Figure 1.
Chemical structures of the C60 fullerene derivatives tested in this study.
The chemical structures of the fullerene derivatives examined in this study are shown. The sources for these structures are described in the Materials and Methods. No. 1, 1,4-dihydro-6,7-dihydroxy [60]fullerenonaphthlene; no. 2, [60]fullerenodicyclopropane-1,1,1′,1′-tetracarboxylic acid; no. 3: [60]fullerenopyrrolidine-2,5-dicarboxylic acid; no. 4, 1-carboxymethyl [60]fullerenopyrrolidine-2,5-dicarboxylic acid; no. 5, 5-isopropyl [60]fullerenopyrrolidine-2-carboxylic acid; no. 6∶1,1,1′,1′-tetramethyl [60]fullerenodipyrrolidinium diiodide; no. 7, [60]fullerenopiperazine-1,4-diacetic acid; no. 8: [60]fullerenotricyclopropane-1,1,1′,1′,1′′,1′′-hexacarboxylic acid; no. 9, 1-ethyl [60]fullerenopyrrolidine-2,5-dicarboxylic acid; no. 10, 1-ethoxycarbonylmethyl [60]fullerenopyrrolidine-2,5-dicarboxylic acid 2-ethyl ester; no. 11, 5-phenyl [60]fullerenopyrrolidine-2-carboxylic acid; and no. 12, 4-(1′-methyl [60]fullerenopyrrolidin-2′-yl)-1-methylpyridinium iodide.
Figure 2.
Inhibition of the activity of the PA endonuclease domain by the fullerene derivatives.
(A) Schematic of the PA subunit of influenza RNA polymerase. (B) Purification of the bacterially expressed PA endonuclease domain using a HiTrap-Q column. The arrow indicates the PA endonuclease domain. (C) The effects of the various fullerene derivatives on the endonuclease activity of the PA N-terminal domain of the influenza A RNA polymerase were tested. The recombinant PA N-terminal domain protein was added to each reaction at a concentration of 0.25 µg/100 µL. A zero control (i.e., no PA domain added) was also assayed. The fullerene derivatives were added at a dose of 1 or 10 µM, and M13 mp18 was used as the substrate.
Figure 3.
Inhibition of the activity of full-length PA endonuclease by the fullerene derivatives.
(A) Schematic of the constructed plasmid, baculovirus expression, and purification of full-length PA protein. (B) Purification of full-length PA protein using a HiTrap-Q column. The numbers indicate the fractions. The arrow indicates full-length PA protein. (C) The effects of the various fullerene derivatives on the endonuclease activity of full-length PA protein of influenza A RNA polymerase were tested. Recombinant full-length PA protein was added to each reaction at a concentration of 0.25 µg/100 µL. A zero control (i.e., no PA protein added) was also assayed. The fullerene derivatives were added at a dose of 10 µM and M13 mp18 was used as the substrate.
Figure 4.
Nuclease activity of the fullerene derivatives.
The method was the essentially same as that of Figs. 2 & 3, except the condition of the absence of PA protein. The fullerene derivatives were added at a dose of 10 µM and M13 mp18 was used as the substrate. The digestion of the substrate was examined by agarose electrophoresis.
Figure 5.
Docking simulation of C60 fullerene with influenza PA endonuclease.
(A) Docking simulation analysis of C60 fullerene with the PA endonuclease domain of influenza A RNA polymerase. The fullerene is shown as a sphere. The surface of the pocket of PA endonuclease is shown in green and purple. The pink ball indicates the carbon atoms in the fullerene. (B) The fitting of the fullerene to the active pocket of PA endonuclease. PA endonuclease is depicted as a ribbon structure. The α-helix and β-strands are shown in red and yellow, respectively. The fullerene is shown as a gray stick structure. The manganese ions in PA endonuclease are behind the fullerene. (C) Two-dimensional analysis of the interactions between fullerene and PA endonuclease. The fullerene is shown in the center with the key and with the interacting amino acids shown around it. MN indicates the Mn2+ ions. The modes of interaction are shown at the bottom. The arene of the fullerene interacts with 2 Mn2+ ions and the amino acids, e.g., lysine and histidine, in PA endonuclease.
Figure 6.
Toxicity of the fullerene derivatives against MDCK cells.
(A) Various concentrations (12.5–100 µM) of the fullerene derivatives (n = 4) were added to cultures of MDCK cells. DMSO and ME were used as negative and positive controls for anti-influenza activity, respectively. At 24 h post-incubation, cell viability was determined using an MTT cell proliferation assay. Data represent the mean ± standard error of the mean (S.E.M.). (B) Various concentrations (0.8–100 µM) of the fullerene derivatives were added to cultures of MDCK cells. ME was used as positive control for cytotoxicity. At 24 h post-incubation, the cells were fixed and viable cells were stained with a naphthol blue black solution.
Figure 7.
Immunostaining of influenza A virus-infected cells.
Various concentrations of the fullerene derivatives (25–100 µM) and an MOI of 1 influenza A virus (A/PR/8/34 (H1N1) (n = 3) (A and C) or A/Aichi (H3N2) (n = 4) (B and D)) were mixed and added to cultures of MDCK cells. At 24 h post-infection, influenza A NP-immunostaining of the treated cells was performed. The wells were photographed under a microscope (×4) (A and B), and the stained cells were counted (C and D). DMSO (n = 4) and ME (n = 4) were used as negative and positive controls for the inhibitory effect of influenza A virus infection, respectively. Data represent the mean ± S.E.M. *p<0.05, **p<0.01.
Table 1.
IC50 of the fullerene derivatives against influenza A virus H1N1 and H3N2 strains.
Figure 8.
Expression levels of influenza A viral proteins.
We mixed 100 µM of the fullerene derivatives or ME and an MOI of 1 influenza A virus (A/PR/8/34 (H1N1)) and added the mixture to cultures of MDCK cells. At 4, 8, 12 (A), and 24 h (B) postinfection, the expression levels of influenza A NP and NS1 proteins in treated-cell lysates were analyzed by western blotting, and β-actin was analyzed as an internal control. The experiments were performed three times and the results were reproducible.