RAPID: Effect of Avidity on Association of Fusion Inhibitory Peptides with the HRN Domain of SARS-CoV-2 Spike Protein

Coronaviruses are enclosed within a membrane and have protruding protein spikes. The spikes help the virus anchor to the cell. Then, the viral membrane fuses with the cell membrane and the infection begins. Introducing a molecule that will bind to the protein spikes can reduce or prevent attachment of the virus particle to the cell. Small protein chains, called peptides, are particularly effect at inhibiting infection for these viruses. If we understand how these peptides attach to each other and to the spike proteins, and how those actions influence their effectiveness, we will be in a better position to design highly effective inhibitors. This could have a significant impact on the design of inhibitors of the SARS-CoV-2 virus, which gives rise to COVID-19 disease, and aid in the recovery of infected patients.

In this project, the effect of avidity on the association of C-peptides with the N-terminal heptad repeat (HRN) domain of the SARS-CoV-2 spike protein will be investigated. This project will test the hypothesis that multimerization can decrease the off-rate and thus increase the residence time of the C-peptide on the HRN domain of the spike protein. Furthermore, this project will evaluate how the structure (e.g. linker flexibility and length) of the multivalent analogs affect six-helix bundle (6HB) formation with SARS-CoV-2 HRN by measuring the kinetic and thermodynamic parameters associated with binding. These parameters will be obtained via a time-resolved Forster resonance energy transfer (TR-FRET) assay. The latter will use a known combination of Lumi4-Terbium fluorophore (Lumi4-Tb) conjugated to the C-peptides as the donor and fluorescein conjugated to the SARS-CoV-2 HRN domain as the acceptor. This combination has a proximity limit of 10-15nm that is more than sufficient for detection of the 6HB. The kinetic rate constants and equilibrium binding constants obtained from this study will inform how fast the C-peptide-HRN system responds to changes in concentration of the C-peptide as well as its residence time on HRN. We expect the results of this project to provide a mechanistic understanding of how avidity can be used to tune the interaction of C-peptides with the SARS-CoV-2 spike protein fusion machinery.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.