Structure-based design of a SARS-CoV-2 Omicron-specific inhibitor.

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) introduced a relatively large number of mutations, including three mutations in the highly conserved heptad repeat 1 (HR1) region of the spike glycoprotein (S) critical for its membrane fusion activity. We show that one of these mutations, N969K induces a substantial displacement in the structure of the heptad repeat 2 (HR2) backbone in the HR1HR2 postfusion bundle. Due to this mutation, fusion-entry peptide inhibitors based on the Wuhan strain sequence are less efficacious. Here, we report an Omicron-specific peptide inhibitor designed based on the structure of the Omicron HR1HR2 postfusion bundle. Specifically, we inserted an additional residue in HR2 near the Omicron HR1 K969 residue to better accommodate the N969K mutation and relieve the distortion in the structure of the HR1HR2 postfusion bundle it introduced. The designed inhibitor recovers the loss of inhibition activity of the original longHR2_42 peptide with the Wuhan strain sequence against the Omicron variant in both a cell-cell fusion assay and a vesicular stomatitis virus (VSV)-SARS-CoV-2 chimera infection assay, suggesting that a similar approach could be used to combat future variants. From a mechanistic perspective, our work suggests the interactions in the extended region of HR2 may mediate the initial landing of HR2 onto HR1 during the transition of the S protein from the prehairpin intermediate to the postfusion state.

Nanomolar inhibition of SARS-CoV-2 infection by an unmodified peptide targeting the prehairpin intermediate of the spike protein.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available coronavirus disease 2019 vaccines and monoclonal antibody therapies through epitope change on the receptor binding domain of the viral spike glycoprotein. 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 so-called heptad repeat 1 and 2 (HR1HR2) six-helix bundle of the SARS-CoV-2 spike (S) protein and thus interfere with viral membrane fusion. We performed structural studies of the HR1HR2 bundle, revealing an extended, well-folded N-terminal region of HR2 that interacts with the HR1 triple helix. Based on this structure, we designed an extended HR2 peptide that achieves single-digit nanomolar inhibition of SARS-CoV-2 in cell-based and virus-based assays without the need for modifications such as lipidation or chemical stapling. The peptide also strongly inhibits all major SARS-CoV-2 variants to date. This extended peptide is ∼100-fold more potent than all previously published short, unmodified HR2 peptides, and it has a very long inhibition lifetime after washout in virus infection assays, suggesting that it targets a prehairpin intermediate of the SARS-CoV-2 S protein. Together, these results suggest that regions outside the HR2 helical region may offer new opportunities for potent peptide-derived therapeutics for SARS-CoV-2 and its variants, and even more distantly related viruses, and provide further support for the prehairpin intermediate of the S protein.

Structural conservation among variants of the SARS-CoV-2 spike postfusion bundle.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) challenge currently available COVID-19 vaccines and monoclonal antibody therapies due to structural and dynamic changes of the viral spike glycoprotein (S). The heptad repeat 1 (HR1) and heptad repeat 2 (HR2) domains of S drive virus­host membrane fusion by assembly into a six-helix bundle, resulting in delivery of viral RNA into the host cell. We surveyed mutations of currently reported SARS-CoV-2 variants and selected eight mutations, including Q954H, N969K, and L981F from the Omicron variant, in the postfusion HR1HR2 bundle for functional and structural studies. We designed a molecular scaffold to determine cryogenic electron microscopy (cryo-EM) structures of HR1HR2 at 2.2­3.8 Å resolution by linking the trimeric N termini of four HR1 fragments to four trimeric C termini of the Dps4 dodecamer from Nostoc punctiforme. This molecular scaffold enables efficient sample preparation and structure determination of the HR1HR2 bundle and its mutants by single-particle cryo-EM. Our structure of the wild-type HR1HR2 bundle resolves uncertainties in previously determined structures. The mutant structures reveal side-chain positions of the mutations and their primarily local effects on the interactions between HR1 and HR2. These mutations do not alter the global architecture of the postfusion HR1HR2 bundle, suggesting that the interfaces between HR1 and HR2 are good targets for developing antiviral inhibitors that should be efficacious against all known variants of SARS-CoV-2 to date. We also note that this work paves the way for similar studies in more distantly related viruses.