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)
489007	Confirmation Assay for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1)	confirmatory	Confirmation Assay 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)

Assay Overview:

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). See below for assay protocol.

This is assay was used to validate select hits from the related primary AlphaScreen assay (AID: 485290)

NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
Assay Provider: Yves Pommier and Christophe Marchand, National Cancer Institute

5'-32P-labeled DNA substrate (1 nmol/L; N14Y; 5'-GATCTAAAAGACTT-pY-3') was incubated with 0.2 nmol/L recombinant Tdp1 in the absence or presence of inhibitor for 15 min at room temperature in an assay buffer containing 1X PBS, pH 7.4, 80 mmol/L KCl and 0.01% Tween-20. Reactions were terminated by the addition of 1 volume of gel loading buffer [99.5% (v/v) formamide, 5 mmol/L EDTA, 0.01% (w/v) xylene cyanol, and 0.01% (w/v) bromophenol blue]. Samples were subjected to a 16% denaturing PAGE and gels were exposed after drying to a PhosphorImager screen (GE Healthcare). Gel images were scanned using a Typhoon 8600 (GE Healthcare) and densitometric analyses were performed using the ImageQuant software (GE Healthcare).

Compounds that showed significant inhibition at any tested concentration were considered active. Compounds that gave basal response were considered as inactive. PUBCHEM_ACTIVITY_SCORE was 0 for all inactive compounds. For Active compounds, -10*LogIC50 was used (rounded to closest whole number) as the activity score.

Grant Number: MH089814-01