Influenza RNA-dependent RNA polymerase is a heterotrimeric complex and has an essential role in the virus life cycle. It is responsible for viral replication and transcription. One of its subunits, the PA, interacts with the PB1 subunit via a crucial protein-protein interaction at its C-terminus. This 310 helix-mediated intersubunit interaction is required for the whole heterotrimer assembly (Fig. A) [1, 2]. The 14 amino acids long peptide from the PB1 N-terminus was identified as a nanomolar inhibitor of the PA-PB1 protein-protein interaction [3]. Furthermore, the peptide array identified several introduced mutations as beneficial for peptide binding, though without an X-ray structure [4]. We have structurally and thermodynamically characterized the PA C-ter interacting with optimized minimal peptide-based inhibitors derived from the PB1 N-terminus (Fig. B). Additionally, the N-terminal domain of PA contains the active site for endonuclease. A key characteristic of endonuclease inhibitors is their ability to bind to Mn2+ and Mg2+ ions that are embedded in the catalytic site of the enzyme. We have explored the inhibitory potency of flavonoids and their derivatives against the endonuclease domain [5, 6]. Using X-ray protein crystallography, we have described the binding modes of those inhibitors in the PA endonuclease active site (Fig. C). Using two different assays and structural analysis, we have been able to identify and characterize the molecular mode of action of flavonoids in cells infected with the influenza virus. We have successfully identified the target protein of these compounds, which are commonly used as supplements during influenza viral infections.
This work was supported by the European Regional Development Fund, OP RDE, Project: “Chemical biology for drugging undruggable targets (ChemBioDrug)" (No. CZ.02.1.01/0.0/0.0/16_019/0000729) and the European Union - Next Generation EU, The project National Institute of Virology and Bacteriology (Programme EXCELES, ID Project No. LX22NPO5103).