A high-throughput screening assay for the identification of flavivirus NS5 capping enzyme GTP-binding inhibitors: implications for antiviral drug development

J Biomol Screen. 2011 Sep;16(8):852-61. doi: 10.1177/1087057111412183. Epub 2011 Jul 25.

Abstract

There are no effective antivirals currently available for the treatment of flavivirus infection in humans. As such, the identification and characterization of novel drug target sites are critical to developing new classes of antiviral drugs. The flavivirus NS5 N-terminal capping enzyme (CE) is vital for the formation of the viral RNA cap structure, which directs viral polyprotein translation and stabilizes the 5' end of the viral genome. The structure of the flavivirus CE has been solved, and a detailed understanding of the CE-guanosine triphosphate (GTP) and CE-RNA cap interactions is available. Because of the essential nature of the interaction for viral replication, disrupting CE-GTP binding is an attractive approach for drug development. The authors have previously developed a robust assay for monitoring CE-GTP binding in real time. They adapted this assay for high-throughput screening and performed a pilot screen of 46 323 commercially available compounds. A number of small-molecule inhibitors capable of displacing a fluorescently labeled GTP in vitro were identified, and a second functional assay was developed to identify false positives. The results presented indicate that the flavivirus CE cap-binding site is a valuable new target site for antiviral drug discovery and should be further exploited for broad-spectrum anti-flaviviral drug development.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Antiviral Agents / chemistry*
  • Antiviral Agents / pharmacology
  • Binding Sites
  • Binding, Competitive
  • Crystallography, X-Ray
  • Enzyme Inhibitors / chemistry*
  • Enzyme Inhibitors / pharmacology
  • Flavivirus / chemistry
  • Flavivirus / enzymology*
  • Flavivirus / genetics
  • Flavivirus Infections / drug therapy*
  • Flavivirus Infections / virology
  • Fluorescence
  • Guanosine Triphosphate / chemistry
  • Guanosine Triphosphate / metabolism*
  • High-Throughput Screening Assays*
  • Humans
  • Models, Molecular
  • Protein Binding
  • Protein Structure, Tertiary
  • RNA Caps / chemistry
  • RNA Caps / metabolism*
  • Viral Nonstructural Proteins / antagonists & inhibitors
  • Viral Nonstructural Proteins / chemistry
  • Viral Nonstructural Proteins / genetics
  • Viral Nonstructural Proteins / metabolism*
  • Virus Replication / drug effects

Substances

  • Antiviral Agents
  • Enzyme Inhibitors
  • NS5 protein, flavivirus
  • RNA Caps
  • Viral Nonstructural Proteins
  • Guanosine Triphosphate