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BioAssay: AID 652116

FRET-based HTS for detection of RAD52 Inhibitors Measured in Biochemical System Using Plate Reader - 7018-01_Inhibitor_Dose_CherryPick_Activity

Assay Overview: The primary assay is a prompt fluorescent gain of signal biochemical assay based on a quenched substrate. Two DNA substrates are provided to recombinant purified His tagged RAD52; a quenched dual labeled double stranded DNA with a single strand tail and a homologous unlabeled single strand. The labels are Black Hole Quencher (BHQ) and Fluorescein (FL) which are close together more ..
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 Tested Compounds
 Tested Compounds
All(996)
 
 
Active(636)
 
 
Inactive(180)
 
 
Inconclusive(180)
 
 
 Tested Substances
 Tested Substances
All(996)
 
 
Active(636)
 
 
Inactive(180)
 
 
Inconclusive(180)
 
 
AID: 652116
Data Source: Broad Institute (7018-01_Inhibitor_Dose_CherryPick_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-03-15

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 636
Related Experiments
AIDNameTypeComment
651668Broad Institute Development of RAD52 inhibitors to induce synthetic lethality of BRCA2-deficient Inhibitor Probe ProjectSummarydepositor-specified cross reference: Summary assay
651660FRET-based HTS for detection of RAD52 Inhibitors Measured in Biochemical System Using Plate Reader - 7018-01_Inhibitor_SinglePoint_HTS_ActivityScreeningsame project related to Summary assay
651710FRET-based HTS for detection of RAD52 Inhibitors Measured in Biochemical System Using Plate Reader - 7018-01_Inhibitor_SinglePoint_HTS_Activity_Set2Screeningsame project related to Summary assay
652128RAD52: DNA binders Measured in Biochemical System Using Plate Reader - 7018-02_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
743275Effect of RAD 52 Inhibitors on Growth of CAPAN-1 Cells Measured in Cell-Based System Using Imaging - 7018-03_Inhibitor_Dose_DryPowder_Activity_Set2Confirmatorysame project related to Summary assay
743276Effect of RAD 52 Inhibitors on Growth of CAPAN-1 Cells Measured in Cell-Based System Using Imaging - 7018-03_Inhibitor_Dose_DryPowder_Activity_Set3Confirmatorysame project related to Summary assay
Description:
Keywords: Homologous Recombination, DNA repair, RAD52

Assay Overview: The primary assay is a prompt fluorescent gain of signal biochemical assay based on a quenched substrate. Two DNA substrates are provided to recombinant purified His tagged RAD52; a quenched dual labeled double stranded DNA with a single strand tail and a homologous unlabeled single strand. The labels are Black Hole Quencher (BHQ) and Fluorescein (FL) which are close together on the double stranded DNA. The enzyme catalyzes annealing of the unlabeled single strand to the FL labeled strand with concomitant displacement of the BHQ labeled strand. Once displaced, the distance between FL and BHQ increases which causes an increase in FL fluorescence.

Expected Outcome: RAD52 inhibitors will cause decreased fluorescence.
Protocol
Assay Buffer = 25 mM Tris Acetate pH 7.5, 100 ug/mL BSA, 1 mM DTT, 0.01% Pluronic F-68
Enzyme Solution = 50 nM RAD52 in Assay Buffer
Substrate Solution = 15 nM 265-55 single stranded DNA 15 nM 337F/1337BHQ double stranded DNA (oligoes provided by Mazin lab)
2 uL Enzyme Solution + 2 uL Substrate Solution are added to a Greiner 1536 well black plate using a Beckman BioRaptor dispenser. No Enzyme control wells contain 2 uL Assay Buffer + 2 uL Substrate Solution. The plate contains predispensed DMSO solutions of the compounds to be screened. The reaction is allowed to proceed for 30 minutes followed by reading an endpoint flourescence (485 nm excitation 535 nm emission) measurement using a Perkin Elmer Envision plate reader.
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=128) and positive control wells (PC; n=128) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).
NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral control wells was set to a normalized activity value of 0.
The median raw signal of the intraplate positive control wells was set to a normalized activity value of -100.
Experimental wells values were scaled to this range.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.
PUBCHEM_ACTIVITY_SCORE:
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
DATA AGGREGATION:
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:
1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.
2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.
3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.
Note:
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Biochemical
From ChEMBL:
Assay Type: Functional
Result Definitions
Show more
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_Qualifier'>','=', or '<'String
2AC50_uM*The concentration at which activity reaches 50% of the maximumFloatμM
3pAC50_MEqual to -1*log10(AC50)String
4ACTIVITY_OUTCOME_TEST1The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
5ACTIVITY_SCORE_TEST1The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
6ASSAYDATA_COMMENT_TEST1Comments relevant to the subset of data included in the indicated Test number.String
7AC50_Qualifier_TEST1'>','=', or '<'String
8AC50_uM_TEST1The concentration at which activity reaches 50% of the maximumFloatμM
9pAC50_M_TEST1Equal to -1*log10(AC50)String
10Hill_Slope_TEST1The slope at AC50Float
11S0_(%)_TEST1The fitted activity value at zero concentrationFloat%
12Sinf_(%)_TEST1The fitted activity value at infinite concentrationFloat%
13Num_Points_TEST1The number of data points used to generate the plotInteger
14Max_Activity_(%)_TEST1The maximum activity value observed, based on mean of replicates per concentrationFloat%
15Max_Activity_Conc_uM_TEST1The concentration at which the maximum activity is observedFloatμM
16Max_Concentration_uM_TEST1Maximum valid test concentrationFloatμM
17Activity_at_2.35uM_(%)_TEST1 (2.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_4.6uM_(%)_TEST1 (4.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_6uM_(%)_TEST1 (6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_9uM_(%)_TEST1 (9μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_12uM_(%)_TEST1 (12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_18uM_(%)_TEST1 (18μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_19.5uM_(%)_TEST1 (19.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_23.5uM_(%)_TEST1 (23.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25ACTIVITY_OUTCOME_TEST2The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
26ACTIVITY_SCORE_TEST2The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
27ASSAYDATA_COMMENT_TEST2Comments relevant to the subset of data included in the indicated Test number.String
28AC50_Qualifier_TEST2'>','=', or '<'String
29AC50_uM_TEST2The concentration at which activity reaches 50% of the maximumFloatμM
30pAC50_M_TEST2Equal to -1*log10(AC50)String
31Hill_Slope_TEST2The slope at AC50Float
32S0_(%)_TEST2The fitted activity value at zero concentrationFloat%
33Sinf_(%)_TEST2The fitted activity value at infinite concentrationFloat%
34Num_Points_TEST2The number of data points used to generate the plotInteger
35Max_Activity_(%)_TEST2The maximum activity value observed, based on mean of replicates per concentrationFloat%
36Max_Activity_Conc_uM_TEST2The concentration at which the maximum activity is observedFloatμM
37Max_Concentration_uM_TEST2Maximum valid test concentrationFloatμM
38Activity_at_2.35uM_(%)_TEST2 (2.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
39Activity_at_4.6uM_(%)_TEST2 (4.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
40Activity_at_6uM_(%)_TEST2 (6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
41Activity_at_9uM_(%)_TEST2 (9μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
42Activity_at_12uM_(%)_TEST2 (12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
43Activity_at_18uM_(%)_TEST2 (18μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
44Activity_at_19.5uM_(%)_TEST2 (19.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
45Activity_at_23.5uM_(%)_TEST2 (23.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
46ACTIVITY_OUTCOME_TEST3The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
47ACTIVITY_SCORE_TEST3The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
48ASSAYDATA_COMMENT_TEST3Comments relevant to the subset of data included in the indicated Test number.String
49AC50_Qualifier_TEST3'>','=', or '<'String
50AC50_uM_TEST3The concentration at which activity reaches 50% of the maximumFloatμM
51pAC50_M_TEST3Equal to -1*log10(AC50)String
52Hill_Slope_TEST3The slope at AC50Float
53S0_(%)_TEST3The fitted activity value at zero concentrationFloat%
54Sinf_(%)_TEST3The fitted activity value at infinite concentrationFloat%
55Num_Points_TEST3The number of data points used to generate the plotInteger
56Max_Activity_(%)_TEST3The maximum activity value observed, based on mean of replicates per concentrationFloat%
57Max_Activity_Conc_uM_TEST3The concentration at which the maximum activity is observedFloatμM
58Max_Concentration_uM_TEST3Maximum valid test concentrationFloatμM
59Activity_at_0.15uM_(%)_TEST3 (0.15μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
60Activity_at_0.285uM_(%)_TEST3 (0.285μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
61Activity_at_0.3uM_(%)_TEST3 (0.3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
62Activity_at_0.6uM_(%)_TEST3 (0.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
63Activity_at_1.2uM_(%)_TEST3 (1.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
64Activity_at_2.35uM_(%)_TEST3 (2.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
65Activity_at_4.6uM_(%)_TEST3 (4.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
66Activity_at_9uM_(%)_TEST3 (9μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
67Activity_at_18uM_(%)_TEST3 (18μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
68Activity_at_19.5uM_(%)_TEST3 (19.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
69ACTIVITY_OUTCOME_TEST4The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
70ACTIVITY_SCORE_TEST4The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
71ASSAYDATA_COMMENT_TEST4Comments relevant to the subset of data included in the indicated Test number.String
72AC50_Qualifier_TEST4'>','=', or '<'String
73AC50_uM_TEST4The concentration at which activity reaches 50% of the maximumFloatμM
74pAC50_M_TEST4Equal to -1*log10(AC50)String
75Hill_Slope_TEST4The slope at AC50Float
76S0_(%)_TEST4The fitted activity value at zero concentrationFloat%
77Sinf_(%)_TEST4The fitted activity value at infinite concentrationFloat%
78Num_Points_TEST4The number of data points used to generate the plotInteger
79Max_Activity_(%)_TEST4The maximum activity value observed, based on mean of replicates per concentrationFloat%
80Max_Activity_Conc_uM_TEST4The concentration at which the maximum activity is observedFloatμM
81Max_Concentration_uM_TEST4Maximum valid test concentrationFloatμM
82Activity_at_0.15uM_(%)_TEST4 (0.15μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
83Activity_at_0.285uM_(%)_TEST4 (0.285μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
84Activity_at_0.3uM_(%)_TEST4 (0.3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
85Activity_at_0.6uM_(%)_TEST4 (0.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
86Activity_at_1.2uM_(%)_TEST4 (1.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
87Activity_at_2.35uM_(%)_TEST4 (2.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
88Activity_at_4.6uM_(%)_TEST4 (4.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
89Activity_at_9uM_(%)_TEST4 (9μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
90Activity_at_18uM_(%)_TEST4 (18μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
91Activity_at_19.5uM_(%)_TEST4 (19.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
92ACTIVITY_OUTCOME_TEST5The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
93ACTIVITY_SCORE_TEST5The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
94ASSAYDATA_COMMENT_TEST5Comments relevant to the subset of data included in the indicated Test number.String
95AC50_Qualifier_TEST5'>','=', or '<'String
96AC50_uM_TEST5The concentration at which activity reaches 50% of the maximumFloatμM
97pAC50_M_TEST5Equal to -1*log10(AC50)String
98Hill_Slope_TEST5The slope at AC50Float
99S0_(%)_TEST5The fitted activity value at zero concentrationFloat%
100Sinf_(%)_TEST5The fitted activity value at infinite concentrationFloat%
101Num_Points_TEST5The number of data points used to generate the plotInteger
102Max_Activity_(%)_TEST5The maximum activity value observed, based on mean of replicates per concentrationFloat%
103Max_Activity_Conc_uM_TEST5The concentration at which the maximum activity is observedFloatμM
104Max_Concentration_uM_TEST5Maximum valid test concentrationFloatμM
105Activity_at_0.15uM_(%)_TEST5 (0.15μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
106Activity_at_0.285uM_(%)_TEST5 (0.285μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
107Activity_at_0.3uM_(%)_TEST5 (0.3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
108Activity_at_0.6uM_(%)_TEST5 (0.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
109Activity_at_1.2uM_(%)_TEST5 (1.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
110Activity_at_2.35uM_(%)_TEST5 (2.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
111Activity_at_4.6uM_(%)_TEST5 (4.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
112Activity_at_9uM_(%)_TEST5 (9μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
113Activity_at_18uM_(%)_TEST5 (18μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
114Activity_at_19.5uM_(%)_TEST5 (19.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
115ACTIVITY_OUTCOME_TEST6The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
116ACTIVITY_SCORE_TEST6The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
117ASSAYDATA_COMMENT_TEST6Comments relevant to the subset of data included in the indicated Test number.String
118AC50_Qualifier_TEST6'>','=', or '<'String
119AC50_uM_TEST6The concentration at which activity reaches 50% of the maximumFloatμM
120pAC50_M_TEST6Equal to -1*log10(AC50)String
121Hill_Slope_TEST6The slope at AC50Float
122S0_(%)_TEST6The fitted activity value at zero concentrationFloat%
123Sinf_(%)_TEST6The fitted activity value at infinite concentrationFloat%
124Num_Points_TEST6The number of data points used to generate the plotInteger
125Max_Activity_(%)_TEST6The maximum activity value observed, based on mean of replicates per concentrationFloat%
126Max_Activity_Conc_uM_TEST6The concentration at which the maximum activity is observedFloatμM
127Max_Concentration_uM_TEST6Maximum valid test concentrationFloatμM
128Activity_at_0.15uM_(%)_TEST6 (0.15μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
129Activity_at_0.285uM_(%)_TEST6 (0.285μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
130Activity_at_0.3uM_(%)_TEST6 (0.3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
131Activity_at_0.6uM_(%)_TEST6 (0.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
132Activity_at_1.2uM_(%)_TEST6 (1.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
133Activity_at_2.35uM_(%)_TEST6 (2.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
134Activity_at_4.6uM_(%)_TEST6 (4.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
135Activity_at_9uM_(%)_TEST6 (9μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
136Activity_at_18uM_(%)_TEST6 (18μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
137Activity_at_19.5uM_(%)_TEST6 (19.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: 1R03MH0957512-01

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