To explore the benefits of multivalency10
(i.e., in the present case, covalent attachment of multiple copies of zanamivir to the same polymeric chain) with respect to drug-resistant strains, herein we have selected three influenza A viruses carrying NA mutations at position 119: two in vitro
avian A/turkey/MN/833/80 (turkey/MN; H4N2) E119D and E119G mutants and a clinically isolated oseltamivir-resistant A/Wuhan/359/95 (Wuhan; H3N2) E119V mutant, which is transmissible in ferrets.17
We have synthesized water-soluble polymeric derivatives of 1
in which the drug is conjugated to poly-l
-glutamine through a flexible tether ().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 (), as well as of other zanamivir derivatives modified at the same position.18,19
Antiviral Activities of Zanamivir (1), As Well As of Its Monomeric (2) and Polymeric (3-5) Derivatives, Against Human Wuhan and Avian Turkey/MN Influenza Strains, As Determined by the Plaque Reduction Assay
Inhibition of influenza A/Yamagata (H1N1) is most effective with a 10% loading of zanamivir on poly-l
; 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 (). 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
Figure 3 Antiviral activity enhancements of polymeric 4 over small-molecule 2 against wild-type and drug-resistant influenza strains. To determine the underlying IC50 values, zanamivir-based inhibitors and viruses were incubated together prior to infection of (more ...)
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 (). Importantly, 4 is also up to 20 times more potent than either 3 or 5 at inhibiting the drug-resistant strains (). 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 Ki for both 4 and 2, using the NA enzyme inhibition assay. The results presented in 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 (). Thus, presentation of zanamivir on the polymer completely compensates for weakened binding in zanamivir-resistant strains.
Inhibition Constants (Ki) of Viral Neuraminidases by Small-Molecule (2) and Polymeric (4) Zanamivir Derivatives Against Both Wild-Type and Drug-Resistant Human and Avian Influenza Strains
Multivalent drug species, such as 4
, exhibit better virus inhibition because of steric effects and increased affinity.10
Flexible linkers, such as that employed in this study, can promote improved ligand–protein binding by reducing steric obstacles and increasing effective ligand concentration21
(as is schematically illustrated in ). Both free and polymer-bound 1
should have similar rotational and translational entropic costs for the first interaction with viral surface proteins but polymer-bound 1
should have lower costs for subsequent ones, thereby providing entropic benefits of multivalency.22
Herein, we have demonstrated that a multivalent presentation of 1
conjugated to a resorbable polymeric backbone overcomes binding deficiencies and leads to potent inhibition not only of wild-type but also of drug-resistant human and avian influenza strains, thus promising safe and more efficacious therapeutics.
Figure 4 Schematic illustration of the proposed interactions of target viral surface proteins (gray claws) with complementary drugs (black ovals) in rigid (a) and flexible (b) drug–polymer conjugates. The large gray spheres represent influenza virus particles (more ...)