Inhibition of Coronavirus Entry In Vitro and Ex Vivo by a Lipid-Conjugated Peptide Derived from the SARS-CoV-2 Spike Glycoprotein HRC Domain

Abstract

The emergence of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), the etiological agent of the 2019 coronavirus disease (COVID-19), has erupted into a global pandemic that has led to tens of millions of infections and hundreds of thousands of deaths worldwide. The development of therapeutics to treat infection or as prophylactics to halt viral transmission and spread is urgently needed. SARS-CoV-2 relies on structural rearrangements within a spike (S) glycoprotein to mediate fusion of the viral and host cell membranes. Here, we describe the development of a lipopeptide that is derived from the C-terminal heptad repeat (HRC) domain of SARS-CoV-2 S that potently inhibits infection by SARS-CoV-2. The lipopeptide inhibits cell-cell fusion mediated by SARS-CoV-2 S and blocks infection by live SARS-CoV-2 in Vero E6 cell monolayers more effectively than previously described lipopeptides. The SARS-CoV-2 lipopeptide exhibits broad-spectrum activity by inhibiting cell-cell fusion mediated by SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV) and blocking infection by live MERS-CoV in cell monolayers. We also show that the SARS-CoV-2 HRC-derived lipopeptide potently blocks the spread of SARS-CoV-2 in human airway epithelial (HAE) cultures, an ex vivo model designed to mimic respiratory viral propagation in humans. While viral spread of SARS-CoV-2 infection was widespread in untreated airways, those treated with SARS-CoV-2 HRC lipopeptide showed no detectable evidence of viral spread. These data provide a framework for the development of peptide therapeutics for the treatment of or prophylaxis against SARS-CoV-2 as well as other coronaviruses.IMPORTANCE SARS-CoV-2, the causative agent of COVID-19, continues to spread globally, placing strain on health care systems and resulting in rapidly increasing numbers of cases and mortalities. Despite the growing need for medical intervention, no FDA-approved vaccines are yet available, and treatment has been limited to supportive therapy for the alleviation of symptoms. Entry inhibitors could fill the important role of preventing initial infection and preventing spread. Here, we describe the design, synthesis, and evaluation of a lipopeptide that is derived from the HRC domain of the SARS-CoV-2 S glycoprotein that potently inhibits fusion mediated by SARS-CoV-2 S glycoprotein and blocks infection by live SARS-CoV-2 in both cell monolayers (in vitro) and human airway tissues (ex vivo). Our results highlight the SARS-CoV-2 HRC-derived lipopeptide as a promising therapeutic candidate for SARS-CoV-2 infections.

Keywords: SARS-CoV-2; fusion inhibitor; lipopeptide; spike protein.

FIG 1

SARS-CoV-2 spike (S) glycoprotein domain architecture and structure. (A) Simplified schematic diagram of SARS-CoV-2 S. The N-terminal domain (NTD), receptor-binding domain (RBD), fusion peptide (FP), N-terminal heptad repeat (HRN), C-terminal heptad repeat (HRC), transmembrane (TM), and cytoplasmic tail (CP) domains are depicted. (B) Prefusion conformation of SARS-CoV-2 S (PDB accession number 6VSB). (C) Model of postfusion conformation of SARS-CoV-2 S based on homology with HCoV-229E S (PDB 6B3O) and SARS-CoV S (PDB 1WYY). (D) Sequences of SARS-CoV-2 HRC, MERS-CoV HRC, and EK1 peptides.

FIG 2

Fusion inhibition assay: inhibiting SARS-CoV-2, SARS-CoV-1, and MERS S-protein-mediated fusion. (A and B) Inhibition of S-mediated fusion in 293T cells with high concentration of ACE-2 receptor (A) or low concentration of ACE-2 receptor (B) by lipopeptides derived from SARS-CoV-2 HRC (red), MERS-CoV HRC (orange), EK1 (blue), or HPIV3 HRC (black). (C) Inhibition of fusion mediated by SARS-CoV-2 S mutants, MERS-CoV S, and SARS-CoV-1 S. Percent inhibition was calculated as the ratio of relative luminescence units in the presence of a specific concentration of inhibitor and the relative luminescence units in the absence of inhibitor and corrected for background luminescence as follows: percent inhibition = 100 × [1 − (luminescence at X − background)/(luminescence in the absence of inhibitor – background)]. Data are means ± standard errors (SE) (error bars) from three separate experiments with the curve representing a four-parameter dose-response model.

FIG 3

Virus inhibition assay: inhibiting SARS-CoV-2 and MERS-CoV virus infection. (A and B) Inhibition of infection by live SARS-CoV-2 (A) or MERS-CoV (B) by lipopeptides derived from SARS-CoV-2 HRC (red), MERS-CoV HRC (orange), EK1 (blue), or HPIV3 HRC (black). Percent inhibition was calculated as the ratio of PFU in the presence of a specific concentration of inhibitor and the PFU in the absence of inhibitor. Data are means ± SE from three separate experiments with the curve representing a four-parameter dose-response model.

FIG 4

SARS-CoV-2-derived cholesterol-conjugated peptides block SARS-CoV-2-mNeonGreen viral spread in human airway epithelial cells (HAE). (A) HAE cells were infected with SARS-CoV-2 (2,000 PFU/well for a multiplicity of infection of ∼0.02) for 90 min before adding SARS-CoV-2 peptide. Fluid was collected from the apical or basolateral surfaces daily for 7 days (as shown in the schematic in panel A). (B) Spread of fluorescent virus is shown at the indicated days with or without peptide treatment. (C) Viral genome copies in apical or basolateral fluids were determined by RT-qPCR at the indicated time points (days postinfection [DPI]). (D) Infectious viruses released were quantified by titration from the apical or basolateral spaces. The median values are represented by horizontal bars, and the detection limits are indicated by the dotted lines. RT-qPCR and viral titration were performed on supernatant fluids sequentially collected from the same HAE wells the pictures were taken. Data were from three separate wells for infection treated and two separate wells for infection untreated.