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BioAssay: AID 485290

qHTS Assay for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1)

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, more ..
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 Tested Compounds
 Tested Compounds
All(344455)
 
 
Active(978)
 
 
Inactive(338207)
 
 
Inconclusive(5543)
 
 
 Tested Substances
 Tested Substances
All(352260)
 
 
Active(986)
 
 
Inactive(345663)
 
 
Inconclusive(5611)
 
 
AID: 485290
Data Source: NCGC (TDP1826)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-09-28

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 978
Depositor Specified Assays
AIDNameTypeComment
489007Confirmation Assay for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1)confirmatory
492990Gel-Based Assay for Inhibitors of HIV Integrase: Counterscreen for TDP1confirmatory
504474Gel-Based Assay for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1): Hit Validationconfirmatory
485312Probe Development Summary for Inhibitors of Tyrosyl-DNA Phosphodiesterase (TDP1)summary
504464Gel-Based Assay for Inhibitors of HIV Integrase: Hit Validation Counterscreen for TDP1 Inhibitorsconfirmatory
Description:
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)

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). 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.
Protocol
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.
Comment
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.
Result Definitions
Show more
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
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.0000657143 uM (6.57143e-05μM**)% Activity at given concentration.Float%
15Activity at 0.0001462857 uM (0.000146286μM**)% Activity at given concentration.Float%
16Activity at 0.0003270475 uM (0.000327048μM**)% Activity at given concentration.Float%
17Activity at 0.0007314286 uM (0.000731429μM**)% Activity at given concentration.Float%
18Activity at 0.00146 uM (0.00146286μM**)% Activity at given concentration.Float%
19Activity at 0.00163 uM (0.00163429μM**)% Activity at given concentration.Float%
20Activity at 0.00395 uM (0.00394793μM**)% Activity at given concentration.Float%
21Activity at 0.00648 uM (0.00648283μM**)% Activity at given concentration.Float%
22Activity at 0.016 uM (0.0157966μM**)% Activity at given concentration.Float%
23Activity at 0.021 uM (0.0213095μM**)% Activity at given concentration.Float%
24Activity at 0.039 uM (0.0391105μM**)% Activity at given concentration.Float%
25Activity at 0.094 uM (0.093537μM**)% Activity at given concentration.Float%
26Activity at 0.147 uM (0.146762μM**)% Activity at given concentration.Float%
27Activity at 0.290 uM (0.289616μM**)% Activity at given concentration.Float%
28Activity at 0.505 uM (0.504673μM**)% Activity at given concentration.Float%
29Activity at 0.902 uM (0.902363μM**)% Activity at given concentration.Float%
30Activity at 2.283 uM (2.28313μM**)% Activity at given concentration.Float%
31Activity at 3.337 uM (3.33656μM**)% Activity at given concentration.Float%
32Activity at 5.473 uM (5.47305μM**)% Activity at given concentration.Float%
33Activity at 12.29 uM (12.2875μM**)% Activity at given concentration.Float%
34Activity at 20.98 uM (20.9837μM**)% Activity at given concentration.Float%
35Activity at 56.06 uM (56.0605μM**)% Activity at given concentration.Float%
36Activity at 96.01 uM (96.0067μM**)% Activity at given concentration.Float%
37Activity at 129.9 uM (129.875μM**)% Activity at given concentration.Float%
38Activity at 234.2 uM (234.24μM**)% Activity at given concentration.Float%
39Activity at 373.1 uM (373.143μM**)% Activity at given concentration.Float%
40Compound QCSource of compound QCString

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

Data Table (Concise)
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