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

Kasprzyk, Renata; Fido, Mateusz; Mamot, Adam; Wanat, Przemyslaw; Smietanski, Miroslaw; Kopcial, Michal; Cowling, Victoria; Kowalska, Joanna; Jemielity, Jacek

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 analogs (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 properties of a turn‐off probe for all three tested N7‐MTases (human, parasite, and viral). Using this compound, a high‐throughput N7‐MTase inhibitor assay was developed and used for screening of synthetic substrate analogs and a commercial library. Several inhibitors with nanomolar activities were identified.