title: Aptamer Displacement Screen for Flaviviral RNA Methyltransferase Inhibitors.
abstract: 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 Kd= 231 nM. The binding was reversed by a 250-nucleotide YFV messenger RNA (mRNA) transcript and by the triphenylmethane dye aurintricarboxylic acid (ATA). The EC50for ATA displacement was 1.54 µM. 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.
authors.affiliation: Department of Medicine, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA.
authors.forename: Shaun P
authors.initials: SP
authors.lastname: Falk
authors.affiliation: Department of Medicine, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA weisblum@wisc.edu.
authors.forename: Bernard
authors.initials: B
authors.lastname: Weisblum
mesh_terms: D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology; D052157:Aptamers, Nucleotide; D001312:Aurintricarboxylic Acid / Q000494:pharmacology; D001665:Binding Sites; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005416:Flavivirus / Q000201:enzymology*; D005454:Fluorescence Polarization; D006160:Guanosine Triphosphate / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods; D008780:Methyltransferases / Q000037:antagonists & inhibitors*; D058990:Molecular Targeted Therapy / Q000379:methods; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D012315:RNA Caps; D012333:RNA, Messenger; D054852:Small Molecule Libraries / Q000494:pharmacology; D017361:Viral Nonstructural Proteins / Q000378:metabolism; D015005:Yellow fever virus / Q000235:genetics / Q000378:metabolism
chemical_list: D000998:Antiviral Agents; D052157:Aptamers, Nucleotide; D004791:Enzyme Inhibitors; D009838:Oligodeoxyribonucleotides; D012315:RNA Caps; D012333:RNA, Messenger; D054852:Small Molecule Libraries; D017361:Viral Nonstructural Proteins; C096493:decathymidylic acid; D001312:Aurintricarboxylic Acid; D006160:Guanosine Triphosphate; D008780:Methyltransferases
keywords: antiviral screen; aptamer displacement; flavivirus; fluorescence polarization; methyltransferase