Binding of Glycans to the SARS CoV-2 Spike Protein, an Open Question: NMR Data on Binding Site Localization, Affinity, and Selectivity.

We have used NMR experiments to explore the binding of selected glycans and glycomimetics to the SARS CoV-2 spike glycoprotein (S-protein) and to its receptor binding domain (RBD). STD NMR experiments confirm the binding of sialoglycans to the S-protein of the prototypic Wuhan strain virus and yield dissociation constants in the millimolar range. The absence of STD effects for sialoglycans in the presence of the Omicron/BA.1 S-protein reflects a loss of binding as a result of S-protein evolution. Likewise, no STD effects are observed for the deletion mutant Δ143-145 of the Wuhan S-protein, thus supporting localization of the binding site in the N-terminal domain (NTD). The glycomimetics Oseltamivir and Zanamivir bind weakly to the S-protein of both virus strains. Binding of blood group antigens to the Wuhan S-protein cannot be confirmed by STD NMR. Using 1 H,15 N TROSY HSQC-based chemical shift perturbation (CSP) experiments, we excluded binding of any of the ligands studied to the RBD of the Wuhan S-protein. Our results put reported data on glycan binding into perspective and shed new light on the potential role of glycan-binding to the S-protein.

NMR Experiments Provide Insights into Ligand-Binding to the SARS-CoV-2 Spike Protein Receptor-Binding Domain.

We have used chemical shift perturbation (CSP) and saturation transfer difference (STD) NMR experiments to identify and characterize the binding of selected ligands to the receptor-binding domain (RBD) of the spike glycoprotein (S-protein) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We also subjected full-length S-protein to STD NMR experiments, allowing correlations with RBD-based results. CSPs reveal the binding sites for heparin and fondaparinux, and affinities were measured using CSP titrations. We then show that α-2,3-sialyllactose binds to the S-protein but not to the RBD. Finally, combined CSP and STD NMR experiments show that lifitegrast, a compound used for the treatment of dry eye, binds to the linoleic acid (LA) binding pocket with a dissociation constant in the µM range. This is an interesting finding, as lifitegrast lends itself well as a blueprint for medicinal chemistry, eventually furnishing novel entry inhibitors targeting the highly conserved LA binding site.