Aptamer Displacement Screen for Flaviviral RNA Methyltransferase Inhibitors

作者：Falk Shaun P; Wei**lum Bernard^*

来源：Journal of Biomolecular Screening, 2014, 19(8): 1147-1153.

DOI：10.1177/1087057114533147

• antiviral screen
• methyltransferase
• flavivirus
• fluorescence polarization
• aptamer displacement

RNA-protein interactions are vital to the replication of the flaviviral genome. Discovery focused on small molecules that disrupt these interactions represent a viable path for identification of new inhibitors. The viral RNA (vRNA) cap methyltransferase (MTase) of the flaviviruses has been validated as a suitable drug target. Here we report the development of a high-throughput screen for the discovery of compounds that target the RNA binding site of flaviviral protein NS5A. The assay described here is based on displacement of an MT-bound polynucleotide aptamer, decathymidylate derivatized at its 5' end with fluorescein (FL-dT10). Based on the measurement of fluorescence polarization, FL-dT10 bound to yellow fever virus (YFV) MTase in a saturable manner with a K-d = 231 nM. The binding was reversed by a 250-nucleotide YFV messenger RNA (mRNA) transcript and by the triphenylmethane dye aurintricarboxylic acid (ATA). The EC50 for ATA displacement was 1.54 mu M. The MTase cofactors guanosine-5'-triphosphate and S-adenosyl-methionine failed to displace FL-dT10. Analysis by electrophoretic mobility shift assay (EMSA) suggests that ATA binds YFV MTase so as to displace the vRNA. The assay was determined to have a Z' of 0.83 and was successfully used to screen a library of known bioactives.

• 出版日期2014-9