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BioAssay: AID 652128

RAD52: DNA binders Measured in Biochemical System Using Plate Reader - 7018-02_Inhibitor_Dose_CherryPick_Activity

The goal of this project is to develop specific small-molecule inhibitors of human RAD52, one of the key homologous recombination (HR) proteins. Previously we ran a primary screen and identified putative RAD52 inhibitors. To rule out DNA binding as an undesired mechanism of action, we tested the ability of these compounds to bind DNA in this assay. Basically, compounds in varied doses were more ..
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 Tested Compounds
 Tested Compounds
All(998)
 
 
Active(85)
 
 
Inactive(877)
 
 
Inconclusive(36)
 
 
 Tested Substances
 Tested Substances
All(998)
 
 
Active(85)
 
 
Inactive(877)
 
 
Inconclusive(36)
 
 
 Related BioAssays
 Related BioAssays
AID: 652128
Data Source: Broad Institute (7018-02_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 ):           Active    All
BioActive Compounds: 85
Depositor Specified Assays
AIDNameTypeComment
651668Broad Institute Development of RAD52 inhibitors to induce synthetic lethality of BRCA2-deficient Inhibitor Probe ProjectsummarySummary assay
Description:
Keywords:
Homologous Recombination, DNA repair, RAD52

Assay Overview:
The goal of this project is to develop specific small-molecule inhibitors of human RAD52, one of the key homologous recombination (HR) proteins. Previously we ran a primary screen and identified putative RAD52 inhibitors. To rule out DNA binding as an undesired mechanism of action, we tested the ability of these compounds to bind DNA in this assay. Basically, compounds in varied doses were pinned into reaction mix of acridine orange and DNA on 384-well plates, and the reactions were incubated at room temperature for 20 minutes before fluorescence polorization was read on EnVision (Perkin Elmer) at ex/em 480/535 S/P nm.

Expected Outcome:
DNA binding of test compounds will result in loss of signal.
Protocol
Dispense 30 uL reaction mix and positive control with combi and combi nL into specific wells on 384-well plates based on plate design.
Pin 100 nL/well of compounds.
Dispense 1.5 nL postive control (Mitoxantrone) with combi nL to get 10 uM final conc.
Incubate at room temperature for 20 minutes.

Read on Envision at 480/535 S and P nm with D505fp/D535 dichroic mirror.


Reaction mix: 6 ug/mL Salmon sperm DNA and 50nM acridine orange in HEN buffer (10 mM HEPES, pH 7.5; 1 mM EDTA pH 7.5; 100 mM NaCl)

Positive control: 10 uM of Mitoxantrone in reaction mix
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC; n=32) and positive control wells (PC; n=32) 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.
Result Definitions
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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_0.26uM_(%)_TEST1 (0.26μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_0.5uM_(%)_TEST1 (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_1uM_(%)_TEST1 (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_2.1uM_(%)_TEST1 (2.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_4.2uM_(%)_TEST1 (4.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_8uM_(%)_TEST1 (8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_16uM_(%)_TEST1 (16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_35uM_(%)_TEST1 (35μM**)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_10uM_(%)_TEST2 (10μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
39Activity_at_16uM_(%)_TEST2 (16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
40Activity_at_21uM_(%)_TEST2 (21μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
41Activity_at_35uM_(%)_TEST2 (35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
42Activity_at_42uM_(%)_TEST2 (42μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
43Activity_at_80uM_(%)_TEST2 (80μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
44ACTIVITY_OUTCOME_TEST3The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
45ACTIVITY_SCORE_TEST3The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
46ASSAYDATA_COMMENT_TEST3Comments relevant to the subset of data included in the indicated Test number.String
47AC50_Qualifier_TEST3'>','=', or '<'String
48AC50_uM_TEST3The concentration at which activity reaches 50% of the maximumFloatμM
49pAC50_M_TEST3Equal to -1*log10(AC50)String
50Hill_Slope_TEST3The slope at AC50Float
51S0_(%)_TEST3The fitted activity value at zero concentrationFloat%
52Sinf_(%)_TEST3The fitted activity value at infinite concentrationFloat%
53Num_Points_TEST3The number of data points used to generate the plotInteger
54Max_Activity_(%)_TEST3The maximum activity value observed, based on mean of replicates per concentrationFloat%
55Max_Activity_Conc_uM_TEST3The concentration at which the maximum activity is observedFloatμM
56Max_Concentration_uM_TEST3Maximum valid test concentrationFloatμM
57Activity_at_4.2uM_(%)_TEST3 (4.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
58Activity_at_8uM_(%)_TEST3 (8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
59Activity_at_10uM_(%)_TEST3 (10μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
60Activity_at_16uM_(%)_TEST3 (16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
61Activity_at_21uM_(%)_TEST3 (21μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
62Activity_at_35uM_(%)_TEST3 (35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
63Activity_at_80uM_(%)_TEST3 (80μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
64ACTIVITY_OUTCOME_TEST4The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
65ACTIVITY_SCORE_TEST4The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
66ASSAYDATA_COMMENT_TEST4Comments relevant to the subset of data included in the indicated Test number.String
67AC50_Qualifier_TEST4'>','=', or '<'String
68AC50_uM_TEST4The concentration at which activity reaches 50% of the maximumFloatμM
69pAC50_M_TEST4Equal to -1*log10(AC50)String
70Hill_Slope_TEST4The slope at AC50Float
71S0_(%)_TEST4The fitted activity value at zero concentrationFloat%
72Sinf_(%)_TEST4The fitted activity value at infinite concentrationFloat%
73Num_Points_TEST4The number of data points used to generate the plotInteger
74Max_Activity_(%)_TEST4The maximum activity value observed, based on mean of replicates per concentrationFloat%
75Max_Activity_Conc_uM_TEST4The concentration at which the maximum activity is observedFloatμM
76Max_Concentration_uM_TEST4Maximum valid test concentrationFloatμM
77Activity_at_0.26uM_(%)_TEST4 (0.26μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
78Activity_at_0.5uM_(%)_TEST4 (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
79Activity_at_1uM_(%)_TEST4 (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
80Activity_at_2.1uM_(%)_TEST4 (2.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
81Activity_at_4.2uM_(%)_TEST4 (4.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
82Activity_at_8uM_(%)_TEST4 (8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
83Activity_at_16uM_(%)_TEST4 (16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
84Activity_at_35uM_(%)_TEST4 (35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
85ACTIVITY_OUTCOME_TEST5The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
86ACTIVITY_SCORE_TEST5The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
87ASSAYDATA_COMMENT_TEST5Comments relevant to the subset of data included in the indicated Test number.String
88AC50_Qualifier_TEST5'>','=', or '<'String
89AC50_uM_TEST5The concentration at which activity reaches 50% of the maximumFloatμM
90pAC50_M_TEST5Equal to -1*log10(AC50)String
91Hill_Slope_TEST5The slope at AC50Float
92S0_(%)_TEST5The fitted activity value at zero concentrationFloat%
93Sinf_(%)_TEST5The fitted activity value at infinite concentrationFloat%
94Num_Points_TEST5The number of data points used to generate the plotInteger
95Max_Activity_(%)_TEST5The maximum activity value observed, based on mean of replicates per concentrationFloat%
96Max_Activity_Conc_uM_TEST5The concentration at which the maximum activity is observedFloatμM
97Max_Concentration_uM_TEST5Maximum valid test concentrationFloatμM
98Activity_at_0.26uM_(%)_TEST5 (0.26μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
99Activity_at_0.5uM_(%)_TEST5 (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
100Activity_at_1uM_(%)_TEST5 (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
101Activity_at_2.1uM_(%)_TEST5 (2.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
102Activity_at_4.2uM_(%)_TEST5 (4.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
103Activity_at_8uM_(%)_TEST5 (8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
104Activity_at_16uM_(%)_TEST5 (16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
105Activity_at_35uM_(%)_TEST5 (35μM**)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)
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