedrug target against Mpro. The N-terminal sequence can helpdevelop a potential inhibitor.
Abstract
The main protease (Mpro) of SARS-CoV-2 cleaves 11 sites ofviral polypeptide chains and generates essential non-structuralproteins for viral replication. Mpro is an important drug targetagainst COVID-19. In this study, we developed a real-time fluorometricturn-on assay system to evaluate Mpro proteolyticactivity for a substrate peptide between NSP4 and NSP5. Itproduced reproducible and reliable results suitable for HTSinhibitor assays. Thus far, most inhibitors against Mpro targetthe active site for substrate binding. Mpro exists as a dimer,which is essential for its activity. We investigated the potentialof the Mpro dimer interface to act as a drug target. The dimerinterface is formed of domain II and domain III of each protomer,in which N-terminal ten amino acids of the domain Iare bound in the middle as a sandwich. The N-terminal partprovides approximately 39% of the dimer interface between twoprotomers. In the real-time fluorometric turn-on assay system,peptides of the N-terminal ten amino acids, N10, can inhibitthe Mpro activity. The dimer interface could be a prospectivedrug target against Mpro. The N-terminal sequence can helpdevelop a potential inhibitor.
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