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DNA-linked inhibitor antibody assay (DIANA) as a new method for screening influenza neuraminidase inhibitors

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  • DNA-linked inhibitor antibody assay (DIANA) as a new method for screening influenza neuraminidase inhibitors

    Biochem J. 2018 Nov 7. pii: BCJ20180764. doi: 10.1042/BCJ20180764. [Epub ahead of print]
    DNA-linked inhibitor antibody assay (DIANA) as a new method for screening influenza neuraminidase inhibitors.

    Ko???ek M1, Navr?til V2, Roj?kov? K2, Pokorn? J2, Albiňana CB2, Pachl P2, Zemanov? J2, Machara A2, ??cha P2, Hudlick? J3, C?sařov? I3, Řez?čov? P2, Konvalinka J2.
    Author information

    Abstract

    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 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 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.


    KEYWORDS:

    DIANA; Influenza neuraminidase; assay; crystallography

    PMID: 30404922 DOI: 10.1042/BCJ20180764
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