An RNA Hybridization Assay for Screening Influenza A Virus Polymerase Inhibitors Using the Entire Ribonucleoprotein Complex
| Autor: | Guo-Qing Tang, Stephen Cusack, Helmut Buschmann, Georg Hinterkörner, Franz-Ferdinand Roch, Andrea Wolkerstorfer, John Menke, Barbara Butzendobler, Kausiki Datta |
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| Rok vydání: | 2015 |
| Předmět: |
Rna hybridization
Drug Evaluation Preclinical DNA-Directed RNA Polymerases Biology medicine.disease_cause Virus Replication Virology Antiviral Agents Virus Ribonucleoprotein complex Viral replication Ribonucleoproteins Influenza A virus Heterotrimeric G protein Drug Discovery medicine biology.protein Molecular Medicine Humans Polymerase Ribonucleoprotein |
| Zdroj: | Assay and drug development technologies. 13(8) |
| ISSN: | 1557-8127 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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