title: Generation of assays and antibodies to facilitate the study of human 5'-tyrosyl DNA phosphodiesterase.
abstract: Topoisomerases regulate DNA topology by the transient cleavage and religation of DNA during transcription and replication. Topoisomerase II (Topo II) poisons such as etoposide can induce abortive DNA strand breaks in which Topo II remains covalently bound to a 5' DNA strand terminus via a phosphotyrosyl linker. Tyrosyl DNA phosphodiesterase 2 (Tdp2) is a recently discovered human 5'-tyrosyl DNA phosphodiesterase that repairs this topoisomerase-mediated DNA damage, thereby playing a central role in maintaining normal DNA topology in cells. Cellular depletion of Tdp2 has been shown to result in increased susceptibility and sensitivity to Topo II-induced DNA double-strand breaks, thereby revealing Tdp2 as a potentially attractive anticancer target. No drug-like inhibitors of Tdp2 have been identified to date, and assays suitable for high-throughput screening (HTS) have not been widely reported. Here we have identified a new and effective chromogenic substrate for Tdp2 and developed a homogeneous and robust HTS assay. A second novel Tdp2 assay was also developed to cross-validate hit matter identified from an HTS. In addition, a new and specific Tdp2 antibody is described. Together, these new tools will aid in the identification of novel Tdp2 inhibitors and the investigation of the role of Tdp2 in cancer.
authors: Cancer Research UK Drug Discovery Unit, Paterson Institute for Cancer Research, University of Manchester, Manchester M20 4BX, UK.
authors: Graeme
authors: G
authors: Thomson
authors: Amanda
authors: A
authors: Watson
authors: Keith
authors: K
authors: Caldecott
authors: Olive
authors: O
authors: Denneny
authors: Paul
authors: P
authors: Depledge
authors: Nicola
authors: N
authors: Hamilton
authors: Gemma
authors: G
authors: Hopkins
authors: Allan
authors: A
authors: Jordan
authors: Christopher
authors: C
authors: Morrow
authors: Ali
authors: A
authors: Raoof
authors: Ian
authors: I
authors: Waddell
authors: Donald
authors: D
authors: Ogilvie
mesh_terms: D000906:Antibodies / Q000276:immunology*; D001483:Base Sequence; D045744:Cell Line, Tumor; D004353:Drug Evaluation, Preclinical / Q000379:methods; D055785:Gene Knockdown Techniques; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008969:Molecular Sequence Data; D009596:Nitrophenols / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D010727:Phosphoric Diester Hydrolases / Q000032:analysis* / Q000235:genetics / Q000276:immunology*; D015203:Reproducibility of Results
chemical_list: D000906:Antibodies; D009596:Nitrophenols; D010726:Phosphodiesterase Inhibitors; C058183:4-nitrophenyl phenylphosphonate; D010727:Phosphoric Diester Hydrolases; C122147:tyrosyl-DNA phosphodiesterase