Mechanistic analysis of polymer-attached inhibitors of influenza virus and their effect on minimizing drug resistance

Abstract

With the emergence of the 2009 A(H1N1) pandemic influenza virus and the rapid spread of drug resistance in recent years, the need to develop new anti-influenza drugs that can reduce the emergence of new resistant viruses is both urgent and important. This thesis explores the use of polymer-attached inhibitors as a new approach in the development of anti-influenza drugs, with particular focus on polymer-attached zanamivir (ZA). We have previously shown that covalently conjugating multiple copies of ZA via a flexible linker to poly-L-glutamine greatly enhances antiviral potency. In the first study, we have elucidated the mechanism of this phenomenon. Like ZA itself, the polymer-attached inhibitor binds specifically to viral neuraminidase and inhibits both its enzymatic activity and the release of newly synthesized virions from infected cells. In contrast to monomeric ZA, however, the polymer-attached drug also synergistically inhibits virus-endosome fusion, thus contributing to the dramatically increased antiviral potency. Next, we went on to investigate polymer-attached ZA's effect on the emergence of drug resistance. We found that viruses adapted rapidly to growing in high concentrations of monomeric ZA, whereas viral growth remained inhibited by low concentrations of polymer-attached ZA even after 23 passages in cell culture. Sequencing analysis established the emergence of an amino acid substitution known to confer ZA resistance (E119G in neuraminidase) after 8 passages of monomeric ZA selection. In contrast, virus grown in polymer-attached ZA remained free of substitutions in E119, and other known resistance-associated residues. We instead found novel substitutions in hemagglutinin (R220G, D241G) and neuraminidase (G111D), which emerged during passages 14-17. Importantly, although the drug-selected variants were resistant to monomeric ZA, the viruses remained susceptible to low pM concentrations of polymer-attached ZA itself. Taken together, these data demonstrate that attaching the drug to a polymeric chain (i) confers a new mechanism of antiviral action; (ii) significantly delays the emergence of drug resistance; and (iii) enhances potency against the selected ZA-resistant variants. The studies presented in this thesis provide further impetus for the use of polymer-attached inhibitors as influenza therapy, and as tools for better understanding of influenza biology.