title: Development of an oligonucleotide-based fluorescence assay for the identification of tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors.
abstract: Topoisomerase 1 (TOP1) generates transient nicks in the DNA to relieve torsional stress encountered during the cellular processes of transcription, replication, and recombination. At the site of the nick there is a covalent linkage of TOP1 with DNA via a tyrosine residue. This reversible TOP1-cleavage complex intermediate can become trapped on DNA by TOP1 poisons such as camptothecin, or by collision with replication or transcription machinery, thereby causing protein-linked DNA single- or double-strand breaks and resulting in cell death. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a key enzyme involved in the repair of TOP1-associated DNA breaks via hydrolysis of 3'-phosphotyrosine bonds. Inhibition of TDP1 is therefore an attractive strategy for targeting cancer cells in conjunction with TOP1 poisons. Existing methods for monitoring the phosphodiesterase activity of TDP1 are generally gel based or of high cost. Here we report a novel, oligonucleotide-based fluorescence assay that is robust, sensitive, and suitable for high-throughput screening of both fragment and small compound libraries for the detection of TDP1 inhibitors. We further validated the assay using whole cell extracts, extending its potential application to determine of TDP1 activity in clinical samples from patients undergoing chemotherapy.
authors.affiliation: Translational Drug Discovery Group, School of Life Sciences, University of Sussex, Brighton BN1 9QJ, UK.
authors.forename: Sarah
authors.initials: S
authors.lastname: Walker
authors.affiliation: Wellcome Trust DNA Repair Group, Genome Damage and Stability Centre, University of Sussex, Brighton BN1 9RQ, UK.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Meisenberg
authors.affiliation: Cancer Research UK DNA Repair Enzymes Group, Genome Damage and Stability Centre, University of Sussex, Science Park Road, Brighton BN1 9RQ, UK.
authors.forename: Rachel A
authors.initials: RA
authors.lastname: Bibby
authors.affiliation: Translational Drug Discovery Group, School of Life Sciences, University of Sussex, Brighton BN1 9QJ, UK.
authors.forename: Trevor
authors.initials: T
authors.lastname: Askwith
authors.affiliation: Translational Drug Discovery Group, School of Life Sciences, University of Sussex, Brighton BN1 9QJ, UK.
authors.forename: Gareth
authors.initials: G
authors.lastname: Williams
authors.affiliation: Gyrasol Technologies, 2029 Becker Drive, Lawrence, KS 66047, USA.
authors.forename: Frauke H
authors.initials: FH
authors.lastname: Rininsland
authors.affiliation: Cancer Research UK DNA Repair Enzymes Group, Genome Damage and Stability Centre, University of Sussex, Science Park Road, Brighton BN1 9RQ, UK.
authors.forename: Laurence H
authors.initials: LH
authors.lastname: Pearl
authors.affiliation: Cancer Research UK DNA Repair Enzymes Group, Genome Damage and Stability Centre, University of Sussex, Science Park Road, Brighton BN1 9RQ, UK.
authors.forename: Antony W
authors.initials: AW
authors.lastname: Oliver
authors.affiliation: Wellcome Trust DNA Repair Group, Genome Damage and Stability Centre, University of Sussex, Brighton BN1 9RQ, UK; Mammalian Genome Stability Group, Kreb's Institute, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK. Electronic address: s.el-khamisy@sheffield.ac.uk.
authors.forename: Sherif
authors.initials: S
authors.lastname: El-Khamisy
authors.affiliation: Translational Drug Discovery Group, School of Life Sciences, University of Sussex, Brighton BN1 9QJ, UK. Electronic address: Simon.Ward@sussex.ac.uk.
authors.forename: Simon
authors.initials: S
authors.lastname: Ward
authors.affiliation: Translational Drug Discovery Group, School of Life Sciences, University of Sussex, Brighton BN1 9QJ, UK. Electronic address: J.Atack@sussex.ac.uk.
authors.forename: John R
authors.initials: JR
authors.lastname: Atack
mesh_terms: D001483:Base Sequence; D003362:Cost-Benefit Analysis; D004353:Drug Evaluation, Preclinical / Q000191:economics / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D007700:Kinetics; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000191:economics / Q000379:methods*
chemical_list: D004791:Enzyme Inhibitors; D009841:Oligonucleotides; D010727:Phosphoric Diester Hydrolases; C498508:TDP1 protein, human
keywords: Assay; Fragment; High-throughput screen; Inhibitor; TDP1; Topoisomerase