6yem
H1N1 2009 PA Endonuclease in complex with Quambalarine B
Structural highlights
FunctionC3W5X6_9INFA Plays an essential role in viral RNA transcription and replication by forming the heterotrimeric polymerase complex together with PB1 and PB2 subunits. The complex transcribes viral mRNAs by using a unique mechanism called cap-snatching. It consists in the hijacking and cleavage of host capped pre-mRNAs. These short capped RNAs are then used as primers for viral mRNAs. The PB2 subunit is responsible for the binding of the 5' cap of cellular pre-mRNAs which are subsequently cleaved after 10-13 nucleotides by the PA subunit that carries the endonuclease activity.[HAMAP-Rule:MF_04063] Publication Abstract from PubMedThe 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 A resolution and quambalarine B at 2.5 A 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. Unraveling the anti-influenza effect of flavonoids: Experimental validation of luteolin and its congeners as potent influenza endonuclease inhibitors.,Zima V, Radilova K, Kozisek M, Albinana CB, Karlukova E, Brynda J, Fanfrlik J, Flieger M, Hodek J, Weber J, Majer P, Konvalinka J, Machara A Eur J Med Chem. 2020 Aug 22;208:112754. doi: 10.1016/j.ejmech.2020.112754. PMID:32883638[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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