Sunflower trypsin monocyclic inhibitor selected for the main protease of SARS-CoV-2 by phage display

Main protease (Mpro), also known as 3-chymotrypsin-like protease (3CLpro), is a nonstructural protein (NSP5) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the cleavage of virus polyproteins during viral replication at 11 sites, which generates 12 functional proteins. Mpro is a cysteine protease that presents specificity for the amino acid residue glutamine (Gln) at the P1 position of the substrate. Due to its essential role in processing the viral polyprotein for viral particle formation (assembly), Mpro inhibition has become an important tool to control coronavirus disease 2019 (COVID-19), since Mpro inhibitors act as antivirals. In this work, we proposed to identify specific inhibitors of the Mpro of SARSCoV- 2 using a monocyclic peptide (sunflower trypsin inhibitor (SFTI)) phage display library. Initially, we expressed, purified and activated recombinant Mpro. The screening of the mutant SFTI phage display library using recombinant Mpro as a receptor resulted in the five most frequent SFTI mutant sequences. Synthetized mutant SFTIs did not inhibit Mpro protease using the fluorogenic substrate. However, the mutant SFTI 4 efficiently decreased the cleavage of recombinant human prothrombin as a substrate by Mpro, as confirmed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Additionally, SFTI 4 presented a dissociation constant (KD) of 21.66 _ 6.66 μM for Mpro by surface plasmon resonance. Finally, 0.1 μM SFTI 4 reduced VERO cell infection by SARS-CoV-2 wt after 24 and 48 h. In conclusion, we successfully screened a monocyclic peptide library using phage display for the Mpro of SARS-CoV-2, suggesting that this methodology can be useful in identifying new inhibitors of viral enzymes.