Characteristic comparison of WT and MDR NAs.

<p>Binding site residues of (A) MDR and (B) WT NAs. The binding site was divided into the 5 subsites S1 (R118, R293, and R368), S2 (E119, D151, W179, and E228), S3 (R152, W179, and I223), S4 (I223, R225, and S247), and S5 (S247 and E277). The negative/positive, polar, hydrophobic, and mixed hydrophobic and polar subsites are shown as red, green, gray, and orange curves, respectively. These residues are shown in N1 numbering. Molecular surfaces represented by electrostatic potentials of (C) MDR and (D) WT NAs. The negative, positive, and neutral/hydrophobic potentials are colored red, blue, and white, respectively.</p

Inhibition of influenza infection and replication by RB19 in MDCK cells.

<p>(A) RB19 inhibits the influenza-induced cytopathic effect. In the antiviral neutralization test, MDCK cells were lysed 64 h after A/WSN/33 infection, as shown in the VC (virus control) column. The agent RB19 was added to A/WSN/33-infected cells by two-fold serial dilution starting with a concentration of 50 µM (leftmost column). (B) Reduction in viral yields from infected cells treat w/o RB19 at different concentrations. MDCK cells were infected with MOI 0.001 A/WSN/33 (H1N1) and various concentrations of RB19 were added at the adsorption stage of the A/WSN/33 replication cycle. At 48 h post infection, culture supernatants were collected for virus titration using neuraminidase activity to monitor the viral yield. (C) Inhibition of influenza virus plaque formation by RB19. Approximately 50–100 PFU/well of A/WSN/33 (H1N1) or A/Udorn/72 (H3N2) of influenza A virus was used to infect MDCK cells in 6-well plates. After the viral adsorption stage, 3 ml of agar was overlayed on the media containing various concentrations of RB19. The concentration of RB19 is indicated at the top.</p

Interaction preference of mutant site.

<p>Interacting atom distributions of compounds on (A) MDR and (B) WT NAs. The interacting atoms are shown as grids if interacting with the binding site by electrostatic (yellow), hydrogen-bonding (dark green), and van der Waals (gray) interactions. (C) Protein-compound interaction profiles. An entry is colored green if the screening compound yielded interactions with the residues; conversely, the entry color is black. The red frame shows the major difference of interaction preferences between WT and MDR NAs. (D) Interaction percentages of residues in the profiles.</p

Parallel Screening of Wild-Type and Drug-Resistant Targets for Anti-Resistance Neuraminidase Inhibitors

<div><p>Infection with influenza virus is a major public health problem, causing serious illness and death each year. Emergence of drug-resistant influenza virus strains limits the effectiveness of drug treatment. Importantly, a dual H275Y/I223R mutation detected in the pandemic influenza A 2009 virus strain results in multidrug resistance to current neuraminidase (NA) drugs. Therefore, discovery of new agents for treating multiple drug-resistant (MDR) influenza virus infections is important. Here, we propose a parallel screening strategy that simultaneously screens wild-type (WT) and MDR NAs, and identifies inhibitors matching the subsite characteristics of both NA-binding sites. These may maintain their potency when drug-resistant mutations arise. Initially, we analyzed the subsite of the dual H275Y/I223R NA mutant. Analysis of the site-moiety maps of NA protein structures show that the mutant subsite has a relatively small volume and is highly polar compared with the WT subsite. Moreover, the mutant subsite has a high preference for forming hydrogen-bonding interactions with polar moieties. These changes may drive multidrug resistance. Using this strategy, we identified a new inhibitor, Remazol Brilliant Blue R (RB19, an anthraquinone dye), which inhibited WT NA and MDR NA with IC₅₀ values of 3.4 and 4.5 µM, respectively. RB19 comprises a rigid core scaffold and a flexible chain with a large polar moiety. The former interacts with highly conserved residues, decreasing the probability of resistance. The latter forms van der Waals contacts with the WT subsite and yields hydrogen bonds with the mutant subsite by switching the orientation of its flexible side chain. Both scaffolds of RB19 are good starting points for lead optimization. The results reveal a parallel screening strategy for identifying resistance mechanisms and discovering anti-resistance neuraminidase inhibitors. We believe that this strategy may be applied to other diseases with high mutation rates, such as cancer and human immunodeficiency virus type 1.</p> </div

Inhibitions of RB19, GS4071, and zanamivir on wild-type and mutant NAs.

a<p>NA with different mutations. Bac-NA^WT, Bac-NA^I223R, Bac-NA^H275Y, and Bac-NA^H275Y&I223R were generated by expressing the wild-type, H275Y, I223R, H275Y and I223R mutants of NIBRG14 (H5N1) NA originating, respectively.</p

Comparison of binding conformations of RB19, zanamivir, and GS4071.

<p>(A) Docking conformation of RB19 on WT NA with hydrogen-bonding interactions represented as light green dashes. (B) Conformations of GS4071 (dark green) and zanamivir (white) in WT (yellow) and MDR (purple) NAs. The GS4071 and zanamivir conformations were derived by superimposing N1 crystal structures (PDB code 3B7E <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056704#pone.0056704-Xu1" target="_blank">[37]</a> and 2HU4 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056704#pone.0056704-Russell1" target="_blank">[67]</a>). (C) Fold changes in IC₅₀ of RB19, zanamivir, and GS4071 when the dual H275Y/I223R mutation arises. (D) Docked conformation of RB19 on MDR NA.</p

Flowchart of the parallel screening strategy.

<p>(A) Parallel screening against WT and MDR NAs. Compounds of the compound library were docked into both NAs using GEMDOCK. (B) Characterization of the mutant subsite by site-moiety map analysis. In the NA site-moiety map, the hydrogen-bonding anchor (colored green) represents a polar environment interacting with polar moieties. (C) Selection of potential anti-resistance inhibitors. Compounds that simultaneously matched characteristics of the 5 subsites for the WT and MDR NAs were selected. (D) Bioassay for verifying the effects of selected compounds on WT and MDR NAs.</p

Residues, moiety preferences, and interaction types of anchors in (A) the mutant subsite and (B) the WT subsite.

<p>Anchors contain conserved interacting residues, moiety preferences, and interaction types. The hydrogen-bonding anchor (green) indicates that the mutant subsite is polar and prefers to form hydrogen bonds with polar moieties. In contrast, the WT subsite has a van der Waals anchor (gray).</p