Limits...
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.

Show MeSH

Related in: MedlinePlus

High-throughput electrochemiluminescene assay developed to identify novel Tdp1 inhibitors. (A) Generation of the electrochemiluminescent (ECL) substrate (BV-14Y). The ruthenium-containing tag (NHS ester BV-Tag; from BioVeris Corp.) is coupled to the 3′-end of the tyrosyl-containing DNA substrate [14Y (sequence as in 3A) linked to a biotin at its 5′ end]. After coupling, the BV Tag is attached to the phosphotyrosine of the 14Y DNA forming the BV-14Y DNA after the release of a succinimide group. (B) Processing of the ECL substrate by Tdp1. The ECL substrate BV-14Y bound to magnetic beads was generated as described in the Materials and Methods section. Upon addition of Tdp1, the tyrosine-BV-Tag group is hydrolyzed away leaving behind a 3′ phosphate on the DNA bound to the beads. After stopping the reactions, the samples are analyzed by the ECL analyzer. During the analysis, only the magnetic beads are retained in the analysis chamber by magnetic field while the rest of the sample is washed away. In the absence of Tdp1, the ECL signal is maximum as the BV-14Y DNA is retained on the magnetic beads. Upon addition of Tdp1, the tyrosine-BV-Tag group is cleaved away resulting in the loss of ECL signal (arrow). Potential Tdp1 inhibitors prevent this loss of signal. (C) Signal response curve in the presence of increasing concentrations of Tdp1. The ECL signal is lost when the Tdp1 concentration is increased.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1935015&req=5

Figure 1: High-throughput electrochemiluminescene assay developed to identify novel Tdp1 inhibitors. (A) Generation of the electrochemiluminescent (ECL) substrate (BV-14Y). The ruthenium-containing tag (NHS ester BV-Tag; from BioVeris Corp.) is coupled to the 3′-end of the tyrosyl-containing DNA substrate [14Y (sequence as in 3A) linked to a biotin at its 5′ end]. After coupling, the BV Tag is attached to the phosphotyrosine of the 14Y DNA forming the BV-14Y DNA after the release of a succinimide group. (B) Processing of the ECL substrate by Tdp1. The ECL substrate BV-14Y bound to magnetic beads was generated as described in the Materials and Methods section. Upon addition of Tdp1, the tyrosine-BV-Tag group is hydrolyzed away leaving behind a 3′ phosphate on the DNA bound to the beads. After stopping the reactions, the samples are analyzed by the ECL analyzer. During the analysis, only the magnetic beads are retained in the analysis chamber by magnetic field while the rest of the sample is washed away. In the absence of Tdp1, the ECL signal is maximum as the BV-14Y DNA is retained on the magnetic beads. Upon addition of Tdp1, the tyrosine-BV-Tag group is cleaved away resulting in the loss of ECL signal (arrow). Potential Tdp1 inhibitors prevent this loss of signal. (C) Signal response curve in the presence of increasing concentrations of Tdp1. The ECL signal is lost when the Tdp1 concentration is increased.

Mentions: To discover inhibitors of Tdp1, we developed a novel ECL high-throughput assay (for details see Materials and Methods section and Figure 1). An ECL substrate for Tdp1 was generated after coupling a ruthenium-containing tag (BV-Tag) to the 3′-end of a 14-mer oligonucleotide with a 5′-biotin and a 3′-tyrosyl moiety (Figure 1A) (3,7). In the presence of Tdp1, the tyrosyl-bound BV-Tag is hydrolyzed from the ECL substrate (BV-14Y DNA) and washed away, leading to a loss of the electroluminescent signal (Figure 1B and C). In this assay, a potential Tdp1 inhibitor would be detected as preventing this loss of signal with a level of signal retention reflective of the potency of the putative inhibitor. Thus, in our high-throughput ECL assay, active compounds were identified as restoring to the control levels (without Tdp1) the signal lost in the presence of Tdp1.Figure 1.


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)

High-throughput electrochemiluminescene assay developed to identify novel Tdp1 inhibitors. (A) Generation of the electrochemiluminescent (ECL) substrate (BV-14Y). The ruthenium-containing tag (NHS ester BV-Tag; from BioVeris Corp.) is coupled to the 3′-end of the tyrosyl-containing DNA substrate [14Y (sequence as in 3A) linked to a biotin at its 5′ end]. After coupling, the BV Tag is attached to the phosphotyrosine of the 14Y DNA forming the BV-14Y DNA after the release of a succinimide group. (B) Processing of the ECL substrate by Tdp1. The ECL substrate BV-14Y bound to magnetic beads was generated as described in the Materials and Methods section. Upon addition of Tdp1, the tyrosine-BV-Tag group is hydrolyzed away leaving behind a 3′ phosphate on the DNA bound to the beads. After stopping the reactions, the samples are analyzed by the ECL analyzer. During the analysis, only the magnetic beads are retained in the analysis chamber by magnetic field while the rest of the sample is washed away. In the absence of Tdp1, the ECL signal is maximum as the BV-14Y DNA is retained on the magnetic beads. Upon addition of Tdp1, the tyrosine-BV-Tag group is cleaved away resulting in the loss of ECL signal (arrow). Potential Tdp1 inhibitors prevent this loss of signal. (C) Signal response curve in the presence of increasing concentrations of Tdp1. The ECL signal is lost when the Tdp1 concentration is increased.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: High-throughput electrochemiluminescene assay developed to identify novel Tdp1 inhibitors. (A) Generation of the electrochemiluminescent (ECL) substrate (BV-14Y). The ruthenium-containing tag (NHS ester BV-Tag; from BioVeris Corp.) is coupled to the 3′-end of the tyrosyl-containing DNA substrate [14Y (sequence as in 3A) linked to a biotin at its 5′ end]. After coupling, the BV Tag is attached to the phosphotyrosine of the 14Y DNA forming the BV-14Y DNA after the release of a succinimide group. (B) Processing of the ECL substrate by Tdp1. The ECL substrate BV-14Y bound to magnetic beads was generated as described in the Materials and Methods section. Upon addition of Tdp1, the tyrosine-BV-Tag group is hydrolyzed away leaving behind a 3′ phosphate on the DNA bound to the beads. After stopping the reactions, the samples are analyzed by the ECL analyzer. During the analysis, only the magnetic beads are retained in the analysis chamber by magnetic field while the rest of the sample is washed away. In the absence of Tdp1, the ECL signal is maximum as the BV-14Y DNA is retained on the magnetic beads. Upon addition of Tdp1, the tyrosine-BV-Tag group is cleaved away resulting in the loss of ECL signal (arrow). Potential Tdp1 inhibitors prevent this loss of signal. (C) Signal response curve in the presence of increasing concentrations of Tdp1. The ECL signal is lost when the Tdp1 concentration is increased.
Mentions: To discover inhibitors of Tdp1, we developed a novel ECL high-throughput assay (for details see Materials and Methods section and Figure 1). An ECL substrate for Tdp1 was generated after coupling a ruthenium-containing tag (BV-Tag) to the 3′-end of a 14-mer oligonucleotide with a 5′-biotin and a 3′-tyrosyl moiety (Figure 1A) (3,7). In the presence of Tdp1, the tyrosyl-bound BV-Tag is hydrolyzed from the ECL substrate (BV-14Y DNA) and washed away, leading to a loss of the electroluminescent signal (Figure 1B and C). In this assay, a potential Tdp1 inhibitor would be detected as preventing this loss of signal with a level of signal retention reflective of the potency of the putative inhibitor. Thus, in our high-throughput ECL assay, active compounds were identified as restoring to the control levels (without Tdp1) the signal lost in the presence of Tdp1.Figure 1.

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