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

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
Number of pages11
JournalChemistry: a European Journal
Early online date7 Apr 2020
DOIs
Publication statusE-pub ahead of print - 7 Apr 2020

Keywords

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

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