Monomeric inhibitors of influenza neuraminidase enhance the hemagglutination inhibition activities of polyacrylamides presenting multiple C-sialoside groups

Background: Influenza viruses use hemagglutinin (HA) arrays to bind to sialic acid moieties on the surface of cells; crosslinking of erythrocytes by this mechanism leads to hemagglutination. A number of synthetic polymers containing multiple sialic acid (Neu5Ac) groups as side chains are potent inhibitors of this process. Inhibition may be due to two mechanisms: polyvalent binding of the inhibitor's multiple Neu5Ac side chains to multiple HA sites on the viral surface, or steric stabilization of the viral particle by a layer of the adsorbed, water-swollen polymer, which prevents adhesion to the erythrocyte. The balance between these two effects is not yet known.Results: Polyacrylamides with multiple C-sialosides (PA(Neu5Ac)) were 2–20 fold more effective as inhibitors of virally mediated hemagglutination when assayed in the presence of Neu2en-NH₂, a potent monomeric inhibitor of influenza neuraminidase (NA). The ability of monomeric inhibitors of NA to enhance the inhibition of hemagglutination in this assay correlated with the affinity of the monomer for NA.Conclusions: We propose that inhibitors of NA act by competing with the C-sialosides of PA(Neu5Ac) for binding to the active sites of the NA. Competitive displacement of Neu5Ac causes an expansion of the layer of polymeric gel adsorbed to the virus, enhancing its inhibitory effect. This study provides an example of synergy between two ligands directed toward the active sites of two different proteins, and reinforces the conclusion that steric stabilization is important for the activity of polyvalent inhibitors.