Our previous screening of 50 240 structurally diverse compounds led to the identification of 39 influenza A virus infection inhibitors (Kao R.Y

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A previous screening of more than 50,000 compounds led to the identification of a pool of bioactive small molecules with inhibitory effect on the influenza A virus. One of these compounds, now widely known as nucleozin, is a small molecule that targets the influenza A virus nucleoprotein. Here we identify and characterize two structurally different novel fusion inhibitors of the influenza A virus group 1 hemagglutinin (HA), FA-583 and FA-617, with low nanomolar activities. Escape mutants that are highly resistant to each of these compounds were generated, and both were found to carry mutations localized in close proximity to the B-loop of the hemagglutinin 2 protein, which plays a crucial role in the virion-host cell fusion process. Recombinant virus, generated through reverse genetics, confirmed the resistance phenotype. In addition, the proposed binding pockets predicted by molecular docking studies are in accordance with the resistance-bearing mutation sites. We show through mechanistic studies that FA-583 and FA-617 act as fusion inhibitors by prohibiting the low-pH-induced conformational change of hemagglutinin. Our study has offered concrete biological and mechanistic explorations for the strategic development of novel fusion inhibitors of influenza A viruses. IMPORTANCE Here we report two structurally distinctive novel fusion inhibitors of influenza