Mucin-inspired, high molecular weight virus binding inhibitors show biphasic binding behavior to influenza A viruses

Multivalent virus binding inhibitors are a promising new class of antivirals, preventing virus infection of cells by inhibiting the first step in the viral infection cycle - binding of viruses to the cell surface. The design of multivalent virus binding inhibitors is complex as many properties, such as inhibitor size and functionalization with virus attachment factors, have a strong impact on the inhibition efficiency. In this study, we synthesized virus binding inhibitors, the design of which has been inspired by mucins, which are naturally occurring glycosylated proteins with molecular weights in the MDa range and which show high affinity in the interaction with various viruses. Hyperbranched polyglycerols (hPG), serving as polymeric scaffolds, were functionalized with sialic acids and sulfate groups at degrees of functionalization as suggested from the structure of mucins. The molecular weights of the hPG-based inhibitors ranged between 10 and 2600 kDa, thereby hitting the size of mucins (MDa scale) and allowing for comparing the inhibition efficiency of the largest, mucin-sized inhibitor (2600 kDa) with related inhibitors of lower molecular weight. Inhibition efficiencies were determined by various methods based on the inhibition of influenza A virus (IAV) binding to lipid membranes, including an assay based on total internal reflection fluorescence (TIRF) microscopy that allows for probing the interaction of IAV with its native attachment factor, sialic acid. Potent inhibition is observed in all assays already at pM concentrations for the mucin-sized inhibitor, while decreasing the inhibitor's molecular weight also decreased its inhibition efficiency. In addition, a biphasic binding behavior of the inhibitors to IAV is observed, which is attributed to differences in the binding affinity to two IAV envelope proteins, neuraminidase and hemagglutinin.