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-(33)P]-AMP from the complex Lig4-[α-(33)P]-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.
authors.affiliation: Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10583, USA.
authors.forename: Hui-Min
authors.initials: HM
authors.lastname: Tseng
authors.forename: David
authors.initials: D
authors.lastname: Shum
authors.forename: Bhavneet
authors.initials: B
authors.lastname: Bhinder
authors.forename: Sindy
authors.initials: S
authors.lastname: Escobar
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Veomett
authors.forename: Alan E
authors.initials: AE
authors.lastname: Tomkinson
authors.forename: David Y
authors.initials: DY
authors.lastname: Gin
authors.forename: Hakim
authors.initials: H
authors.lastname: Djaballah
authors.forename: David A
authors.initials: DA
authors.lastname: Scheinberg
mesh_terms: D001665:Binding Sites; D004247:DNA / Q000737:chemistry*; D000072481:DNA Ligase ATP; D011088:DNA Ligases / Q000032:analysis* / Q000737:chemistry*; D004789:Enzyme Activation; D006801:Humans; D007553:Isotope Labeling; D011485:Protein Binding; D011869:Radioligand Assay / Q000379:methods*; D012588:Scintillation Counting / Q000379:methods*
chemical_list: C000607893:LIG4 protein, human; D004247:DNA; D011088:DNA Ligases; D000072481:DNA Ligase ATP