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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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Binding of furamidine to a 495 response units surface of a stem-loop duplex oligonucleotide (A) and 504 response units surface of a single-stranded oligonucleotide (B). The equilibrium level of binding was determined for each furamidine concentration for the duplex oligonucleotide (C) or the single-stranded oligonucleotides (D). The graphs represent a fit using a two binding-site model for the stem-loop duplex oligonucleotide (C) or a single binding-site model for the single-stranded oligonucleotide (D). Twofold increments of furamidine concentration (0.097, 0.19, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5 and 25 µM) were used.
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Figure 4: Binding of furamidine to a 495 response units surface of a stem-loop duplex oligonucleotide (A) and 504 response units surface of a single-stranded oligonucleotide (B). The equilibrium level of binding was determined for each furamidine concentration for the duplex oligonucleotide (C) or the single-stranded oligonucleotides (D). The graphs represent a fit using a two binding-site model for the stem-loop duplex oligonucleotide (C) or a single binding-site model for the single-stranded oligonucleotide (D). Twofold increments of furamidine concentration (0.097, 0.19, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5 and 25 µM) were used.

Mentions: Binding experiments were performed on a Biacore 2000 instrument (Biacore Inc., Piscataway NJ, USA). 5′ biotinylated stem-loop (biotin-GATCTAAAAGACTTTCTCAAGTCTTTTAGATC) and single-stranded oligonucleotides (biotin-GATCTAAAAGACTT) were synthesized by IDT (Coralville, IA, USA). Stem-loop oligonucleotides were annealed by heating to 90°C for 5 min followed by snap cooling on ice for 15 min. Biotinylated oligonucleotides were immobilized to neutravidin-coated sensor chips as described previously (22). Approximately 5000 RU's of neutravidin was attached to all flow cells on the sensor chips. Oligonucleotides were reconstituted in buffer consisting of 10 mM Tris, pH 7.5, 300 mM NaCl and 1 mM EDTA. Single-stranded and stem-loop oligonucleotides were injected over flow cell 2 and 4, respectively until approximately 500 RU's of oligonucleotide were captured on the chip surface. Furamidine was diluted into running buffer [10 mM MES, 100 mM NaCl, 1 mM EDTA, 5% DMSO (v/v) pH 6.25] and injected over all flow cells at 20 µl/min at 25°C. Following compound injections, the surface was regenerated with a 10 s 1 M NaCl injection followed by a 10 s running buffer injection. A DMSO calibration curve was included to correct for refractive index mismatches between the running buffer and compound dilution series. Data was analyzed using the Scrubber software version 2 (David Myszka, University of Utah) and the equilibrium binding of furamidine was fit to either a single-site or two-site steady state binding model (Figure 4C and D). The equation used to fit the single-site steady state binding model was:where B = bound complex, Rmax = total concentration of ligand, F = total concentration of analyte, NS = non-specific binding and KD = equilibrium dissociation constant. The equation used to fit the two-site steady state binding model was:where B = sum of bound species for the two types of complex, Rmax1 and Rmax2 = total concentration to the two species of ligand, F = total concentration of analyte, KD1 and KD2 = equilibrium dissociation constants for the two types of bound complex and NS = non-specific binding.


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)

Binding of furamidine to a 495 response units surface of a stem-loop duplex oligonucleotide (A) and 504 response units surface of a single-stranded oligonucleotide (B). The equilibrium level of binding was determined for each furamidine concentration for the duplex oligonucleotide (C) or the single-stranded oligonucleotides (D). The graphs represent a fit using a two binding-site model for the stem-loop duplex oligonucleotide (C) or a single binding-site model for the single-stranded oligonucleotide (D). Twofold increments of furamidine concentration (0.097, 0.19, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5 and 25 µM) were used.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1935015&req=5

Figure 4: Binding of furamidine to a 495 response units surface of a stem-loop duplex oligonucleotide (A) and 504 response units surface of a single-stranded oligonucleotide (B). The equilibrium level of binding was determined for each furamidine concentration for the duplex oligonucleotide (C) or the single-stranded oligonucleotides (D). The graphs represent a fit using a two binding-site model for the stem-loop duplex oligonucleotide (C) or a single binding-site model for the single-stranded oligonucleotide (D). Twofold increments of furamidine concentration (0.097, 0.19, 0.39, 0.78, 1.56, 3.125, 6.25, 12.5 and 25 µM) were used.
Mentions: Binding experiments were performed on a Biacore 2000 instrument (Biacore Inc., Piscataway NJ, USA). 5′ biotinylated stem-loop (biotin-GATCTAAAAGACTTTCTCAAGTCTTTTAGATC) and single-stranded oligonucleotides (biotin-GATCTAAAAGACTT) were synthesized by IDT (Coralville, IA, USA). Stem-loop oligonucleotides were annealed by heating to 90°C for 5 min followed by snap cooling on ice for 15 min. Biotinylated oligonucleotides were immobilized to neutravidin-coated sensor chips as described previously (22). Approximately 5000 RU's of neutravidin was attached to all flow cells on the sensor chips. Oligonucleotides were reconstituted in buffer consisting of 10 mM Tris, pH 7.5, 300 mM NaCl and 1 mM EDTA. Single-stranded and stem-loop oligonucleotides were injected over flow cell 2 and 4, respectively until approximately 500 RU's of oligonucleotide were captured on the chip surface. Furamidine was diluted into running buffer [10 mM MES, 100 mM NaCl, 1 mM EDTA, 5% DMSO (v/v) pH 6.25] and injected over all flow cells at 20 µl/min at 25°C. Following compound injections, the surface was regenerated with a 10 s 1 M NaCl injection followed by a 10 s running buffer injection. A DMSO calibration curve was included to correct for refractive index mismatches between the running buffer and compound dilution series. Data was analyzed using the Scrubber software version 2 (David Myszka, University of Utah) and the equilibrium binding of furamidine was fit to either a single-site or two-site steady state binding model (Figure 4C and D). The equation used to fit the single-site steady state binding model was:where B = bound complex, Rmax = total concentration of ligand, F = total concentration of analyte, NS = non-specific binding and KD = equilibrium dissociation constant. The equation used to fit the two-site steady state binding model was:where B = sum of bound species for the two types of complex, Rmax1 and Rmax2 = total concentration to the two species of ligand, F = total concentration of analyte, KD1 and KD2 = equilibrium dissociation constants for the two types of bound complex and NS = non-specific binding.

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