Characterization of SARS-COV-2 main protease inhibitory peptides from Ulva prolifera proteins

The main protease (M-pro) is essential for the replication of SARS-COV-2 and therefore represents a promising anti-viral target. In this study, we screened M-pro inhibitory peptides from Ulva prolifera protein on in-silico proteolysis. Cytotoxicity analysis using the online toxic prediction tool ToxinPred revealed that all the peptides were non-cytotoxic. The hexapeptide (SSGFID) exhibited high M-pro inhibitory activity in molecular docking and its IC50 value was 139.40 & PLUSMN;0.82 & mu;mol/L in vitro according to fluorescence resonance energy transfer assay (FRET). Quantitative real-time (qRT-) PCR results show that SSGFID could stimulate the expression of mitosis-related factors, including nuclear factor-& kappa;B, cyclin D1, and cyclin-dependent kinase 4, to promote the proliferation of mice splenocytes. Stability study revealed that SSGFID showed resistance against pepsin and trypsin but lost D (Asp) after pretreatment at 121 & DEG;C for 15 min. Besides, SSGFID was mainly transported through the Caco-2 cell monolayer by the peptide transporter PepT1 and passive-mediated transport during the transport study. Unfortunately, the peptide was also degraded by Caco-2 intracellular enzymes, and the transfer rate of intact peptide was 4.2%. Furthermore, Lineweaver-Burk plots demonstrated that SSGFID possessed a mixed inhibitory characteristic with M-pro. Our study indicated the potential of Ulva prolifera as antiviral and immune-enhancing functional food ingredients and nutraceuticals.