Non-covalent cyclic peptides simultaneously targeting Mpro and NRP1 are highly effective against Omicron BA.2.75

Abstract

Available vaccine-based immunity may at high risk of being evaded due to substantial mutations in the variant Omicron. The main protease (Mpro) of SARS-CoV-2 and human neuropilin-1 (NRP1), two less mutable proteins, have been reported to be crucial for SARS-CoV-2 replication and entry into host cells, respectively. Their dual blockade may avoid vaccine failure caused by continuous mutations of the SARS-CoV-2 genome and exert synergistic antiviral efficacy. Herein, four cyclic peptides non-covalently targeting both Mpro and NRP1 were identified using virtual screening. Among them, MN-2 showed highly potent affinity to Mpro (K _d = 18.2 ± 1.9 nM) and NRP1 (K _d = 12.3 ± 1.2 nM), which was about 3,478-fold and 74-fold stronger than that of the positive inhibitors Peptide-21 and EG3287. Furthermore, MN-2 exhibited significant inhibitory activity against Mpro and remarkable anti-infective activity against the pseudotyped variant Omicron BA.2.75 without obvious cytotoxicity. These data demonstrated that MN-2, a novel non-covalent cyclic peptide, is a promising agent against Omicron BA.2.75.

Keywords: COVID-19; SARS-CoV-2; main protease; neuropilin-1; virtual screening.

FIGURE 1

Schematic illustration of the mechanism of dual Mpro/NRP1-targeting inhibitors against SARS-CoV-2.

FIGURE 2

Identification of cyclic peptides targeting Mpro/NRP1. (A) Details of the Mpro-based pharmacophore model (F1, F2, and F4 are hydrogen-bond donor features, and F3 is a hydrogen-bond acceptor feature). (B) The flowchart of virtual screening, interaction analysis, and biological assay for identification of dual Mpro/NRP1-targeting peptides. Pharmacophore features of Mpro are represented as spheres. Hydrogen bonds are represented by purple dotted lines. Italics indicate Mpro sub-pockets. Protein secondary structures are shown in line form. The surface of the Mpro and NRP1-BD are plotted by H-bonding (purple), hydrophobicity (green), and mild polar (blue) regions.

FIGURE 3

Predicted docking poses of MNs 1-4 at the Mpro active site. (A), (C), (E), and (G) are MNs 1-4, respectively, and (B), (D), (F), and (H) are their corresponding surface plots. Peptides are represented by different colors (orange for MN-1, pink for MN-2, blue for MN-3, and red for MN-4), and Mpro is color-coded by cyan-blue. Pharmacophore features of Mpro are represented as spheres. The hydrogen bonds were indicated by purple dotted lines. The surface of the Mpro is plotted by H-bonding (purple), hydrophobicity (green), and mild polar (blue) regions.

FIGURE 4

Predicted docking poses of MNs 1-4 at the NRP1-BD. (A), (C), (E), and (G) are MNs 1-4, respectively, and (B), (D), (F), and (H) are their corresponding surface plots. Peptides are represented by different colors (orange for MN-1, pink for MN-2, blue for MN-3, and red for MN-4), and NRP1-BD is color-coded by yellow. The hydrogen bonds were indicated by purple dotted lines. The surface of NRP1-BD is plotted by H-bonding (purple), hydrophobicity (green), and mild polar (blue) regions.

FIGURE 5

RMSD of Cα atoms of Mpro-peptide and NRP1-peptide complex atoms with respect to the initial structures obtained from docking. (A–D) RMSD in Mpro-peptide complexes, and (E–H) RMSD in NRP1-peptide complexes. In all panels the color code is MN-1 (green), MN-2 (blue), MN-3 (brown), and MN-4 (yellow).

FIGURE 6

RMSF of Cα atoms of Mpro residues in Mpro-peptide complexes (A–D) and NRP1 in NRP1-peptide complexes (E–H). In all panels the color code is MN-1 (green), MN-2 (blue), MN-3 (brown), and MN-4 (yellow).

FIGURE 7

Binding affinity, anti-pseudovirus infection, and cytotoxicity of MNs 1-4. (A) Sequences and binding affinities of peptides. ^aMST data shown represent the mean ± SD (n = 3). ^bPeptide-21 and EG3287 served as the positive controls. MNs 1-4 were cyclized through a disulfide bond formed by two cysteines. (B) The cytotoxicity effects of MNs 1-4 on 293T cells using MTT assay. (C) Infection rate of the screened peptides MNs 1-4, EG3287, Peptide-21, and the combination of EG3287 and Peptide-21 against pseudotyped SARS-CoV-2 Omicron BA.2.75 at a concentration of 2 μM in 293T cells. (D–G) The cytotoxicity effects of MNs 1-4 on A549 cells detected using MTT assay. Cells were treated with different concentrations (0–50 μM) of peptides for 48 h. The results are represented as mean ± SD (n = 3). *p < 0.05, **p < 0.01 means a significant difference versus Peptide-21.