Structural and Functional Analysis of Laninamivir and its Octanoate Prodrug Reveals Group Specific Mechanisms for Influenza NA Inhibition

Abstract

The 2009 H1N1 influenza pandemic (pH1N1) led to record sales of neuraminidase (NA) inhibitors, which has contributed significantly to the recent increase in oseltamivir-resistant viruses. Therefore, development and careful evaluation of novel NA inhibitors is of great interest. Recently, a highly potent NA inhibitor, laninamivir, has been approved for use in Japan. Laninamivir is effective using a single inhaled dose via its octanoate prodrug (CS-8958) and has been demonstrated to be effective against oseltamivir-resistant NA in vitro. However, effectiveness of laninamivir octanoate prodrug against oseltamivir-resistant influenza infection in adults has not been demonstrated. NA is classified into 2 groups based upon phylogenetic analysis and it is becoming clear that each group has some distinct structural features. Recently, we found that pH1N1 N1 NA (p09N1) is an atypical group 1 NA with some group 2-like features in its active site (lack of a 150-cavity). Furthermore, it has been reported that certain oseltamivir-resistant substitutions in the NA active site are group 1 specific. In order to comprehensively evaluate the effectiveness of laninamivir, we utilized recombinant N5 (typical group 1), p09N1 (atypical group 1) and N2 from the 1957 pandemic H2N2 (p57N2) (typical group 2) to carry out in vitro inhibition assays. We found that laninamivir and its octanoate prodrug display group specific preferences to different influenza NAs and provide the structural basis of their specific action based upon their novel complex crystal structures. Our results indicate that laninamivir and zanamivir are more effective against group 1 NA with a 150-cavity than group 2 NA with no 150-cavity. Furthermore, we have found that the laninamivir octanoate prodrug has a unique binding mode in p09N1 that is different from that of group 2 p57N2, but with some similarities to NA-oseltamivir binding, which provides additional insight into group specific differences of oseltamivir binding and resistance.

Structural and functional characterization of neuraminidase-like molecule N10 derived from bat influenza A virus

“…Influenza A virus NAs can be grouped based on their primary sequences (group 1 and group 2) and featured by variant structural properties (open or closed state) of the 150-loop (24). As demonstrated by an in vitro fluorescence-based activity assay, typical group 2 N2, atypical group 1 09N1, and typical group 1 N5 displayed high sialidase activity and similar K m values (20.3-36.1 μM) as reported previously (25,26).…”

“…The preparation procedures for recombinant 09N1, N2, and N5 were as described in our previous reports (18,20,24). Recombinant N10 protein was prepared using our established baculovirus expression system.…”

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

“…AX4 cells, MDCK cells with increased levels of sialyl-a2,6-galactose moieties, were grown and maintained following previous protocols 36,37 Expression and purification of influenza NAs. Recombinant NAs were purified to homogeneity after being expressed in a baculovirus expression system, based on the original method by Xu et al [38][39][40][41] with modifications. For rN2-Tyr406Asp and 2009 pandemic rN1-His274Tyr, point mutations were introduced by site-directed mutagenesis based upon the corresponding wild-type NAs (Generay, China).…”

“…40), we were unable to obtain enough quality N1 crystals for the soaking experiments carried out in this study. Instead, quality rN2 crystals were obtained as previously described 41 , and rN2-Tyr406Asp crystals were grown by vapour diffusion in hanging drops that consist of 1 ml concentrated protein (6 mg ml À 1 ) and 1 ml reservoir solution (0.1 M MES monohydrate pH 6.0, 14% w/v polyethylene glycol 4000). rN2 and rN2-Tyr406Asp crystals were soaked in the corresponding mother liquor containing 10 mM of 2a,3ax-difluoro-Neu5Ac2 at 4°C for 45 min and 10 mM 3 0 -sialyllactose at 18°C for 180 min, respectively.…”

Resistance to Mutant Group 2 Influenza Virus Neuraminidases of an Oseltamivir-Zanamivir Hybrid Inhibitor

“…So far, the structures of all subtypes have been solved (13)(14)(15)(16)(17)(18)(19). Influenza viruses containing N1 and N2 (i.e., H1N1, H3N2, and H2N2) are the most common types that infect humans and are therefore a great threat to public health (20)(21)(22)(23).…”