Zanamivir derivative 2 (Fig. 1) was synthesized as described previously.¹² To prepare polymer conjugates 3–5 (Fig. 1), 2 was reacted with the benzotriazole ester of poly-l-glutamic acid, followed by quenching with NH₄OH (Fig. 2)¹¹. Bare poly-l-glutamine was synthesized analogously.¹¹ Zanamivir content in 3–5 was quantified by ¹H NMR.

Plaque assays were performed in 12-well plates using a modified literature procedure.^12,13 First, 55-μL aliquots of virus preparation (~800 pfu/mL in PBS) were incubated with equal volumes of inhibitor solutions (serially 10-fold diluted in PBS) for 1 h at room temperature. After washing, MDCK cells grown to confluency were infected at room temperature for 1 h with 100 μL of the virus–inhibitor mixture. The inoculum was removed by aspiration, and the cells were overlaid with 1 mL of plaque medium¹² [2×F12 medium, 0.01% DEAE-dextran, 0.1% NaHCO₃, 100 units/mL of penicillin G, 100 μg/mL of streptomycin, 4 μg/mL of trypsin, and 0.6% purified agar (L28; Oxoid Co., Basingstoke, UK)]. Plaques were counted after 3 to 4 days of incubation at 37°C.

We have synthesized water-soluble polymeric derivatives of 1 in which the drug is conjugated to poly-l-glutamine through a flexible tether (Fig. 1).^11,12 The introduction of the linker group did not drastically affect the anti-influenza or NA inhibitory activity. The inhibitory potency of 2 is within an order of magnitude of 1’s (Table 1), as well as of other zanamivir derivatives modified at the same position.^18,19

Inhibition of influenza A/Yamagata (H1N1) is most effective with a 10% loading of zanamivir on poly-l-glutamine¹¹; other multivalent systems reach a plateau in efficacy between 10% and 30% modification.²⁰ Therefore, we have selected 10 mol% loading for our initial experiments as well. Using the plaque reduction assay, we have demonstrated that the poly-l-glutamine-attached drug 4 is not only about 20,000-fold more effective than 2 against another human influenza strain, Wuhan, but also some 200-fold more potent against an avian strain, turkey/MN.

Importantly, both oseltamivir- and zanamivir-resistant mutants of these viral strains are also far more susceptible to polymeric 4 than to small-molecule inhibitor 2: some 6000-fold improvement against oseltamivir-resistant Wuhan E119V mutant and a 2000-fold more enhancement against zanamivir-resistant turkey/MN mutants have been observed (Fig. 3). In comparison, maximal improvements for novel monomeric inhibitors to date have not exceeded 1000 times against oseltamivir-resistant influenza strains⁹ and 250 times against zanamivir-resistant ones.⁶

Next, we have examined how the amount of 2 conjugated to poly-l-glutamine affects inhibitory potency. Polymeric 4 has been found to be 10-fold more potent than either 3 or 5 against wild-type strains of both human Wuhan and avian turkey/MN viruses (Table 1). Importantly, 4 is also up to 20 times more potent than either 3 or 5 at inhibiting the drug-resistant strains (Table 1). The observation of the same optimal degree of loading for all the viral strains tested by us suggests that beyond a certain point the benefits of multivalency are counteracted by steric hindrances—presumably, too many ligand molecules attached to the same polymer chain interfere with each other’s action.

To gain further mechanistic insights, we have determined inhibition constants K_i for both 4 and 2, using the NA enzyme inhibition assay. The results presented in Table 2 afford several important conclusions. First, as seen in the last two lines of the table, the enzymes of zanamivir-resistant influenza strains expectedly bind 2 at least two orders of magnitude weaker than the wild-type and oseltamivir-resistant strains. Second, in all instances, the polymer-attached drug is a substantially more potent enzyme inhibitor than its small-molecule parent. Third, while the binding affinity enhancements caused by conjugation to poly-l-glutamine are relatively modest for wild-type human and avian strains, as well as for the oseltamivir-resistant human one, zanamivir-resistant turkey/MN mutants bind 4 2000 times more stronger than 2. As a result of this strengthened binding, the zanamivir-resistant mutants become as sensitive to 4 (in contrast to 2) as the others (Table 2). Thus, presentation of zanamivir on the polymer completely compensates for weakened binding in zanamivir-resistant strains.