A ricin-based peptide BRIP from Hordeum vulgare inhibits Mpro of SARS-CoV-2

Abstract

COVID-19 pandemic caused by SARS-CoV-2 led to the research aiming to find the inhibitors of this virus. Towards this world problem, an attempt was made to identify SARS-CoV-2 main protease (M^pro) inhibitory peptides from ricin domains. The ricin-based peptide from barley (BRIP) was able to inhibit M^pro in vitro with an IC₅₀ of 0.52 nM. Its low and no cytotoxicity upto 50 µM suggested its therapeutic potential against SARS-CoV-2. The most favorable binding site on M^pro was identified by molecular docking and steered molecular dynamics (MD) simulations. The M^pro-BRIP interactions were further investigated by evaluating the trajectories for microsecond timescale MD simulations. The structural parameters of M^pro-BRIP complex were stable, and the presence of oppositely charged surfaces on the binding interface of BRIP and M^pro complex further contributed to the overall stability of the protein-peptide complex. Among the components of thermodynamic binding free energy, Van der Waals and electrostatic contributions were most favorable for complex formation. Our findings provide novel insight into the area of inhibitor development against COVID-19.

Figure 1

The multiple sequence alignment of type-I RIPs; an antifungal RIP1 from barley; Hordeum vulgare (accession number KAE8814914.1), trichosanthin from Trichosanthes kirilowii (accession number AAA34207.1), saporin from Saponaria officinalis (accession number CAA41948.1), PAP-S1 from Phytolacca americana (accession number KT630652.1.) with conserved ricin/shiga toxin domain (predicted by ExPASY PROSITE) boxed (a). (b) The sequences of ricin- peptides retrieved from barley RIP (BRIP), trichosanthin (TRI), saporin (SAP) and PAP-S1 (PAP).

Figure 2

Effect of BRIP on inhibition of M^pro activity and determination of cytotoxicity. (a) The IC₅₀ value (0.52 nM) of BRIP was determined by studying the inhibition of M^pro at different test concentrations, as described in the Methods section. (b) Percent hemolysis of rat erythrocytes by BRIP at different concentrations. Data represent mean ± SE, n = 3 independent replicates.

Figure 3

Detection of the protein-peptide binding site. (a) BRIP docked on the top five predicted binding sites on M^pro. (b) The pull force profiles of BRIP attached to the binding pockets of M^pro. The pull force trajectories are colored according to the binding poses shown in panel a. (c) The M^pro-BRIP interactions at the most favorable binding site. The solid blue color lines represent hydrogen bonds, while the striped lines denote non-bonded interactions. The residue color coding scheme: positive (blue), negative (red), neutral (green), aliphatic (gray), aromatic (purple), proline and glycine (orange).

Figure 4

The net charge distribution on surface of M^pro and BRIP at different time intervals during the simulation. (a) 250 ns, (b) 500 ns, (c) 750 ns, and (d) 1000 ns.

Figure 5

The Gibbs free energy landscape of M^pro-BRIP complex showing the metastable conformation at the lowest energy basin.

Figure 6

The key residues involved in M^pro-BRIP interactions. (a) The interaction profiles of static poses extracted at different time intervals during the simulation. The solid blue and red color lines represents hydrogen bonds and salt bridges respectively, while the striped lines denote non bonded interactions. The residue color coding scheme is as follows: positive (blue), negative (red), neutral (green), aliphatic (gray), aromatic (purple), proline and glycine (orange). (b) The per residue contribution energy of M^pro and BRIP observed for the entire simulation run by the MM-PBSA method.