3.1 Screening of compound libraries for inhibitors of influenza A virus
To develop better or novel antiviral drugs against influenza virus infection, we screened compound libraries through the MDCK cell-based screening system using A/PR/8/34 (H1N1) virus, the standard strain of human IAV. We tested a total of 1,280 compounds and found 13 anti-influenza candidates. Aprotinin, a serine protease inhibitor, was selected as a final candidate because both aprotinin samples from this library and from that of another company showed inhibitory effects on the virus (data not shown). Virus inhibition assay in MDCK cells showed that aprotinin inhibits A/PR/8/34 (H1N1) in a dose-dependent manner (Fig. 1A). Aprotinin could also inhibit A/CA/04/09 (H1N1), A/PH/2/82 (H3N2), A/AB/Kor/CN05/09 (H6N5), A/Ck/Kor/01310/01 (H9N2), A/Bris/10/07 (H3N2), and B/Seoul/32/11 in a dose-dependent manner (Fig. 1B-G). Based on the results of the viral inhibition assay, we applied the lowest effective concentration against all the tested influenza strains (60 nM) for subsequent experiments.
We next compared the antiviral activity of aprotinin against A/PR/8/34 (H1N1) virus with that of oseltamivir (100 μM). Aprotinin showed corresponding or superior antiviral activity to oseltamivir against PR8 virus infection (Fig. 2A). Colorimetric cytotoxicity assay revealed that there was no cytotoxicity in the range of aprotinin concentrations tested in this study (≤200 nM) (Fig. 2B).