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).