TY - JOUR
T1 - Direct High-Throughput Screening Assay for mRNA Cap Guanine-N7 Methyltransferase Activity
AU - Kasprzyk, Renata
AU - Fido, Mateusz
AU - Mamot, Adam
AU - Wanat, Przemyslaw
AU - Smietanski, Miroslaw
AU - Kopcial, Michal
AU - Cowling, Victoria H.
AU - Kowalska, Joanna
AU - Jemielity, Jacek
N1 - Funding Information:
We thank Dr. Mikolaj Chrominski for providing reagents for the study. This work was financially supported by the National Science Centre (grant numbers UMO‐2016/23/N/ST4/03169 and UMO‐2019/32/T/ST4/00091 to R.K. and 2015/18/E/ST5/00555 to J.K.) and by the Foundation for Polish Science (TEAM/2016‐2/13 to J.J.).
Publisher Copyright:
© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
PY - 2020/9/1
Y1 - 2020/9/1
N2 - 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.
AB - 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.
KW - cap
KW - fluorescent probes
KW - high-throughput screening
KW - methyltransferases
KW - pyrenes
UR - http://www.scopus.com/inward/record.url?scp=85089138584&partnerID=8YFLogxK
U2 - 10.1002/chem.202001036
DO - 10.1002/chem.202001036
M3 - Article
C2 - 32259329
VL - 26
SP - 11266
EP - 11275
JO - Chemistry: a European Journal
JF - Chemistry: a European Journal
SN - 0947-6539
IS - 49
ER -