With regular influenza epidemics and the prevalence of drug-resistant influenza virus strains, it is extremely crucial to develop effective and low-toxicity anti-influenza A virus drugs that act on conserved sites of novel targets. Here, we found a new anti-influenza virus compound, 1,3-dihydroxy-6-benzo[ c]chromene (D715-2441), from a library of 8026 small-molecule compounds by cell-based MTT assay and explored the underlying mechanisms. Our results revealed that D715-2441 possessed antiviral activities against multiple subtypes of influenza A viruses (IAVs) strains, including H1N1, H5N1, H7N9, H3N2, the clinical isolate 690 (H3), and oseltamivir-resistant strains with the H274Y NA mutation, and suppressed the early steps in the virus replication cycle. Further mechanistic studies indicated that D715-2441 clearly inhibited viral polymerase activity and directly influenced the location of the PB2 protein. Moreover, binding affinity analyses confirmed that D715-2441 bound specifically to the PB2cap protein. Further, protein sequence alignment and a computer-aided molecular docking indicated that highly conserved amino acid residues in the cap-binding pocket of PB2cap were possible binding sites for D715-2441, which indicates that D715-2441 might be employed as a cap-binding competitor. Moreover, the combination of D715-2441 and zanamivir possessed a remarkable synergistic antiviral effect, with an FICI value of 0.40. In conclusion, these results strongly suggest that D715-2441 has potential as a promising candidate against IAV infection. More importantly, our work offers novel options for the strategic development of PB2cap inhibitors of IAV.
Keywords: PB2; PB2 cap-binding inhibitor; influenza A virus; viral polymerase; virus replication.