title: An RNA Hybridization Assay for Screening Influenza A Virus Polymerase Inhibitors Using the Entire Ribonucleoprotein Complex.
abstract: Novel antiviral drugs, which are less prone to resistance development, are desirable alternatives to the currently approved drugs for the treatment of potentially serious influenza virus infections. The viral polymerase is highly conserved and serves as an attractive target for antiviral drugs since potent inhibitors would directly stop viral replication at an early stage. Recent structural studies on the functional domains of the heterotrimeric influenza polymerase, which comprises subunits PA, PB1, and PB2, opened the way to a structure-based approach for optimizing inhibitors of viral replication. These strategies, however, are limited by the use of isolated protein fragments instead of employing the entire ribonucleoprotein complex (RNP), which represents the functional form of the influenza polymerase in infected cells. In this study, we have established a screening assay for efficient and reliable analysis of potential influenza polymerase inhibitors of various molecular targets such as monoselective polymerase inhibitors targeting the endonuclease site, the cap-binding domain, and the polymerase active site, respectively. By utilizing whole viral RNPs and a radioactivity-free endpoint detection with the capability for efficient compound screening while offering high-content information on potential inhibitors to drive medicinal chemistry program in a reliable manner, this biochemical assay provides significant advantages over the currently available conventional assays. We propose that this assay can eventually be adapted for coinstantaneous analysis and subsequent optimization of two or more different chemical scaffold classes targeting multiple active sites within the polymerase complex, thus enabling the evaluation of drug combinations and characterization of molecules with dual functionality.
authors.affiliation: 1 Savira Pharmaceuticals GmbH , Vienna, Austria .
authors.forename: Franz-Ferdinand
authors.initials: FF
authors.lastname: Roch
authors.affiliation: 1 Savira Pharmaceuticals GmbH , Vienna, Austria .
authors.forename: Georg
authors.initials: G
authors.lastname: Hinterkörner
authors.affiliation: 2 Virology Discovery, Hoffmann-La Roche, Inc. , Nutley, New Jersey.
authors.forename: John
authors.initials: J
authors.lastname: Menke
authors.affiliation: 2 Virology Discovery, Hoffmann-La Roche, Inc. , Nutley, New Jersey.
authors.forename: Guo-Qing
authors.initials: GQ
authors.lastname: Tang
authors.affiliation: 3 Grenoble Outstation, European Molecular Biology Laboratory , Grenoble, France .
authors.forename: Stephen
authors.initials: S
authors.lastname: Cusack
authors.affiliation: 1 Savira Pharmaceuticals GmbH , Vienna, Austria .
authors.forename: Barbara
authors.initials: B
authors.lastname: Butzendobler
authors.affiliation: 1 Savira Pharmaceuticals GmbH , Vienna, Austria .
authors.forename: Helmut
authors.initials: H
authors.lastname: Buschmann
authors.affiliation: 2 Virology Discovery, Hoffmann-La Roche, Inc. , Nutley, New Jersey.
authors.forename: Kausiki
authors.initials: K
authors.lastname: Datta
authors.affiliation: 1 Savira Pharmaceuticals GmbH , Vienna, Austria .
authors.forename: Andrea
authors.initials: A
authors.lastname: Wolkerstorfer
mesh_terms: D000998:Antiviral Agents / Q000032:analysis* / Q000494:pharmacology; D012321:DNA-Directed RNA Polymerases / Q000032:analysis* / Q000037:antagonists & inhibitors* / Q000235:genetics; D004353:Drug Evaluation, Preclinical / Q000379:methods; D006801:Humans; D009980:Influenza A virus / Q000187:drug effects / Q000201:enzymology*; D012261:Ribonucleoproteins / Q000032:analysis* / Q000235:genetics / Q000494:pharmacology; D014779:Virus Replication / Q000187:drug effects / Q000502:physiology
chemical_list: D000998:Antiviral Agents; D012261:Ribonucleoproteins; D012321:DNA-Directed RNA Polymerases