Comparison of key NA-ligand interactions.

<p>All residues are N2 numbered and distances are given in Å. The 4-guanidino group of zanamivir, laninamivir and CS-8958 is abbreviated as ‘4-guan’. Bond distances are based on the distances between oxygen and nitrogen atoms and do not include hydrogen atoms, which cannot be directly observed using X-ray diffraction. Distances are given for molecule A in the asymmetric unit of each structure and are highly consistent between molecules. The distance of the unique hydrogen bond between the laninamivir octanoate 9-ester-O and p09N1 Asn294 is given for both molecules A and B as it varies significantly. Laninamivir octanoate is listed as CS-8958 to save space.</p><p>*The Asp151 side chain carboxy hydrogen bonds with the 4-N of all the ligands used in this study.</p><p>**The Asp151 and Trp178 backbone carbonyl groups both hydrogen bond with the 4-guanidino group of zanamivir, laninamivir and laninamivir octanoate.</p><p>***The laninamivir octanoate 9-ester-carbonyl forms a hydrogen bond with N2 Arg224.</p

IC₅₀ values and 95% CIs for the inhibition of p57N2, 09N1 and N5.

<p>Laninamivir octanoate is listed as CS-8958 to save space.</p

Binding of laninamivir and zanamivir to p57N2, p09N1 and N5.

<p>In each panel, zanamivir appears as the same color as the respective NA active site and laninamivir appears as turquoise. Acidic and basic side chains of key residues are colored red and blue, respectively. The 4-guanidino group of laninamivir and zanamivir is buried deep beneath the 150-loop where it engages many key interactions with NA residues (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002249#ppat-1002249-t002" target="_blank">Table 2</a>). Although the binding modes of laninamivir and zanamivir are highly similar, the accessibility of the 4-guanidino to its binding site is lowest in p57N2, with a 147–150 salt bridge in its closed 150-loop (A - green) and highest in group 1 N5, which contains a 150-cavity in its uncomplexed structure (C - yellow). Inhibition by zanamivir and laninamivir are highest for N5, and lowest for p57N2. p09N1, with its unique 150-loop characteristics (B - magenta), has intermediate laninamivir inhibition.</p

Comparison of oseltamivir (yellow), laninamivir octanoate (magenta) and laninamivir (turquoise) binding to p09N1.

<p>Laninamivir binds to p09N1 with a similar active site conformation to the uncomplexed structure. Both laninamivir octanoate and oseltamivir binding to p09N1 induces rotation of Glu276 toward Arg224 where they form a salt bridge. This Glu276 rotation creates a hydrophobic pocket that accommodates the hydrophobic pentyl ether side chain of oseltamivir, however results in a weaker overall binding mode of laninamivir octanoate. The terminal carbon of the oseltamivir side chain is 3.73 Å from the hydrophobic Glu276 Cβ.</p

Crystallographic X-ray diffraction and refinement statistics.

<p>Laninamivir octanoate is listed as CS-8958 to save space.</p

Binding of the laninamivir octanoate prodrug (CS-8958) to p57N2 and p09N1.

<p>A) Binding of laninamivir octanoate to p57N2, which is similar to laninamivir/zanamivir NA binding, except with a novel hydrogen bond between the 9-ester carbonyl and Arg224. B) Unique binding mode of laninamivir octanoate to p09N1. Glu276 is rotated to form a salt bridge with Arg224 and there is a novel hydrogen bond between the laninamivir octanoate 9-ester-O and Asn294. C) Comparison of laninamivir octanoate binding to p57N2 and p09N1. Key residues are labeled. Tyr252 is group 1 specific and takes part in a hydrogen bond network with His274 and Glu276. The less bulky Thr252 (compared to group 1 Tyr252) is group 2 specific and allows for greater movement of residue 274 away from Glu276. The His274Tyr substitution results in group 1 specific oseltamivir-resistance also via Glu276 interactions.</p

Comparison of the active sites of p57N2, p09N1 and N5.

<p>Group 2 p57N2 has a 150-cavity deficient active site with a salt bridge between Asp147 and His150 which stabilizes the closed conformation of its 150-loop (upper left - green). p09N1 is an atypical group 1 structure and also has a 150-cavity deficient active site similar to many group 2 structures (upper right - magenta). N5 is a typical group 1 NA and displays a 150-cavity in its uncomplexed structure (lower left - yellow) that closes upon inhibitor binding (lower right - yellow: N5-laninamivir complex).</p

The chemical structures of influenza NA inhibitors used in this study.

<p>1, Neu5Ac2en (NA transition state analogue); 2, zanamivir; 3, laninamivir; 4, laninamivir octanoate (CS-8958); and 5, oseltamivir.</p