Aurintricarboxylic acid inhibits influenza virus neuraminidase

Abstract

There is a continuing threat that the highly pathogenic avian influenza virus will cause future influenza pandemics. In this study, we screened a library of compounds that are biologically active and structurally diverse for inhibitory activity against influenza neuraminidase (NA). We found that aurintricarboxylic acid (ATA) is a potent inhibitor of NA activity of both group-1 and group-2 influenza viruses with IC(50)s (effective concentration to inhibit NA activity by 50%) values at low micromolar concentrations. ATA was equally potent in inhibiting the NA activity derived from wild-type NA and its H274Y mutant which renders NA resistance to inhibition by oseltamivir. Although ATA is structurally distinct from sialic acid, molecular modeling experiments suggested that ATA binds to NA at the enzyme's substrate binding site. These results indicate that ATA may be a good starting material for the design of a novel class of NA inhibitors for the treatment influenza viruses.

Fig. 1

Inhibition of NAs derived from A/WSN/33 (H1N1), A/Udorn/72 (H3N2), and NIBRG-14 (H5N1) by ATA. Formaldehyde-treated virus suspensions were used in the MU-NANA fluorogenic substrate method with various concentrations of ATA. The squares, triangles, and circles represent A/WSN/33 (H1N1), A/Udorn/72 (H3N2), and NIBRG-14 (H5N1) NA, respectively.

Fig. 2

Inhibition of influenza virus plaque formation units by ATA (A) and reduction in viral yields from infected cells treated with ATA (B) and GS4071 (C) at different concentrations. (A) Approximately 50–100 PFU/well of three different subtypes of influenza A virus, A/WSN/33 (H1N1), A/Udorn/72 (H3N2), or NIBRG-14 (H5N1), was used to infect MDCK cells in 6-well plates. After the viral adsorption stage, 3 ml of agar overlay media containing various concentrations of ATA was added to the cells. The concentration of ATA is indicated at the top. MDCK cells were infected with MOI 0.001 A/WSN/33 (H1N1) and various concentrations of ATA (B) and GS4071 (C) were added to the infected cells at the adsorption stage of A/WSN/33 replication cycle. After 48 post-infection hours, the culture supernatant were collected for virus titration by plaque forming assay.

Fig. 3

Effects of ATA on wild-type and mutant H274Y NA activity. Insect cells were infected with Bac-NA^WT(WSN), Bac-NA^H274Y(WSN), Bac-NA^WT(H5N1), or Bac-NA^H274Y(H5N1) for 72 h. The NA enzymatic activity of cell lysates containing 2 μg protein treated with the indicated concentrations of ATA was detected by the MU-NANA fluorogenic substrate method. (A) The NA^WT and NA^H274Y were derived from influenza A/WSN/33 (H1N1). (B) The NA^WT and NA^H274Y were derived from influenza NIBRG-14 (H5N1).

Fig. 4

Docked conformations of aurintricarboxylic acid (ATA) (green), GS4071 (gray), and zanamivir (blue) in H5N1 avian influenza neuraminidase. The docked conformation of ATA is obtained by using GEMDOCK and the binding pocket of N1 neuraminidase is derived from Protein Data Bank (PDB code 2HU4). The main contact residues (yellow) of N1 neuraminidase are labeled and hydrogen bonds (dash with green line) between ATA (green) and the neuraminidase are indicated. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 5

Computational conformation of aurintricarboxylic acid (ATA) in H5N1 avian influenza neuraminidase. The conformations of ATA and zanamivir are docked by using GEMDOCK, and the conformation of zanamivir is obtained from the X-ray structure (PDB code 2HU4). The main contact residues (yellow) are labeled and hydrogen bonds (dash with green line) between ATA and the neuraminidase are indicated. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)