Unraveling the anti-influenza effect of flavonoids: Experimental validation of luteolin and its congeners as potent influenza endonuclease inhibitors

• Published in: European journal of medicinal chemistry Vol. 208; p. 112754
• Main Authors: Zima, Václav, Radilová, Kateřina, Kožíšek, Milan, Albiñana, Carlos Berenguer, Karlukova, Elena, Brynda, Jiří, Fanfrlík, Jindřich, Flieger, Miroslav, Hodek, Jan, Weber, Jan, Majer, Pavel, Konvalinka, Jan, Machara, Aleš
• Format: Journal Article
• Language: English
• Published: ISSY-LES-MOULINEAUX Elsevier Masson SAS 15.12.2020; Elsevier
• Subjects: Chemistry, Medicinal; Influenza; Life Sciences & Biomedicine; Neuraminidase; PAN endonuclease Inhibitors; Pharmacology & Pharmacy; Polyphenols; RNA polymerase; Science & Technology; MM; PA-Nter; SPR; SAR; Influenza; Neuraminidase; Polyphenols; RNA polymerase; QM; PAN endonuclease Inhibitors; PDB; PA(N) endonuclease Inhibitors; PA ENDONUCLEASE; COMPLEXES; IONS; IDENTIFICATION; VIRUS REPLICATION; DISCOVERY; QUERCETIN; ANTIOXIDANT; CANCER PREVENTION; DERIVATIVES
• ISSN: 0223-5234; 1768-3254
• DOI: 10.1016/j.ejmech.2020.112754

The biological effects of flavonoids on mammal cells are diverse, ranging from scavenging free radicals and anti-cancer activity to anti-influenza activity. Despite appreciable effort to understand the anti-influenza activity of flavonoids, there is no clear consensus about their precise mode-of-action at a cellular level. Here, we report the development and validation of a screening assay based on AlphaScreen technology and illustrate its application for determination of the inhibitory potency of a large set of polyols against PA N-terminal domain (PA-Nter) of influenza RNA-dependent RNA polymerase featuring endonuclease activity. The most potent inhibitors we identified were luteolin with an IC50 of 72 ± 2 nM and its 8-C-glucoside orientin with an IC50 of 43 ± 2 nM. Submicromolar inhibitors were also evaluated by an in vitro endonuclease activity assay using single-stranded DNA, and the results were in full agreement with data from the competitive AlphaScreen assay. Using X-ray crystallography, we analyzed structures of the PA-Nter in complex with luteolin at 2.0 Å resolution and quambalarine B at 2.5 Å resolution, which clearly revealed the binding pose of these polyols coordinated to two manganese ions in the endonuclease active site. Using two distinct assays along with the structural work, we have presumably identified and characterized the molecular mode-of-action of flavonoids in influenza-infected cells. [Display omitted] •A screening assay based on AlphaScreen technology was developed.•A large library of flavonoids was assembled and tested for inhibition of influenza endonuclease.•Luteolin and its congeners were identified as new hit compounds binding through 3′,4′-dihydroxyplenyl moiety to the enzyme.•Detailed explanation of structure-activity relationship.•Structural work also revealed other binding mode of flavonoids to endonuclease.•Screened flavonoids did not inhibit influenza neuraminidase.