title: High-throughput screening identification of poliovirus RNA-dependent RNA polymerase inhibitors.
abstract: Viral RNA-dependent RNA polymerase (RdRP) enzymes are essential for the replication of positive-strand RNA viruses and established targets for the development of selective antiviral therapeutics. In this work we have carried out a high-throughput screen of 154,267 compounds to identify poliovirus polymerase inhibitors using a fluorescence based RNA elongation assay. Screening and subsequent validation experiments using kinetic methods and RNA product analysis resulted in the identification of seven inhibitors that affect the RNA binding, initiation, or elongation activity of the polymerase. X-ray crystallography data show clear density for five of the compounds in the active site of the poliovirus polymerase elongation complex. The inhibitors occupy the NTP binding site by stacking on the priming nucleotide and interacting with the templating base, yet competition studies show fairly weak IC50 values in the low μM range. A comparison with nucleotide bound structures suggests that weak binding is likely due to the lack of a triphosphate group on the inhibitors. Consequently, the inhibitors are primarily effective at blocking polymerase initiation and do not effectively compete with NTP binding during processive elongation. These findings are discussed in the context of the polymerase elongation complex structure and allosteric control of the viral RdRP catalytic cycle.
authors: Department of Biochemistry & Molecular Biology, 1870 Campus Delivery, Colorado State University, Fort Collins, CO 80523-1870, USA.
authors: Grace
authors: G
authors: Campagnola
authors: Peng
authors: P
authors: Gong
authors: Olve B
authors: OB
authors: Peersen
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D001665:Binding Sites; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005453:Fluorescence; D057166:High-Throughput Screening Assays; D006801:Humans; D020128:Inhibitory Concentration 50; D008958:Models, Molecular; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism; D011051:Poliomyelitis / Q000188:drug therapy* / Q000821:virology; D017955:Poliovirus / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects* / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D054852:Small Molecule Libraries / Q000737:chemistry / Q000494:pharmacology*; D014779:Virus Replication / Q000187:drug effects
chemical_list: D004791:Enzyme Inhibitors; D009711:Nucleotides; D012367:RNA, Viral; D054852:Small Molecule Libraries; D012324:RNA-Dependent RNA Polymerase