Figure 1.
Chemical structures of the phytochemicals tested in this study.
Diverse chemical structures of the phytochemicals screened in this study for their anti-influenza A activities. The sources for these structures are described in the materials and methods.
Figure 2.
Screening of anti-influenza phytochemicals using a PA endonuclease-inhibition assay.
Screening of phytochemicals for anti-influenza A activity using a PA endonuclease assay. The effects of the various phytochemicals upon 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 0.35 µg/ 100 µl. A zero control (no PA domain added) was also assayed. Phytochemicals were added at a 1 or 10 µM dose and M13mp18 was used as the substrate.
Figure 3.
Docking simulation of marchantin E with influenza PA endonuclease.
A) Docking simulation analysis of marchantin E with the PA endonuclease domain of the influenza A RNA polymerase. PA endonuclease is depicted as a ribbon structure. The α-helix and β-strands are shown in red and yellow, respectively. Manganese ions in the PA endonuclease are shown as cyan. Marchantin E is shown as a stick structure with oxygen atoms indicated in red. B) The fitting of marchantin E to the active pocket of the PA endonuclease. Marchantin E is displayed in a sphere mode. The surface of the pocket of the PA endonuclease is indicated in green and purple. The grey and red balls indicate carbon and oxygen atoms in marchantin E, respectively. C) Two dimensional analysis of the interaction between marchantin E and PA endonuclease. The chemical structure of marchantin E is shown in the center with the key interacting amino acids shown around it. The dihydroxyphenyl group of marchantin E interacts with two manganese ions. The hydroxy groups of marchantin bind to amino acids in PA endonuclease.
Figure 4.
Anti-influenza activity of marchantins.
Inhibition of the growth of influenza A or B viral by phytochemicals. MDCK cells were treated with influenza A/Hiroshima/52/2006 (H3N2) (A), A/Solomon/3/2006 (H1N1) (B) or B/Malaysia/2506/2004 (C) viruses. The cells were then treated with various concentrations of phytochemicals (6.25–50 µM), some of which suppressed viral-induced cell death. DMSO was used as the phytochemical solvent.
Figure 5.
Decrease of the viral infectivity titer by marchantins.
Decrease of viral infectivity titer of influenza B viral by phytochemicals. MDCK cells were treated with influenza B/Malashia/2506/2004 virus. The cells were then treated with various concentrations of phytochemicals (3.1–50 µM), some of which decreased viral infectivity titer. DMSO was used as the phytochemicals solvent. (A) Viral growth curve under exposure to various concentrations of marchantins. The horizontal axis is the culture time and the vertical axis is the logarithm of virus infectivity titer (focus formation unit (FFU)). (B) Viral growth curve in the presence of marchantins and comparisons of the same concentration. The horizontal axis is the culture time and the vertical axis is the logarithm of virus infectivity titer (focus formation unit (FFU)).
Table 1.
Specific activity of marchantins against viral infectivity titer.
Figure 6.
Docking amino acids are conserved between influenza A and B.
The docking amino acids (Glu23, His41, Glu80, Arg84, Asp108, Glu119 and Lys134) identified in Figure 3 are shown in an alignment of influenza A and B amino acid sequences. This alignment was generated using the clustal X program.
Figure 7.
Consensus structure of chemicals with anti-influenza activity.
Consensus structure of the phytochemicals and synthesized chemicals possessing anti-influenza A activity. The dihydroxyphenethyl group (shown as red) is common to marchantin, catechin and several phthalimide molecules which all inhibit the influenza virus.
Figure 8.
Comparison between marchantins and catechins that bind to PA endonuclease.
Docking simulation analysis of marchantin A (panel A) or EGCG (panel B) with the PA endonuclease domain. Two dimensional analysis of the interactions of marchantin A or EGCG with PA endonuclease is shown. The chemical structures of marchantin A and EGCG is shown in the center. The interacting amino acids of PA endonuclease are shown around them. The dihydroxyphenyl groups of both marchantin A and EGCG interact with two manganese ions.