A high-throughput scintillation proximity-based assay for human DNA ligase IV Journal Article

Authors: Tseng, H. M.; Shum, D.; Bhinder, B.; Escobar, S.; Veomett, N. J.; Tomkinson, A. E.; Gin, D. Y.; Djaballah, H.; Scheinberg, D. A.
Article Title: A high-throughput scintillation proximity-based assay for human DNA ligase IV
Abstract: Ionizing radiation (IR) and certain chemotherapeutic drugs are designed to generate cytotoxic DNA double-strand breaks (DSBs) in cancer cells. Inhibition of the major DSB repair pathway, nonhomologous end joining (NHEJ), will enhance the cytotoxicity of these agents. Screening for inhibitors of the DNA ligase IV (Lig4), which mediates the final ligation step in NHEJ, offers a novel target-based drug discovery opportunity. For this purpose, we have developed an enzymatic assay to identify chemicals that block the transfer of [α- 3P]-AMP from the complex Lig4-[α- 33P]-AMP onto the 5′ end of a double-stranded DNA substrate and adapted it to a scintillation proximity assay (SPA). A screen was performed against a collection of 5,280 compounds. Assay statistics show an average Z′ value of 0.73, indicative of a robust assay in this SPA format. Using a threshold of >20% inhibition, 10 compounds were initially scored as positive hits. A follow-up screen confirmed four compounds with IC 50 values ranging from 1 to 30 μM. Rabeprazole and U73122 were found to specifically block the adenylate transfer step and DNA rejoining; in whole live cell assays, these compounds were found to inhibit the repair of DSBs generated by IR. The ability to screen and identify Lig4 inhibitors suggests that they may have utility as chemo- and radio-sensitizers in combination therapy and provides a rationale for using this screening strategy to identify additional inhibitors. © 2012 MARY ANN LIEBERT, INC.
Keywords: controlled study; unclassified drug; human cell; nonhuman; follow up; animal cell; dna repair; protein binding; enzyme activation; high throughput screening; enzyme activity; cell assay; dna; double stranded dna; isotope labeling; double stranded dna break; binding sites; chemical structure; ic 50; polydeoxyribonucleotide synthase; enzyme assay; dna ligases; adenosine phosphate; adenylation; infrared radiation; scintillation proximity assay; rabeprazole; scintillation counting; radioligand assay; scintillation; 1 [[6 (3 methoxyestra 1,3,5(10) trien 17beta yl)amino]hexyl] 1h pyrrole 2,5 dione; polydeoxyribonucleotide synthase 4
Journal Title: Assay and Drug Development Technologies
Volume: 10
Issue: 3
ISSN: 1540-658X
Publisher: Mary Ann Liebert, Inc  
Date Published: 2012-06-01
Start Page: 235
End Page: 249
Language: English
DOI: 10.1089/adt.2011.0404
PROVIDER: scopus
PMCID: PMC3374410
PUBMED: 22192310
Notes: --- - "Export Date: 2 July 2012" - "CODEN: ADDTA" - "Source: Scopus"
Citation Impact
MSK Authors
  1. Hui-Min Tseng
    2 Tseng
  2. David Y Gin
    41 Gin
  3. Bhavneet Bhinder
    30 Bhinder
  4. David Shum
    52 Shum
  5. Nicholas J Veomett
    10 Veomett