FRET-based assay using a three-way junction DNA substrate to identify inhibitors of human cytomegalovirus pUL89 endonuclease activity

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3 Scopus citations

Abstract

Human cytomegalovirus (HCMV) packages its viral genome using a multi-protein complex often called the terminase complex. The terminase complex promotes the packaging of a single copy of the double-stranded viral DNA genome by cleaving concatemeric genomic DNA during particle assembly. The endonuclease activity necessary to cleave the viral genomic DNA derives from a terminase complex protein, pUL89. The genome cleavage step is necessary for infectious particle assembly and is therefore an interesting target for antiviral intervention. We and others have previously described agarose gel- and enzyme-linked immunosorbent assay-based procedures to detect pUL89 endonuclease activity; however, these approaches were labor intensive and low-throughput. To develop a screening platform for compound libraries, we constructed a fluorescently-labeled three-way junction DNA that continuously reported pUL89 endonuclease activity and was suitable for high-throughput screening. In a pilot screen of 1280 compounds, we identified trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid and (2′Z, 3′E)-6-bromoindirubin-3′-oxime as hits with low micromolar half maximal inhibitory concentration (IC 50 ) values. The latter hit also inhibited HCMV at a late step in virus replication with a 1 μM half maximal effective concentration (EC 50 ). We describe here the development and validation of a sensitive fluorescence resonance energy transfer-based assay for high throughput screening that identified inhibitors of pUL89 endonuclease activity and virus replication.

Original languageEnglish (US)
Pages (from-to)29-37
Number of pages9
JournalEuropean Journal of Pharmaceutical Sciences
Volume127
DOIs
StatePublished - Jan 15 2019

Bibliographical note

Funding Information:
We thank the Zhengqiang Wang lab (University of Minnesota) for synthesis of 10k and BDCRB. Support for this study was provided by the Center for Drug Design at the University of Minnesota .

Publisher Copyright:
© 2018 Elsevier B.V.

Copyright:
Copyright 2019 Elsevier B.V., All rights reserved.

Keywords

  • High-throughput assay
  • Human cytomegalovirus
  • Terminase inhibitor
  • Three-way DNA junction
  • UL89

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