Direct High-Throughput Screening Assay for mRNA Cap Guanine-N7 Methyltransferase Activity

Renata Kasprzyk, Mateusz Fido, Adam Mamot, Przemyslaw Wanat, Miroslaw Smietanski, Michal Kopcial, Victoria H. Cowling, Joanna Kowalska (Lead / Corresponding author), Jacek Jemielity (Lead / Corresponding author)

    Research output: Contribution to journalArticlepeer-review

    4 Citations (Scopus)
    157 Downloads (Pure)

    Abstract

    In eukaryotes, mature mRNA is formed through modifications of precursor mRNA, one of which is 5’ cap biosynthesis, involving RNA cap guanine-N7 methyltransferase (N7-MTase). N7-MTases are also encoded by some eukaryotic viruses and facilitate their replication. N7-MTase inhibitors have therapeutic potential, but their discovery is difficult because long RNA substrates are usually required for activity. Herein, we report a universal N7-MTase activity assay based on small-molecule fluorescent probes. We synthesized 12 fluorescent substrate analogues (GpppA and GpppG derivatives) varying in the dye type, dye attachment site, and linker length. GpppA labeled with pyrene at the 3’-O position of adenosine acted as an artificial substrate with the properties of a turn-off probe for all three tested N7-MTases (human, parasite, and viral). Using this compound, a N7-MTase inhibitor assay adaptable to high-throughput screening was developed and used to screen synthetic substrate analogues and a commercial library. Several inhibitors with nanomolar activities were identified.

    Original languageEnglish
    Pages (from-to)11266-11275
    Number of pages10
    JournalChemistry: a European Journal
    Volume26
    Issue number49
    Early online date7 Apr 2020
    DOIs
    Publication statusPublished - 1 Sept 2020

    Keywords

    • cap
    • fluorescent probes
    • high-throughput screening
    • methyltransferases
    • pyrenes

    ASJC Scopus subject areas

    • Catalysis
    • Organic Chemistry

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