Crystal structures of oseltamivir-resistant influenza virus neuraminidase mutants

Abstract

The potential impact of pandemic influenza makes effective measures to limit the spread and morbidity of virus infection a public health priority. Antiviral drugs are seen as essential requirements for control of initial influenza outbreaks caused by a new virus, and in pre-pandemic plans there is a heavy reliance on drug stockpiles. The principal target for these drugs is a virus surface glycoprotein, neuraminidase, which facilitates the release of nascent virus and thus the spread of infection. Oseltamivir (Tamiflu) and zanamivir (Relenza) are two currently used neuraminidase inhibitors that were developed using knowledge of the enzyme structure. It has been proposed that the closer such inhibitors resemble the natural substrate, the less likely they are to select drug-resistant mutant viruses that retain viability. However, there have been reports of drug-resistant mutant selection in vitro and from infected humans. We report here the enzymatic properties and crystal structures of neuraminidase mutants from H5N1-infected patients that explain the molecular basis of resistance. Our results show that these mutants are resistant to oseltamivir but still strongly inhibited by zanamivir owing to an altered hydrophobic pocket in the active site of the enzyme required for oseltamivir binding. Together with recent reports of the viability and pathogenesis of H5N1 (ref. 7) and H1N1 (ref. 8) viruses with neuraminidases carrying these mutations, our results indicate that it would be prudent for pandemic stockpiles of oseltamivir to be augmented by additional antiviral drugs, including zanamivir.

Influenza neuraminidase operates via a nucleophilic mechanism and can be targeted by covalent inhibitors

“…The similarity of the 3ax-fluoro-Neu5Ac moiety binding mode to the natural NA ligand Neu5Ac is a strong indication that the covalent inhibitor 2a,3ax-difluoro-Neu5Ac should be effective against drug-resistant NA. This is also illustrated by the high similarity between the binding modes of the covalent inhibitor and zanamivir, which remain effective against oseltamivir-resistant His274Tyr NA 32 (Fig. 5).…”

“…However, in order to accommodate oseltamivir, Glu276 must rotate to form a salt bridge with Arg224. The most common oseltamivir-resistant mutations, like His274Tyr, interfere with the rotation of Glu276, which is necessary to accommodate oseltamivir 32 .…”

“…Therefore, we also tested inhibition against oseltamivir-resistant rN1-His274Tyr from the 2009 H1N1 pandemic in comparison to wild-type rN1. Although His274Tyr N1 has been reported to exhibit more than 200-times lower inhibition by oseltamivir than by wild-type N1 32 , the covalent inhibitor 2a,3ax-difluoro-Neu5Ac inhibited recombinant 2009 pandemic N1 (r09N1-His274Tyr) at the same level as wild-type recombinant 2009 pandemic N1 (r09N1) ( Table 3). This demonstrates that 2a,3ax-difluoro-Neu5Ac is an effective inhibitor against the most common oseltamivir-resistant NA.…”

Molecular mechanism of a specific capsid binder resistance caused by mutations outside the binding pocket

“…The pentanyl substituent of oseltamivir forms hydrophobic interactions with the protein. In H274Y variants, the larger Tyr pushes the polar side chain of neighboring E276 farther into the binding site, toward the hydrophobic pentanyl moiety (von Grafenstein et al, 2015;von Itzstein, 2007) (Collins et al, 2008). In search for orally effective neuraminidase inhibitors oseltamivir, comprising a pentanyl substituent was designed.…”

In-silico Virtual Screening and ADMET Study to Find Novel Neuraminidase N1 Inhibitors Extended to the 150-Cavity

“…Zanamivir and oseltamivir were active against NAs from Groups 1 and 2 Influenza A as well as Influenza B viruses (Collins et al, 2008). In-silico drug discovery is considered one of the most promising methods used to accelerate the drug development process (Tollman et al, 2001).…”