Pharmacological interventions targeting PI3K signaling affect EV-A71 replication in SF268 neural cells.

<p>The effect of PI3K (LY294002 and wortmannin), P38 MAPK (SB203580) (A) and GSK3β (AR-A014418 and LiCl) (B) inhibitors as pharmacological interventions on the virus yields of EV-A71-infected cells was assessed. SF268 cells were infected with EV-A71 (moi 0.5), and various concentrations of compounds were added to the infected cells after 1 h viral adsorption. At 48 h post-infection, the culture supernatants and cell lysates were collected for virus titration using plaque assays. Data are displayed as mean ± s.e.m. from at least two independent experiments performed in duplicates.</p

LiCl inhibits EV-A71 replication in SF268 cells (A), (B) and SH-SY5Y cells (C).

<p>Cells were infected with EV-A71 (moi 0.5), and various concentrations of LiCl were added to the infected cells after viral adsorption time. Lysates collected At 8 h (<b>A</b>) and 72 h (<b>B, C</b>) post-infection were analyzed with immunoblotting. Rupintrivir was employed as a positive control because it can effectively inhibit EV-A71 replication. Measurements were made in three independent experiments.</p

Effects of A771726 in combination with LiCl on EV71- induced cytopathic effect.

<p>CalcuSyn analysis provides combination index (CI) values to determine potential drug additivity in this Table. Inhibition of EV71 replication was evaluated in CPE test involving treatment with serial dilutions of A771726 (0, 0.625, 1.25, 2.5, 5 and 10 µM) or LiCl (0, 1.25, 2.5, 5, 10, 20 and 40 mM), or with a combination of the 2 compounds at fixed 2∶1 or 4∶1 ratios. The results show that A771726 combined with LiCl is synergistic, with CI values ranging from 0.20 to 0.49.</p><p>Effects of A771726 in combination with LiCl on EV71- induced cytopathic effect.</p

LiCl suppresses pro-inflammatory IL-6 and IL-1β mRNA expression (A) and IL-6 protein expression (B) in EV-A71-infected SF268 cells.

<p>SF268 cells were infected with EV-A71 (moi 0.5), and various concentrations of LiCl were added to the infected cells after viral adsorption time. At 48 h post-infection, the cell lysates were collected for quantification of the IL-6 and IL-1β mRNA. In a parallel experiment, the IL-6 protein levels in cell culture supernatant were measured using enzyme-linked immunosorbent assay. Data are the mean ± s.e.m. from at least three parallel measurements per experiment.</p

The active metabolite of Leflunomide, A771726, suppress virus production (A) and the expression of the pro-inflammatory cytokine IL-6 (B) in EV-A71-infected SF268 cells.

<p>SF268 cells were infected with EV-A71 (moi 0.5), and various concentrations of A771726 were added to the infected cells after viral adsorption time. At 48 h post-infection, the culture supernatants and cell lysates were collected for virus titration (A). In a parallel experiment, the IL-6 protein levels in cell culture supernatant were measured using ELISA (B). Each virus stock to be titered is serially diluted in10-fold series (dilution fold 10⁻⁴) and added to Vero cells. Data are the mean ± s.e.m. from at least two parallel measurements per experiment.</p

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

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

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

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

Development of an Anti-Influenza Drug Screening Assay Targeting Nucleoproteins with Tryptophan Fluorescence Quenching

Recent studies have shown that NP (nucleoprotein), which possesses multiple functions in the viral life cycle, is a new potential anti-influenza drug target. NP inhibitors reliably induce conformational changes in NPs, and these changes may confer inhibition of the influenza virus. The six conserved tryptophan residues in NP can be used as an intrinsic probe to monitor the change in fluorescence of the tryptophan residues in the protein upon binding to an NP inhibitor. In the present study, we found that the fluorescence of recombinant NP proteins was quenched following the binding of available NP inhibitors (such as nucleozin) in a concentration- and time-dependent manner, which suggests that the inhibitor induced conformational changes in the NPs. The minimal fluorescence-quenching effect and weak binding constant of nucleozin to the swine-origin influenza virus H1N1pdm09 (SOIV) NP revealed that the SOIV is resistant to nucleozin. We have used the fluorescence-quenching property of tryptophans in NPs that were bound to ligands in a 96-well-plate-based drug screen to assess the ability of promising small molecules to interact with NPs and have identified one new anti-influenza drug, CSV0C001018, with a high SI value. This convenient method for drug screening may facilitate the development of antiviral drugs that target viruses other than the influenza virus, such as HIV and HBV