Influenza neuraminidase inhibitors: antiviral action and mechanisms of resistance

Abstract

There are two major classes of antivirals available for the treatment and prevention of influenza, the M2 inhibitors and the neuraminidase inhibitors (NAIs). The M2 inhibitors are cheap, but they are only effective against influenza A viruses, and resistance arises rapidly. The current influenza A H3N2 and pandemic A(H1N1)pdm09 viruses are already resistant to the M2 inhibitors as are many H5N1 viruses. There are four NAIs licensed in some parts of the world, zanamivir, oseltamivir, peramivir, and a long-acting NAI, laninamivir. This review focuses on resistance to the NAIs. Because of differences in their chemistry and subtle differences in NA structures, resistance can be both NAI- and subtype specific. This results in different drug resistance profiles, for example, the H274Y mutation confers resistance to oseltamivir and peramivir, but not to zanamivir, and only in N1 NAs. Mutations at E119, D198, I222, R292, and N294 can also reduce NAI sensitivity. In the winter of 2007-2008, an oseltamivir-resistant seasonal influenza A(H1N1) strain with an H274Y mutation emerged in the northern hemisphere and spread rapidly around the world. In contrast to earlier evidence of such resistant viruses being unfit, this mutant virus remained fully transmissible and pathogenic and became the major seasonal A(H1N1) virus globally within a year. This resistant A(H1N1) virus was displaced by the sensitive A(H1N1)pdm09 virus. Approximately 0.5-1.0% of community A(H1N1)pdm09 isolates are currently resistant to oseltamivir. It is now apparent that variation in non-active site amino acids can affect the fitness of the enzyme and compensate for mutations that confer high-level oseltamivir resistance resulting in minimal impact on enzyme function.

Figure 1

Chemical structures of the neuraminidase inhibitors (A) DANA, (B) zanamivir, (C) oseltamivir carboxylate, (D) peramivir, and (E) laninamivir. Structures are oriented to demonstrate the differences relative to DANA – the C4‐guanidinium group on zanamivir, peramivir, and laninamivir, the C4‐amino group on oseltamivir, and the pentyl side chains on oseltamivir and peramivir.

Figure 2

Structures of the N9 NA with bound inhibitors (A,B) zanamivir (PDB NNC), (C) oseltamivir (PDB QWK), (D) peramivir (PDB IL7F). To minimize overcrowding, only some amino acids which affect NAI binding are labeled in each figure. (B) is slightly rotated from (A) to show Y252 situated behind H274. Residue 252 is Y in N9, but the H252Y difference between clade 1 and clade 2 avian H5N1 NAs reduces binding of oseltamivir. Arrows show rotation of E276 to form a salt bridge to R224, creating the hydrophobic pocket to accommodate pentyl side chains of oseltamivir and peramivir.

Figure 3

Graphs of IC₅₀ kinetics show how changes in IC₅₀ during the enzyme inhibition assay can identify slow and fast binding inhibitors. IC₅₀s are compared either with pre‐incubation with inhibitors (+) or with no pre‐incubation, where virus, inhibitor, and substrate are added simultaneously (−). Pre‐incubation enhances binding, leading to lower IC₅₀s for slow‐binding inhibitors compared with no pre‐incubation. Where inhibitors are no longer slow binding, there is little difference in the IC₅₀s with or without pre‐incubation, for example, wild‐type B virus with oseltamivir, D198E mutant with both inhibitors, and H274Y and E119V only with oseltamivir. The latter two remain sensitive to zanamivir and still demonstrate slow binding. As substrate competes with the inhibitor in pre‐incubation reactions, there is an increase in IC₅₀. For wild‐type viruses, there is a more rapid dissociation of oseltamivir than that of zanamivir.