Influenza neuraminidase is responsible for the escape of new viral particles from the infected cell surface. Several neuraminidase inhibitors are used clinically to treat patients or stockpiled for emergencies. However, the increasing development of viral resistance against approved inhibitors has underscored the need for the development of new antivirals effective against resistant influenza strains. A facile, sensitive, and inexpensive screening method would help achieve this goal. Recently, we described a multiwell plate-based DNA-linked inhibitor antibody assay (DIANA). This highly sensitive method can quantify femtomolar concentrations of enzymes. DIANA also has been applied to high-throughput enzyme inhibitor screening, allowing the evaluation of inhibition constants from a single inhibitor concentration. Here, we report the design, synthesis, and structural characterization of a tamiphosphor derivative linked to a reporter DNA oligonucleotide for the development of a DIANA-type assay to screen potential influenza neuraminidase inhibitors. The neuraminidase is first captured by an immobilized antibody, and the test compound competes for binding to the enzyme with the oligo-linked detection probe, which is then quantified by qPCR. We validated this novel assay by comparing it with the standard fluorometric assay and demonstrated its usefulness for sensitive neuraminidase detection as well as high-throughput screening of potential new neuraminidase inhibitors.

Department of Biochemistry Faculty of Science Charles University Hlavova 8 12800 Prague 2 Czech Republic

Department of Inorganic Chemistry Faculty of Science Charles University Hlavova 8 12843 Prague 2 Czech Republic

Department of Organic Chemistry Faculty of Science Charles University Hlavova 8 12800 Prague 2 Czech Republic

Institute of Molecular Genetics of the Academy of Sciences of the Czech Republic Vídeňská 1083 14000 Prague 4 Czech Republic

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences Gilead Sciences and IOCB Research Center Flemingovo n 2 16610 Prague 6 Czech Republic

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