AID	Name	Type	Comment
485312	Probe Development Summary for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1)	summary	Probe Development Summary for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1)
485290	qHTS Assay for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1)	confirmatory	qHTS Assay for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1)
504474	Gel-Based Assay for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1): Hit Validation	confirmatory
504464	Gel-Based Assay for Inhibitors of HIV Integrase: Hit Validation Counterscreen for TDP1 Inhibitors	confirmatory
492990	Gel-Based Assay for Inhibitors of HIV Integrase: Counterscreen for TDP1	confirmatory
492989	Gel-Based Assay for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1)	confirmatory

Human tyrosyl-DNA phosphodiesterase I (Tdp1) is a rational anticancer target because this enzyme is involved in the repair of DNA lesions created by the trapping of human DNA topoisomerase I (Top1) following treatment by anticancer agents such as camptothecins. Recombinant Tdp1 was expressed and purified from E. coli in the laboratory (Anthony et al, Nucleic Acids Research, 2007, Vol. 35, 4474-4484). A Tdp1 DNA substrate bearing a 5' biotin and a 3' phospho-tyrosine was obtained by custom synthesis. This oligonucleotidic DNA substrate was subsequently coupled to a fluorescein isothiocyanate (FITC) group via the free amino group present on the phospho-tyrosine. Tdp1 was screened against the NCGC small molecule library using the AlphaScreen (AS) detection method in which a streptavidin donor AS bead is bound at the 5' end and an anti-FITC AS acceptor bead is bound at the 3' end of the DNA oligonucleotidic substrate. In the presence of Tdp1, the enzyme cleaves the tyrosine-FITC group, leaving a 3' end phosphate on the DNA substrate. This cleavage reaction results in a loss of signal in AS.

This BioAssay is a confirmation of followup compounds from primary TDP1 screen (AID:485290)

NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
Assay Provider: Yves Pommier and Christophe Marchand, National Cancer Institute
Assay Title: qHTS Assay for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1)

Buffer: 1X PBS, pH 7.4, 80 mM KCl, 0.05% Tween-20

Reagents/Controls:
[1] 3 ul of buffer is dispensed in columns 3 & 4 as negative control (no enzyme and maximum signal),
[2] 3 ul of enzyme (1 nM final) is dispensed in columns 1,2, 5-48,
[3] 1 ul of substrate (15 nM final) is dispensed throughout the plate,
[4] 1 ul of AS donor/acceptor bead mix (10 ug/ml final) is dispensed throughout the plate

Assay steps:
3 ul of Tdp1 enzyme is dispensed to 1536-well Kalypsys black solid bottom plates. Compounds and controls (23 nl) are transferred via Kalypsys Pin Tool. The plates are incubated for 15 min at room temperature, and then 1 ul of substrate is added to start the reaction. After 5 min incubation at room temperature, 1 ul of AS donor/acceptor bead mix is added and the plates are further incubated for 10 min at room temperature. The detection is then performed on a PerkinElmer Envision reader.

Compound Ranking:

1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.

Grant Number: MH089814-01