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BioAssay: AID 743297

Epi Absorbance-based biochemical high throughput dose response assay to identify inhibitors of human tyrosyl-DNA phosphodiesterase 2 (TDP2)

Name: Epi Absorbance-based biochemical high throughput dose response assay to identify inhibitors of human tyrosyl-DNA phosphodiesterase 2 (TDP2). ..more
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 Tested Compounds
 Tested Compounds
All(221)
 
 
Active(59)
 
 
Inactive(162)
 
 
 Tested Substances
 Tested Substances
All(222)
 
 
Active(59)
 
 
Inactive(163)
 
 
AID: 743297
Data Source: The Scripps Research Institute Molecular Screening Center (TDP2_INH_EPIABS_1536_3XIC50 DRUN)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2014-03-03

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 59
Related Experiments
AIDNameTypeComment
720702Epi Absorbance-based biochemical primary high throughput screening assay to identify inhibitors of human tyrosyl-DNA phosphodiesterase 2 (TDP2)Screeningdepositor-specified cross reference: Primary Asasy (TDP2 INH in singlicate)
720732Summary of the probe development effort to identify inhibitors of human tyrosyl-DNA phosphodiesterase 2 (TDP2)Summarydepositor-specified cross reference: Summary
1083212On Hold
743263Epi Absorbance-based biochemical high throughput confirmation assay to identify inhibitors of human tyrosyl-DNA phosphodiesterase 2 (TDP2)Screeningsame project related to Summary assay
Description:
Source (MLPCN Center Name): The Scripps Research Institute Molecular Screening Center (SRIMSC)
Center Affiliation: The Scripps Research Institute (TSRI)
Assay Provider: Sanjay Adhikari, Georgetown University
Network: Molecular Libraries Probe Production Centers Network (MLPCN)
Grant Proposal Number: 1 R03 DA035193-01A1
Grant Proposal PI: Sanjay Adhikari
External Assay ID: TDP2_INH_EPIABS_1536_3XIC50 DRUN

Name: Epi Absorbance-based biochemical high throughput dose response assay to identify inhibitors of human tyrosyl-DNA phosphodiesterase 2 (TDP2).

Description:

The TopoisomeraseII (TopII) family is an important class of topoisomerases that produces transient 5'DNA-protein crosslinks [1, 2]. Drugs targeting these enzymes act by stabilizing the crosslinks, producing a covalent DNA-protein complex [3, 4]. Despite their clinical efficacy, drugs that target TopII (TopII poisons) may lead to negative side-effects, including secondary malignancies. The repair of TopII-DNA complexes is poorly understood. Recently, a human 5?- tyrosine phosphodiesterase (hTDP2) has been identified for the excision of TopII-DNA adducts [5, 6]. As hTDP2 repairs the cytotoxic adducts produced by TopII poisons, it is likely that effective inhibition of hTDP2 can be targeted to improve the efficacy of TopII poisons and reduce the side-effects. It has been shown that the knockdown/knockout of TDP2 activity in A549 (non-small cell lung cancer cell line, NSCLC) and DT40 cells increased sensitivity to the TopII targeting agent etoposide, demonstrating that hTDP2 can be a target for adjuvant chemotherapy [7]. Our hypothesis is effective inhibition of hTDP2 will increase the cancer cell killing efficacy of TopII poisons like etoposide [8]. Therefore, hTDP2 inhibitor screening, especially in a high-throughput manner, will expedite drug discovery.

References:

1. Corbett, K.D. and J.M. Berger, Structure, molecular mechanisms, and evolutionary relationships in DNA topoisomerases. Annu Rev Biophys Biomol Struct, 2004. 33: p. 95-118. PMID 15139806
2. Wang, J.C., Moving one DNA double helix through another by a type II DNA topoisomerase: the story of a simple molecular machine. Q Rev Biophys, 1998. 31(2): p. 107-44. PMID 9794033.
3. Cutts, S.M., et al., The power and potential of doxorubicin-DNA adducts. IUBMB Life, 2005. 57(2): p. 73-81. PMID 16036566
4. Habermeyer, M., et al., Anthocyanidins modulate the activity of human DNA topoisomerases I and II and affect cellular DNA integrity. Chem Res Toxicol, 2005. 18(9): p. 1395-404. PMID 16167831
5. Cortes Ledesma, F., et al., A human 5'-tyrosyl DNA phosphodiesterase that repairs topoisomerase-mediated DNA damage. Nature, 2009. 461(7264): p. 674-8. PMID 19794497.
6. Bahmed, K., K.C. Nitiss, and J.L. Nitiss, UnTTrapping the ends: a new player in overcoming protein linked DNA damage. Cell Res, 2010. 20(2): p. 122-3. PMID 20118967.
7. Zeng, Z., et al., TDP2/TTRAP is the major 5'-tyrosyl DNA phosphodiesterase activity in vertebrate cells and is critical for cellular resistance to topoisomerase II-induced DNA damage. J Biol Chem, 2011. 286(1): p. 403-9. PMID 21030584.
8. Bandele, O.J., S.J. Clawson, and N. Osheroff, Dietary polyphenols as topoisomerase II poisons: B ring and C ring substituents determine the mechanism of enzyme-mediated DNA cleavage enhancement. Chem Res Toxicol, 2008. 21(6): p. 1253-60. PMID 18461976.
9. Adachi, N., et al., Hypersensitivity of nonhomologous DNA end-joining mutants to VP-16 and ICRF-193: implications for the repair of topoisomerase II-mediated DNA damage. J Biol Chem, 2003. 278(38): p. 35897-902. PMID 12842886.

Keywords:

DRUN, Dose Response, Triplicate, end-point, endpoint, TDP2, hTDP2, tyrosyl-DNA phosphodiesterase 2, PRUN, primary, singlicate, biochemical, inhibit, inhibitor, inhibition, inh, p-nitrophenol, nitrophenol, hydrolysis, T5PNP, PNP, p-nitrophenyl-thymidine-5'-phosphate, cancer, DNA repair, excise, topoisomerase, phosphodiesterase, substrate, absorbance, colorimetric, chromogenic, HTS, high throughput screen, 1536, Scripps, Scripps Florida, MLSMR, The Scripps Research Institute
Protocol
Assay Overview:
The purpose of this assay is to determine dose response curves for compounds that confirmed tyrosyl DNA phosphodiesterase 2 (TDP2) inhibitory activity in a set of previous experiments entitled, "Epi Absorbance-based biochemical primary high throughput screening assay to identify inhibitors of human tyrosyl-DNA phosphodiesterase 2 (TDP2)" (AID 720702, 743463). TDP2 is a divalent cation-dependent enzyme that repairs TopII-associated DNA strand breaks. It is hypothesized that inhibitors of TDP2 may serve as useful adjuvants in combination with cancer drugs such as etoposide.
In this biochemical assay, recombinant human TDP2 protein is incubated at 37 degrees Celsius with the T5PNP substrate in the presence of Mg2+-containing assay buffer.T5PNP is a substrate for snake venom phosphodiesterase, as well as a substrate for TDP2. As a substrate for TDP2, T5PNP is used to mimic the TopII-DNA complex. TDP2 cleaves the phosphodiester bond in T5PNP, and the chromogenic p-nitrophenol group is released. As time increases the TDP2 enzyme will increasingly catalyze hydrolysis of the T5PNP substrate, resulting in increased release of p-nitrophenol and detection at 415nM wavelength. Compounds are tested in triplicate using a 10-point 1:3 dilution series starting at a maximum nomimal test concentration of 128 uM.
Protocol Summary:
Prior to the start of the assay, 2 ul of a solution containing T5PNP subtrate (final concentration 5mM) in assay buffer (50mM Tris-HCl pH7.5, 1mM DTT, 1mM MgCl2, 50mM KCl and 100ug/ml BSA) was dispended into a all wells of a 1536 well plate. Next, 39nL of test compound in DMSO or DMSO alone (1% final concentration) was added to the appropriate wells. The assay was started by dispensing 1 ul of a solution contanting TDP2 enzyme (120nM final concentration) in assay buffer to wells in columns 4-48 and 1ul of assay buffer alone to wells in columnes 1-3. Plates were centrifuged and incubated for 2hrs at 37 degrees Celsius at which time fluorescence intensity was measured (Ex. 405nm and Em. 405nm) using a EnVision microplate reader (Perkin Elmer).
Prior to further calculations, the following formula was used to calculate Epi Absorbance (EPIABS):
EPIABS = -log10( sample / background)
Where:
Sample is defined as the fluorescent intensity of wells containing test compounds or DMSO.
Background is defined as the fluorescent intensity of wells containing buffer and T5PNP only.
The % inhibition for each well was then calculated as follows:
%_Inhibition = ( EPIABS_Test_Compound - MedianEPIABS_Low_Control ) / ( MedianEPIABS_High_Control - MedianEPIABS_Low_Control ) * 100
Where:
Low_Control is defined as wells containing DMSO, TDP2 and T5PNP.
Test_Compound is defined as wells containing test compound, TDP2 and T5PNP.
High_Control is defined as wells containing only Buffer and T5PNP.
For each test compound, percent inhibition was plotted against compound concentration. A four parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline using Assay Explorer software (Accelrys Inc). The reported IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% activation level of the Y-intercept value. In cases where the highest concentration tested (i.e. 128 uM) did not result in greater than 50% activation, the IC50 was determined manually as greater than 128 uM.
PubChem Activity Outcome and Score:
Compounds with an IC50 greater than 10 uM were considered inactive. Compounds with an IC50 equal to or less than 10 uM were considered active.
The PubChem Activity Score range for active compounds is 100-1, and for inactive compounds 0-0.
List of Reagents:
TDP2 (supplied by Assay Provider)
Tris Base(Sigma, 93349)
DTT (Sigma, 43815)
BSA (Sigma, A2153)
MgCl2 (Sigma,M2670)
KCl (Sigma, P9333)
T5PNP (Sigma, T4510)
1536 well plates (Corning 7254)
Comment
Due to the increasing size of the MLPCN compound library, this assay may have been run as two or more separate campaigns, each campaign testing a unique set of compounds. In this case the results of each separate campaign were assigned "Active/Inactive" status based upon that campaign's specific compound activity cutoff value. All data reported were normalized on a per-plate basis. Possible artifacts of this assay can include, but are not limited to: dust or lint located in or on wells of the microtiter plate, compounds that modulate well fluorescence. All test compound concentrations reported above and below are nominal; the specific test concentration(s) for a particular compound may vary based upon the actual sample provided by the MLSMR. The MLSMR was not able to provide all compounds selected for testing in this assay.
Categorized Comment - additional comments and annotations
From BioAssay Depositor:
Assay: CurveFit [1]: Equation: =( ( [Maximal Response] * [Concentration]^[Hill Slope] ) / ( [Inflection Point Concentration]^[Hill Slope] + [Concentration]^[Hill Slope] ) ) + [Baseline Response]
Assay: CurveFit [1]: Mask: Excluded Points
Assay: Dictionary: Version: 0.1
Comment Tag: Comment Value
From PubChem:
Assay Format: Biochemical
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Qualifierinhibition Qualifier identifies if the resultant data IC50 came from a fitted curve or was determined manually to be less than or greater than its listed IC50 concentration.String
2IC50*The concentration at which 50 percent of the inhibition in the inhibitor assay is observed; (IC50) shown in micromolar.FloatμM
3LogIC50Log10 of the qualified IC50 (IC50) from the inhibitor assay in M concentrationFloat
4Maximal ResponseThe maximal or asymptotic response above the baseline as concentration increases without bound.Float
5Baseline ResponseAdjustable baseline of the curve fit, minimal response value.Float
6Inflection Point ConcentrationThe concentration value for the inflection point of the curve.FloatμM
7Chi SquareA measure for the 'goodness' of a fit. The chi-square test (Snedecor and Cochran, 1989) is used to test if a sample of data came from a population with a specific distribution.Float
8RsquareThis statistic measures how successful the fit explains the variation of the data; R-square is the square of the correlation between the response values and the predicted response values.Float
9Number of DataPointsOverall number of data points of normalized percent inhibition that was used for calculations (includes all concentration points); in some cases a data point can be excluded as outlier.Integer
10Hill SlopeThe variable HillSlope describes the steepness of the curve. This variable is called the Hill slope, the slope factor, or the Hill coefficient. If it is positive, the curve increases as X increases. If it is negative, the curve decreases as X increases. A standard sigmoid dose-response curve (previous equation) has a Hill Slope of 1.0. When HillSlope is less than 1.0, the curve is more shallow. When HillSlope is greater than 1.0, the curve is steeper. The Hill slope has no units.Float
11Response RangeThe range of Y.Float
12Excluded PointsFlags to indicate which of the dose-response points were excluded from analysis. (1) means the point was excluded and (0) means the point was not excluded.String
13Inhibition at 0.0065 uM [1] (0.0065μM**)Value of % inhibition at 0.0065 uM compound concentration; replicate [1]Float%
14Inhibition at 0.0065 uM [2] (0.0065μM**)Value of % inhibition at 0.0065 uM compound concentration; replicate [2]Float%
15Inhibition at 0.0065 uM [3] (0.0065μM**)Value of % inhibition at 0.0065 uM compound concentration; replicate [3]Float%
16Inhibition at 0.02 uM [1] (0.02μM**)Value of % inhibition at 0.02 uM compound concentration; replicate [1]Float%
17Inhibition at 0.02 uM [2] (0.02μM**)Value of % inhibition at 0.02 uM compound concentration; replicate [2]Float%
18Inhibition at 0.02 uM [3] (0.02μM**)Value of % inhibition at 0.02 uM compound concentration; replicate [3]Float%
19Inhibition at 0.059 uM [1] (0.059μM**)Value of % inhibition at 0.059 uM compound concentration; replicate [1]Float%
20Inhibition at 0.059 uM [2] (0.059μM**)Value of % inhibition at 0.059 uM compound concentration; replicate [2]Float%
21Inhibition at 0.059 uM [3] (0.059μM**)Value of % inhibition at 0.059 uM compound concentration; replicate [3]Float%
22Inhibition at 0.18 uM [1] (0.18μM**)Value of % inhibition at 0.18 uM compound concentration; replicate [1]Float%
23Inhibition at 0.18 uM [2] (0.18μM**)Value of % inhibition at 0.18 uM compound concentration; replicate [2]Float%
24Inhibition at 0.18 uM [3] (0.18μM**)Value of % inhibition at 0.18 uM compound concentration; replicate [3]Float%
25Inhibition at 0.53 uM [1] (0.53μM**)Value of % inhibition at 0.53 uM compound concentration; replicate [1]Float%
26Inhibition at 0.53 uM [2] (0.53μM**)Value of % inhibition at 0.53 uM compound concentration; replicate [2]Float%
27Inhibition at 0.53 uM [3] (0.53μM**)Value of % inhibition at 0.53 uM compound concentration; replicate [3]Float%
28Inhibition at 1.6 uM [1] (1.6μM**)Value of % inhibition at 1.6 uM compound concentration; replicate [1]Float%
29Inhibition at 1.6 uM [2] (1.6μM**)Value of % inhibition at 1.6 uM compound concentration; replicate [2]Float%
30Inhibition at 1.6 uM [3] (1.6μM**)Value of % inhibition at 1.6 uM compound concentration; replicate [3]Float%
31Inhibition at 4.7 uM [1] (4.7μM**)Value of % inhibition at 4.7 uM compound concentration; replicate [1]Float%
32Inhibition at 4.7 uM [2] (4.7μM**)Value of % inhibition at 4.7 uM compound concentration; replicate [2]Float%
33Inhibition at 4.7 uM [3] (4.7μM**)Value of % inhibition at 4.7 uM compound concentration; replicate [3]Float%
34Inhibition at 14.2 uM [1] (14.2μM**)Value of % inhibition at 14.2 uM compound concentration; replicate [1]Float%
35Inhibition at 14.2 uM [2] (14.2μM**)Value of % inhibition at 14.2 uM compound concentration; replicate [2]Float%
36Inhibition at 14.2 uM [3] (14.2μM**)Value of % inhibition at 14.2 uM compound concentration; replicate [3]Float%
37Inhibition at 42.7 uM [1] (42.7μM**)Value of % inhibition at 42.7 uM compound concentration; replicate [1]Float%
38Inhibition at 42.7 uM [2] (42.7μM**)Value of % inhibition at 42.7 uM compound concentration; replicate [2]Float%
39Inhibition at 42.7 uM [3] (42.7μM**)Value of % inhibition at 42.7 uM compound concentration; replicate [3]Float%
40Inhibition at 128 uM [1] (128μM**)Value of % inhibition at 128 uM compound concentration; replicate [1]Float%
41Inhibition at 128 uM [2] (128μM**)Value of % inhibition at 128 uM compound concentration; replicate [1]Float%
42Inhibition at 128 uM [3] (128μM**)Value of % inhibition at 128 uM compound concentration; replicate [3]Float%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1 R03 DA035193-01A1

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