Nucleic Acids Research Advance Access published online on June 18, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm463
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Nucleic Acid Enzymes |
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
1Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, 20892 and 2Protein Chemistry Laboratory, Advanced Technology Program, SAIC-Frederick, Inc., NCI Frederick, Frederick, Maryland, 21702, USA
*To whom correspondence should be addressed. Tel: +1 301 451 8596; Fax: +1 301 402 0752; Email: antonys{at}mail.nih.gov
Received April 11, 2007. Revised May 25, 2007. Accepted May 26, 2007.
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.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors