Design of Potent Membrane Fusion Inhibitors against SARS-CoV-2, an Emerging Coronavirus with High Fusogenic Activity

Abstract

The 2019 coronavirus disease (COVID-19), caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has posed serious threats to global public health and economic and social stabilities, calling for the prompt development of therapeutics and prophylactics. In this study, we first verified that SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) as a cell receptor and that its spike (S) protein mediates high membrane fusion activity. The heptad repeat 1 (HR1) sequence in the S2 fusion protein of SARS-CoV-2 possesses markedly increased α-helicity and thermostability, as well as a higher binding affinity with its corresponding heptad repeat 2 (HR2) site, than the HR1 sequence in S2 of severe acute respiratory syndrome coronavirus (SARS-CoV). Then, we designed an HR2 sequence-based lipopeptide fusion inhibitor, termed IPB02, which showed highly potent activities in inhibiting SARS-CoV-2 S protein-mediated cell-cell fusion and pseudovirus transduction. IPB02 also inhibited the SARS-CoV pseudovirus efficiently. Moreover, the structure-activity relationship (SAR) of IPB02 was characterized with a panel of truncated lipopeptides, revealing the amino acid motifs critical for its binding and antiviral capacities. Therefore, the results presented here provide important information for understanding the entry pathway of SARS-CoV-2 and the design of antivirals that target the membrane fusion step.IMPORTANCE The COVID-19 pandemic, caused by SARS-CoV-2, presents a serious global public health emergency in urgent need of prophylactic and therapeutic interventions. The S protein of coronaviruses mediates viral receptor binding and membrane fusion, thus being considered a critical target for antivirals. Herein, we report that the SARS-CoV-2 S protein has evolved a high level of activity to mediate cell-cell fusion, significantly differing from the S protein of SARS-CoV that emerged previously. The HR1 sequence in the fusion protein of SARS-CoV-2 adopts a much higher helical stability than the HR1 sequence in the fusion protein of SARS-CoV and can interact with the HR2 site to form a six-helical bundle structure more efficiently, underlying the mechanism of the enhanced fusion capacity. Also, importantly, the design of membrane fusion inhibitors with high potencies against both SARS-CoV-2 and SARS-CoV has provided potential arsenals to combat the pandemic and tools to exploit the fusion mechanism.

Keywords: SARS-CoV-2; fusion inhibitor; lipopeptide; membrane fusion.

FIG 1

Schematic diagram of SARS-CoV-2 S protein and its peptide derivatives. (A) Functional domains of the S protein. SP, signal peptide; NTD, N-terminal domain; RBD, receptor-binding domain; SD, subdomain; FP, fusion peptide; HR1, heptad repeat 1; CH, central helix; CD, connector domain; HR2, heptad repeat 2; TM, transmembrane domain; CT, cytoplasmic tail. The S1/S2 cleavage site (685/686) is marked. The HR1 and HR2 sequences and membrane-proximal external sequence (MPES) are listed. (B) HR2-derived fusion inhibitor peptides. chol, cholesterol.

FIG 2

Functional characterization of the SARS-CoV-2 and SARS-CoV S proteins. (A) The infectivity of the SARS-CoV-2 and SARS-CoV pseudoviruses in 293T cells or 293T/ACE2 cells was determined by a single-cycle infection assay. (B) The fusogenic activity of the SARS-CoV-2 and SARS-CoV S proteins with 293T cells or 293T/ACE2 cells as a target was determined by a DSP-based cell fusion assay. (C and D) The fusion activities of S proteins in 293T cells (C) and 293T/ACE2 cells (D) were determined at different time points. The experiments were repeated three times, and the data are expressed as the means ± standard deviations.

FIG 3

Interactions between the HR1 and HR2 peptides derived from the S2 proteins of SARS-CoV-2 and SARS-CoV. (A) Sequence comparison of the HR1 and HR2 domains in SARS-CoV-2 and SARS-CoV. The sequences in the dotted boxes represent the core sites that mediate the helical interaction between the HR1 and HR2 domains. (B to E) The α-helicity and thermostability of the HR1 peptides alone (B and C) or in complexes with an HR2 peptide (D and E) were determined by CD spectroscopy, in which the peptides or peptide mixtures were dissolved in PBS, with the final concentration of each peptide being 10 μM. The experiments were performed two times, and representative data are shown.

FIG 4

Secondary structure and binding stability of fusion inhibitor peptides determined by CD spectroscopy. The α-helicity and thermostability of peptide inhibitors alone (A and B) or in complexes with the SARS-CoV-2 HR1 peptide (C and D) or the SARS-CoV HR1 peptide (E and F) were detected, with the final concentration of each peptide being 10 μM. The experiments were performed two times, and representative data are shown.

FIG 5

Visualization of the interactions between HR1 peptides and inhibitors by N-PAGE analysis. Each of the peptides was used at a final concentration of 40 μM. The positively charged peptides SARS2NP and SARS1NP migrated up and off the gel; thus, no bands appeared. IPB01 or IPB02 alone and their complexes with SARS2NP or SARS1NP displayed specific bands because of their net negative charges. The experiments were repeated two times, and representative data are shown.

FIG 6

Inhibitory activity of IPB01 and IPB02 against SARS-CoV-2 and SARS-CoV. (A) Inhibition of the SARS-CoV-2 S protein-mediated cell-cell fusion by the inhibitors, determined by a DSP-based cell fusion assay. (B to D) The activity of IPB01 and IPB02 in inhibiting SARS-CoV-2 (B), SARS-CoV (C), and control pseudovirus VSV-G (D) was determined by a single-cycle infection assay. The experiments were repeated three times, and the data are expressed as the means ± standard deviations.