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| - Although antiviral drugs are available for the treatment of influenza infection, it is an urgent requirement to develop new antiviral drugs regarding the emergence of drug‐resistant viruses. The nucleoprotein (NP) is conserved among all influenza A viruses (IAVs) and has no cellular equivalent. Therefore, NP is an ideal target for the development of new IAV inhibitors. In this study, we identified a novel anti‐influenza compound, ZBMD‐1, from a library of 20,000 compounds using cell‐based influenza A infection assays. We found that ZBMD‐1 inhibited the replication of H1N1 and H3N2 influenza A virus strains in vitro, with an IC (50) ranging from 0.41–1.14 μM. Furthermore, ZBMD‐1 inhibited the polymerase activity and specifically impaired the nuclear export of NP. Further investigation indicated that ZBMD‐1 binds to the nuclear export signal 3 (NES3) domain and the dimer interface of the NP pocket. ZBMD‐1 also protected mice that were challenged with lethal doses of A/PR/8/1934 (H1N1) virus, effectively relieving lung histopathology changes, as well as strongly inhibiting the expression of pro‐inflammatory cytokines/chemokines, without inducing toxicity effects in mice. These results suggest that ZBMD‐1 is a promising anti‐influenza compound which can be further investigated as a useful strategy against IAVs in the future.
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