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BioAssay: AID 504474

Gel-Based Assay for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1): Hit Validation

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. ..more
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 Tested Compounds
 Tested Compounds
All(3)
 
 
Active(2)
 
 
Inactive(1)
 
 
 Tested Substances
 Tested Substances
All(3)
 
 
Active(2)
 
 
Inactive(1)
 
 
AID: 504474
Data Source: NCGC (TDP1803)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-03-17

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 2
Related Experiments
AIDNameTypeComment
485290qHTS Assay for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1)Confirmatorydepositor-specified cross reference: qHTS Assay for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1)
485312Probe Development Summary for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1)Summarydepositor-specified cross reference: Probe Development Summary for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1)
489007Confirmation Assay for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1)Confirmatorydepositor-specified cross reference: Confirmation Assay for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1)
492989Gel-Based Assay for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1)Confirmatorysame project related to Summary assay
492990Gel-Based Assay for Inhibitors of HIV Integrase: Counterscreen for TDP1Confirmatorysame project related to Summary assay
504464Gel-Based Assay for Inhibitors of HIV Integrase: Hit Validation Counterscreen for TDP1 InhibitorsConfirmatorysame project related to Summary assay
Description:
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
Protocol
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).
Comment
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.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3LogIC50The logarithm of the Potency from a fit of the data to the Hill equation (calculated based on Molar Units).Float
4Compound QCSource of compound QCString

* Activity Concentration.
Additional Information
Grant Number: MH089814-01

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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