HTS procedures for inhibitors of influenza virus transcription complex assembly

University Of Cambridge, Cambridge

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Abstract

[unreadable] DESCRIPTION (provided by applicant): The goal of this research is to develop a scalable screening protocol to search for compounds that block binding of the influenza virus RNA polymerase to the viral genome segments. The polymerase is a highly conserved protein complex essential for virus replication and is a very strong candidate target for antiviral screening. Many pharmaceutical companies have run screens to identify inhibitors of the transcriptional activity of the polymerase but with very little success. A key property of the polymerase that has not been incorporated into previous HT screening assays is its requirement to bind with high affinity and specificity to the 5' terminal 11 nucleotides of the viral genome. These sequences are absolutely conserved, thus an inhibitor of this interaction would be expected to block replication of all existing sub-types. A solid phase in vitro transcription assay for the viral polymerase has been reported. It uses a short RNA oligo containing the polymerase recognition sequence to immobilize recombinantly expressed influenza virus polymerase onto streptavidin coated beads that can then initiate dinucleotide ApG and mRNA primed transcription. Here it is proposed to adapt this assay to a plate format and to develop it as a screen specifically for identifying compounds that inhibit binding. Currently, DELFIA is being used to assay polymerase binding, as this has the advantage of high sensitivity which reduces the amount of polymerase required for the assay (the chief limiting component). An alternative assay uses a tagged RNA oligonucleotide to function as molecular beacon. The RNA is bound to the plate surface and then incubated with the polymerase in the presence of test compounds. Binding of the polymerase separates the fluorophore and quenching moieties of the oligonucleotide, resulting in fluorescence dequenching proportional to the level of polymerase binding. Inhibitors are identified by the decrease in fluorescence intensity. Removal of the reaction components prior to measurement avoids quenching artifacts of the test compounds. A homogenous fluorescence polarization assay to detect binding of fluorophore-labeled ApG primer by the immobilized polymerase will also be investigated. Relevance: The identification of antiviral drugs that can inhibit the replication of a wide range of influenza virus types is vital for preparedness against the next influenza virus pandemic. [unreadable] [unreadable] [unreadable]