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BioAssay: AID 651710

FRET-based HTS for detection of RAD52 Inhibitors Measured in Biochemical System Using Plate Reader - 7018-01_Inhibitor_SinglePoint_HTS_Activity_Set2

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(352070)
 
 
Active(1608)
 
 
Inactive(350548)
 
 
 Tested Substances
 Tested Substances
All(355884)
 
 
Active(1634)
 
 
Inactive(354250)
 
 
AID: 651710
Data Source: Broad Institute (7018-01_Inhibitor_SinglePoint_HTS_Activity_Set2)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-10-26

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 1608
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
652116FRET-based HTS for detection of RAD52 Inhibitors Measured in Biochemical System Using Plate Reader - 7018-01_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame 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) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in decreasing readout signal.

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v7.0.3):
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.
All well activities were then multiplied by -1 to create a positive activity readout value range, to match Pubchem convention.


PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.7.0.3) was applied.

PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized sample replicate activities, rounded to the nearest integer .
The minimum PUBCHEM_ACTIVITY_SCORE required for a compound to be called a hit (the activity threshold, or AT) was set at -50.

PERCENTAGE OF ACTIVE REPLICATES:
For each sample, the percentage of replicates (PCT_ACTIVE_REP) which had activity scores >= AT was determined.
The minimum percentage of replicates required for a compound to be called a hit (PAR_T) was set at 100.

PUBCHEM_ACTIVITY_OUTCOME:
Samples passing BOTH threshold criteria were assigned an outcome of 2 (active):
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP >= PAR_T

Samples passing NEITHER threshold criteria were assigned an outcome of 1 (inactive):
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP < PAR_T

Samples passing AT only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE >= AT, and PCT_ACTIVE_REP < PAR_T

Samples passing PAR_T only were assigned an outcome of 2 (active) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1REPRODUCIBILITY_COSINE_TRANSFORMA measure of how well the activity reproduced across the two samples. Computed as the absolute value of the cosine between the 'replicate vector' (ScoreA, ScoreB ---as well as ScoreC and/or ScoreD where applicable) and the vector (1, 1) representing perfect reproducibility. NULL will appear in this column if a sample was not run in duplicate or if the data produced by one of the replicates was Invalid# Float
2PCT_ACTIVE_REPLICATESThe percentage of replicates which pass the activity threshold.Float
3REPLICATE_A_ACTIVITY_SCORE_18.71uM_(%) (18.71μM**)The calculated activity for the indicated sampleFloat%

** 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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