Discovery of New Fusion Inhibitor Peptides against SARS-CoV-2 by Targeting the Spike S2 Subunit

Abstract

A novel coronavirus, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), caused a worldwide pandemic. Our aim in this study is to produce new fusion inhibitors against SARS-CoV-2, which can be the basis for developing new antiviral drugs. The fusion core comprising the heptad repeat domains (HR1 and HR2) of SARS-CoV-2 spike (S) were used to design the peptides. A total of twelve peptides were generated, comprising a short or truncated 24-mer (peptide #1), a long 36-mer peptide (peptide #2), and ten peptide #2 analogs. In contrast to SARS-CoV, SARS-CoV-2 S-mediated cell-cell fusion cannot be inhibited with a minimal length, 24-mer peptide. Peptide #2 demonstrated potent inhibition of SARS-CoV-2 S-mediated cell-cell fusion at 1 µM concentration. Three peptide #2 analogs showed IC50 values in the low micromolar range (4.7-9.8 µM). Peptide #2 inhibited the SARS-CoV-2 pseudovirus assay at IC50=1.49 µM. Given their potent inhibition of viral activity and safety and lack of cytotoxicity, these peptides provide an attractive avenue for the development of new prophylactic and therapeutic agents against SARS-CoV-2.

Keywords: Antiviral drugs; COVID-19; Fusion inhibitors; SARS-CoV-2.

Fig. 1

The structure of SARS-CoV-2 fusion core and sequence of peptides. (A) Schematic representation of SARS-CoV-2 Spike. The S1 subunit contains the N-terminal domain (NTD) and the receptor-binding domain (RBD). The S2 subunit comprises the fusion protein (FP) and two heptad repeat domains, HR1 and HR2. (B) HR1 and HR2 monomers during the fusion state showing the regions comprising the synthesized. (C) The position of peptides #1 and 2 on HR2. The side chains of amino acids is presented in green dots. (D) The sequences of peptides #1-12, the conserved sequences were highlighted with the same colour.

Fig. 2

The effect of peptides on SARS-CoV-2 S-mediated membrane fusion. (A) The effect of each peptides on the coculture fusion assay using DSP as a reporter. Peptides were tested at different concentrations, and the additional proteins other than reporters (DSPs) transduced into the effector and target cells are indicated below the graph. The relative cell fusion was represented as the DSP value (RL activity measured in RLU) normalized to that of the control assay with DMSO alone. (B) The effect of each peptides on RL measurement. Each peptides was added to cells co-expressing DSP1-7 and DSP8-11 to evaluate its direct inhibitory effects on RL. The relative DSP signal is indicated in the vertical axis by setting the control value with DSP alone as 100%. (C) Dose-response analysis for 5 peptides. The relative cell fusion was represented as the DSP value normalized to that of the control assay with DMSO alone. (D) Calculated IC50 value in figure (C). *p<0.05, **p<0.01 vs. control assay with DMSO.

Fig. 3

The effect of peptide #2 on SARS-CoV-2 S-mediated viral infection. (A) The effect of peptide #2 on infection of Calu-3 cells with SARS-CoV-2 S pseudotyped VSV viral particles. The relative infectivity was represented as the RLU normalized to that of the control assay with DMSO alone. (B) The effect of peptide #2 on infection of Calu-3 cells with VSV-G pseudotyped VSV viral particles. The relative infectivity was represented as the RLU normalized to that of the control assay with DMSO alone. **p<0.01.

Fig. 4

Molecular dynamics simulation of the peptides #2-12 complexes with HR1. (A) RMSD plot showing the changes of the α-carbon atom over the simulation time. (B) RMSF plot showing the fluctuations of the peptidesHR2 complex residues.