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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.

Antony S, Marchand C, Stephen AG, Thibaut L, Agama KK, Fisher RJ, Pommier Y - Nucleic Acids Res. (2007)

Bottom Line: Inhibition of Tdp1 by furamidine is effective both with single- and double-stranded substrates but is slightly stronger with the duplex DNA.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. antonys@mail.nih.gov

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.

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Related in: MedlinePlus

Competitive inhibition of Tdp1 by furamidine. (A) A 100-µl reaction mixture containing 25 nM 14Y and 5 ng of Tdp1 was incubated at pH 8.0 at 25°C in the absence of drug, or in the presence of the indicated concentrations of furamidine. Aliquots were taken at the indicated times (min). Reaction products were analyzed by denaturing PAGE. (B) Densitometry analysis of the gel shown in A. Tdp1 activity measured as the percentage of DNA substrate 14Y converted to 14P (left panel) or substrate 14Y remaining (right panel) as a function of reaction time. (C) Reactions (20 µl) containing 25 nM 14Y and the indicated amounts (ng) of Tdp1 were carried out in the absence or presence of increasing concentrations of furamidine for 20 min. A representative gel is shown. (D) Densitometry analysis of the gel shown in C. Tdp1 activity was calculated as the percentage of DNA substrate 14Y converted to 14P. The horizontal line corresponds to 50% inhibition of Tdp1 activity.
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Figure 7: Competitive inhibition of Tdp1 by furamidine. (A) A 100-µl reaction mixture containing 25 nM 14Y and 5 ng of Tdp1 was incubated at pH 8.0 at 25°C in the absence of drug, or in the presence of the indicated concentrations of furamidine. Aliquots were taken at the indicated times (min). Reaction products were analyzed by denaturing PAGE. (B) Densitometry analysis of the gel shown in A. Tdp1 activity measured as the percentage of DNA substrate 14Y converted to 14P (left panel) or substrate 14Y remaining (right panel) as a function of reaction time. (C) Reactions (20 µl) containing 25 nM 14Y and the indicated amounts (ng) of Tdp1 were carried out in the absence or presence of increasing concentrations of furamidine for 20 min. A representative gel is shown. (D) Densitometry analysis of the gel shown in C. Tdp1 activity was calculated as the percentage of DNA substrate 14Y converted to 14P. The horizontal line corresponds to 50% inhibition of Tdp1 activity.

Mentions: As shown in Figure 7A and B (A, left; and squares in B), 1 ng of Tdp1 converted ∼50% (t1/2) of the 14Y substrate within ∼1.9 min. Thus, we wished to determine how furamidine affected the kinetics of Tdp1 activity. Tdp1 activity was slowed down as the concentration of furamidine increased (Figure 7A). Kinetic plots (Figure 7B) demonstrated that furamidine increased the conversion half-time (t1/2) of the 14Y substrate from 1.9 min in the absence of drug to 2.7 min in the presence of 30 µM furamidine (diamond in Figure 7B) and 4.4 min in the presence of 60 µM furamidine (inverted triangle in Figure 7B). Additionally, increasing Tdp1 concentration was able to overcome Tdp1 inhibition by furamidine (Figure 7C and D). The 50% inhibition of Tdp1 activity observed by 30 µM furamidine with 0.1 ng of Tdp1 was almost completely reversed by increasing the concentration of Tdp1 to 1 ng (Figure 7C and diamond in Figure 7D). Similar effects were seen with 60 µM and 250 µM furamidine (Figure 7C and D). Thus, free Tdp1 competes with furamidine. Together, these results suggest that furamidine produces reversible and competitive inhibition of Tdp1.Figure 7.


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.

Antony S, Marchand C, Stephen AG, Thibaut L, Agama KK, Fisher RJ, Pommier Y - Nucleic Acids Res. (2007)

Competitive inhibition of Tdp1 by furamidine. (A) A 100-µl reaction mixture containing 25 nM 14Y and 5 ng of Tdp1 was incubated at pH 8.0 at 25°C in the absence of drug, or in the presence of the indicated concentrations of furamidine. Aliquots were taken at the indicated times (min). Reaction products were analyzed by denaturing PAGE. (B) Densitometry analysis of the gel shown in A. Tdp1 activity measured as the percentage of DNA substrate 14Y converted to 14P (left panel) or substrate 14Y remaining (right panel) as a function of reaction time. (C) Reactions (20 µl) containing 25 nM 14Y and the indicated amounts (ng) of Tdp1 were carried out in the absence or presence of increasing concentrations of furamidine for 20 min. A representative gel is shown. (D) Densitometry analysis of the gel shown in C. Tdp1 activity was calculated as the percentage of DNA substrate 14Y converted to 14P. The horizontal line corresponds to 50% inhibition of Tdp1 activity.
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Related In: Results  -  Collection

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Figure 7: Competitive inhibition of Tdp1 by furamidine. (A) A 100-µl reaction mixture containing 25 nM 14Y and 5 ng of Tdp1 was incubated at pH 8.0 at 25°C in the absence of drug, or in the presence of the indicated concentrations of furamidine. Aliquots were taken at the indicated times (min). Reaction products were analyzed by denaturing PAGE. (B) Densitometry analysis of the gel shown in A. Tdp1 activity measured as the percentage of DNA substrate 14Y converted to 14P (left panel) or substrate 14Y remaining (right panel) as a function of reaction time. (C) Reactions (20 µl) containing 25 nM 14Y and the indicated amounts (ng) of Tdp1 were carried out in the absence or presence of increasing concentrations of furamidine for 20 min. A representative gel is shown. (D) Densitometry analysis of the gel shown in C. Tdp1 activity was calculated as the percentage of DNA substrate 14Y converted to 14P. The horizontal line corresponds to 50% inhibition of Tdp1 activity.
Mentions: As shown in Figure 7A and B (A, left; and squares in B), 1 ng of Tdp1 converted ∼50% (t1/2) of the 14Y substrate within ∼1.9 min. Thus, we wished to determine how furamidine affected the kinetics of Tdp1 activity. Tdp1 activity was slowed down as the concentration of furamidine increased (Figure 7A). Kinetic plots (Figure 7B) demonstrated that furamidine increased the conversion half-time (t1/2) of the 14Y substrate from 1.9 min in the absence of drug to 2.7 min in the presence of 30 µM furamidine (diamond in Figure 7B) and 4.4 min in the presence of 60 µM furamidine (inverted triangle in Figure 7B). Additionally, increasing Tdp1 concentration was able to overcome Tdp1 inhibition by furamidine (Figure 7C and D). The 50% inhibition of Tdp1 activity observed by 30 µM furamidine with 0.1 ng of Tdp1 was almost completely reversed by increasing the concentration of Tdp1 to 1 ng (Figure 7C and diamond in Figure 7D). Similar effects were seen with 60 µM and 250 µM furamidine (Figure 7C and D). Thus, free Tdp1 competes with furamidine. Together, these results suggest that furamidine produces reversible and competitive inhibition of Tdp1.Figure 7.

Bottom Line: Inhibition of Tdp1 by furamidine is effective both with single- and double-stranded substrates but is slightly stronger with the duplex DNA.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA. antonys@mail.nih.gov

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.

Show MeSH
Related in: MedlinePlus