title: Novel high-throughput electrochemiluminescent assay for identification of human tyrosyl-DNA phosphodiesterase (Tdp1) inhibitors and characterization of furamidine (NSC 305831) as an inhibitor of Tdp1.
abstract: By enzymatically hydrolyzing the terminal phosphodiester bond at the 3'-ends of DNA breaks, tyrosyl-DNA phosphodiesterase (Tdp1) repairs topoisomerase-DNA covalent complexes and processes the DNA ends for DNA repair. To identify novel Tdp1 inhibitors, we developed a high-throughput assay that uses electrochemiluminescent (ECL) substrates. Subsequent to screening of 1981 compounds from the 'diversity set' of the NCI-Developmental Therapeutics Program, here we report that furamidine inhibits Tdp1 at low micromolar concentrations. Inhibition of Tdp1 by furamidine is effective both with single- and double-stranded substrates but is slightly stronger with the duplex DNA. Surface plasmon resonance studies show that furamidine binds both single- and double-stranded DNA, though more weakly with the single-stranded substrate DNA. Thus, the inhibition of Tdp1 activity could in part be due to the binding of furamidine to DNA. However, the inhibition of Tdp1 by furamidine is independent of the substrate DNA sequence. The kinetics of Tdp1 inhibition by furamidine was influenced by the drug to enzyme ratio and duration of the reaction. Comparison with related dications shows that furamidine inhibits Tdp1 more effectively than berenil, while pentamidine was inactive. Thus, furamidine represents the most potent Tdp1 inhibitor reported to date.
authors.affiliation: Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. antonys@mail.nih.gov
authors.forename: Smitha
authors.initials: S
authors.lastname: Antony
authors.forename: Christophe
authors.initials: C
authors.lastname: Marchand
authors.forename: Andrew G
authors.initials: AG
authors.lastname: Stephen
authors.forename: Laurent
authors.initials: L
authors.lastname: Thibaut
authors.forename: Keli K
authors.initials: KK
authors.lastname: Agama
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Fisher
authors.forename: Yves
authors.initials: Y
authors.lastname: Pommier
mesh_terms: D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D001550:Benzamidines / Q000737:chemistry / Q000494:pharmacology*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004133:Diminazene / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004563:Electrochemistry; D006801:Humans; D007700:Kinetics; D008163:Luminescent Measurements; D010419:Pentamidine / Q000737:chemistry / Q000494:pharmacology; D010726:Phosphodiesterase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D013936:Thymidine / Q000737:chemistry
chemical_list: D000970:Antineoplastic Agents; D001550:Benzamidines; D004277:DNA, Single-Stranded; D010726:Phosphodiesterase Inhibitors; D010419:Pentamidine; C033286:furamidine; D004247:DNA; D010727:Phosphoric Diester Hydrolases; C498508:TDP1 protein, human; C003915:diminazene aceturate; D013936:Thymidine; D004133:Diminazene