Our research group has focused on the discovery of new compounds from microbial sources due to their great chemical diversity and interesting biological activity [1-5]. Original assays systems were used to screen our culture collection for bioactive compounds. Over 1,000 microbial samples were screened, and fungal strain BF-0073 was selected for producing an inhibitor of the cap-snatching activity of influenza virus. Seven-day-old culture broth (200 mL) of this strain was extracted with an equal volume of ethanol, and the extract was collected by suction filtration and then evaporated in vacuo to obtain a water fraction. The water fraction was extracted with ethyl acetate (200 mL) and then concentrated in vacuo to yield a crude extract (275.6 mg). The crude extract was dissolved in a small volume of methanol and further purified by HPLC using a reverse-phase C18 column (PEGASIL ODS SP100, i.d. 20×250 mm) under the following conditions: solvent, 25% aq CH3CN containing 0.05% TFA; flow rate, 6.0 mL/min; detection, UV at 210 nm. Under these conditions, the active compound was eluted as a peak with a retention time of 28 min. This fraction was collected, concentrated in vacuo, and lyophilized to dryness to yield pure compound 1 (15.3 mg) as a purple powder. The structure was elucidated by spectroscopic data, including NMR experiments. Compound 1 was identified as oosporein based on comparisons with previously reported data [6] (Figure 1). 1H-NMR (400 MHz, pyridine-d5): δ 1.70 (s, 3H). 13C-NMR (100 MHz, pyridine-d5): δ 8.1, 106.1, 107.4, 171.9, 173.4. ESI-MS: [M-H]−: 305.
The effects of 1 on the cap-snatching activity of influenza virus were evaluated according to previously established methods [7-9]. Briefly, a reaction mixture (25 μL) containing 50 mM Tris-HCl (pH 7.9), 0.1 M ammonium acetate, 1.5 mM MgCl2, 2.5 mM DTT, 0.1% Nonidet P-40, 4U of RNasin, and 30-50 fmol of [32P]Cap 1-GACU32-biot (2 × 105 cpm/pmol) was incubated with influenza virus A virions (1 μg) at 37°C for 1h. The reaction was stopped by incubation withbuffer (200 μL) containing 20 mM Tris-HCl (pH 8.0), 5 mM EDTA, 150 mM NaCl, and streptavidin beads (30 μg) at room temperature for 10 min. The streptavidin beads-bound and unbound fractions were then collected separately, and the radioactivity of each was measured to calculate the corresponding IC50 value. Compound 1 inhibited cap-snatching activity in a dose-dependent manner by suppressing the generation of the cleaved fragment from [32P]Cap 1-GACU32-biot. The calculated IC50 value was 20.0 μg/mL (Figure 2), which was comparable to that of a known inhibitor of cap-snatching activity described in the literature, 2-hydroxy-4-oxo-4-phenyl-2-butenoic acid [10].
In conclusion, we identified oosporein produced by the fungal strain BF-0073 as an inhibitor of influenza virus cap-snatching activity. Terry et al. reported that the compound inhibited DNA polymerase of herpes simplex virus type 1 with an IC50 value of 22.9 μg/mL [11]. Interestingly, oosporein had no observable effect on DNA polymerase of HeLa cells or Escherichia coli, confirming the DNA polymerase selectivity of these organisms. Further study is needed to determine whether inhibition of the cap-snatching activity of influenza virus and DNA polymerase of herpes simplex virus type I occurs via a similar mechanism.
Competing Interests
The authors declare that they have no competing interests.
Acknowledgments
We thank Ms. Aya Niijima, Ms. Shino Masuo, and Ms. Haruka Mio, School of Pharmacy, Kitasato University, for their assistance. We express our thanks to Ms. Noriko Sato and Dr. Kenichiro Nagai, School of Pharmacy, Kitasato University, for NMR and MS analyses, respectively.