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Nanomolar inhibition of SARS-CoV-2 infection by N-terminally extended HR2 peptides

ABSTRACT: Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available COVID-19 vaccines and monoclonal antibody therapies through changes of epitopes on the receptor binding domain of the viral spike glycoprotein (S). Hence, there is a specific urgent need for alternative antivirals that target processes less likely to be affected by mutation, such as the membrane fusion step of viral entry into the host cell. One such antiviral class includes peptide inhibitors which block formation of the HR1HR2 six-helix bundle of the SARS-CoV-2 spike protein and thus interfere with viral membrane fusion; HR2 derived peptides are validated inhibitors of this process. Here, we identify a short N-terminal region that, when appended to the helical region of an HR2 peptide from previous studies, leads to potent inhibition of fusion. The extended peptide shows an ~100-fold increase in efficacy compared with the previously used 36-amino-acid version of HR2, effectively achieving single-digit nanomolar inhibition of SARS-CoV-2 in cell-based fusion, VSV-SARS-CoV-2 chimera, and authentic SARS-CoV-2 infection assays. The peptide also strongly inhibits all major SARS-CoV-2 variants to date. Structural studies of the HR1HR2 bundle reveal the formation of an extended, stabilized N-terminus that interacts with the HR2 triple helix, further stabilizing the HR1HR2 bundle. Together, these results suggest that regions outside the previously studied HR2 helical region may offer new opportunities for potent peptide-derived therapeutics for SARS-CoV-2 and its variants.

ORGANISM(S): Severe acute respiratory syndrome coronavirus 2

PROVIDER: GSE203229 | GEO | 2022/09/21

REPOSITORIES: GEO

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Project description:Despite the clinical success of anti-spike vaccines, the effectiveness of neutralizing antibodies and vaccines by rapidly spreading SARS-CoV-2 variants has been compromised. Viruses can hijack the glycosylation machinery of host cells to shield themselves from the host’s immune response and attenuate antibody efficiency. However, it still remains unclear whether targeting glycosylation on spike can impair SARS-CoV-2 and its variants infectivity. Methods: To assess the binding ability of glycosylated or deglycosylated spike with ACE2, we performed flow cytometry, ELISA, and BioLayer Interferometry methods. Viral entry ability was determined by luciferase intensity, immunoblotting, and immunofluorescence assay. A genome-wide association study (GWAS) was performed to identify the relationship of STT3A and COVID-19 severity. N-glycosylation regulated by NF-kB/STT3A axis was investigated by knockdown approach, chromatin immunoprecipitation, and promoter assay. To specifically target SARS-CoV-2 infected cells, we developed an antibody-drug conjugate coupling non-neutralization anti-spike antibody with NGI-1 (4G10-ADC) on inhibitory effects of SARS-CoV-2 infection. Results: We found receptor binding domain and three SARS-CoV-2 distinct surface Nglycosylation sites in 57,311 spikes retrieved from NCBI-Virus-database are highly evolutionarily conserved (99.67%) and involved in ACE2 interaction. We further identified STT3A as a key glycosyltransferase that catalyzed spike glycosylation and positively correlated with COVID-19 severity. Inhibition of STT3A by N-linked glycosylation inhibitor-1 (NGI-1) impaired SARS-CoV-2 and its variants (B.1.1.7, and B.1.351) infectivity. Most importantly, 4G10-ADC internalized SARS-CoV-2 infected cells and subsequently released NGI-1 to deglycosylate spike protein. Thereby, it reinforces the neutralizing abilities in antibodies, vaccines, or convalescent sera, inhibiting SARS-CoV-2 and its variants’ infectivity. Our results suggest targeting STT3A-mediated evolution conserved glycosylation via ADC can provide a widespread impact on SARS-CoV-2 variants infection. Together, we identified a novel deglycosylation method to eradicate SARS-CoV-2 variants infection.

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Nanomolar inhibition of SARS-CoV-2 infection by N-terminally extended HR2 peptides

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