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BioAssay: AID 602329

Identification of inhibitors of RAD54 Measured in Biochemical System Using Plate Reader - 2159-01_Inhibitor_SinglePoint_HTS_Activity

The goal of this proposal is to develop specific small-molecule inhibitors of human RAD54, one of the key homologous recombination (HR) proteins. Basically, RAD54 and DNA that forms Holiday Junctions are added according to plate design with BioRAPTR (Beckman) to respective wells of 1536-well assay ready plates (Aurora 19180) that contain 2.5 nL/well of 10 mM compound. The reaction is incubated at room temperature for 40 minutes. Fluorescence is then read on Viewlux (Perkin Elmer) at ex/em 480/540 nm. ..more
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 Tested Compounds
 Tested Compounds
All(358496)
 
 
Active(505)
 
 
Inactive(357779)
 
 
Inconclusive(213)
 
 
 Tested Substances
 Tested Substances
All(361408)
 
 
Active(506)
 
 
Inactive(360689)
 
 
Inconclusive(213)
 
 
AID: 602329
Data Source: Broad Institute (2159-01_Inhibitor_SinglePoint_HTS_Activity)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-03-08
Modify Date: 2012-03-28

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 505
Related Experiments
AIDNameTypeComment
602352Broad Institute Identification of Inhibitors of RAD54 Probe ProjectSummarydepositor-specified cross reference
651651Identification of Rad54 Inhibitors - Ruling Out Flurescence Quenchers Measured in Biochemical System Using Plate Reader - 2159-03_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
651657Identification of Inhibitors of RAD54 Measured in Biochemical System Using Plate Reader - 2159-01_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
651673Identification of Rad54 Inhibitors - Ruling Out Compounds That Bind DNA Measured in Biochemical System Using Plate Reader - 2159-04_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
651692Cytotoxicity assay Measured in Cell-Based System Using Plate Reader - 2159-05_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
Description:
Keywords:
RAD54, Branch migration, Holliday Junction, homologous recombination, DNA repair, cancer

Assay Overview:
The goal of this proposal is to develop specific small-molecule inhibitors of human RAD54, one of the key homologous recombination (HR) proteins. Basically, RAD54 and DNA that forms Holiday Junctions are added according to plate design with BioRAPTR (Beckman) to respective wells of 1536-well assay ready plates (Aurora 19180) that contain 2.5 nL/well of 10 mM compound. The reaction is incubated at room temperature for 40 minutes. Fluorescence is then read on Viewlux (Perkin Elmer) at ex/em 480/540 nm.



Expected Outcome: Inhibitors of RAD54 will result in loss of signal.
Protocol
1.#Reaction is started by Dispensing 0.6 uL/well of RAD54 Mix, 0.6 uL/well of DNA Mix, and 1.2 uL/well of Reaction Buffer in respective wells with BioRAPTR (Beckman) according to plate design in 1536-well assay ready plates (Aurora 19180) that contain 2.5 nL/well of 10 mM compound.
2.#Incubate at room temperature for 60 minutes.
3.#Read fluorescence at ex/em 480/540 nm on Viewlux
Reaction Buffer
Concentrations Tris base pH7.525mMMagnesium Acetate3mMBSA0.1mg/mlDTT2mMCreatine Phosphate15mMCreatine Phosphate kianse10Us/mLATP2mM
RAD54 Mix:
120 nM RAD54 (Provided by A Mazin, Drexel University College of Medicine) in Reaction Buffer
DNA Mix:
120 nM of the annealed Oligos (33/1337 and 265/1265) in Reaction Buffer. The oligos are as follows.
AVM#337-F, 60-mer: /56-FAM/CAC TGT GAT GCA CGA TGA TCG ACG ACA GTA GTC AGT GCT GGG TCA ACA TCT GTA TGC AGG
AVM #1337-BHQ: AGC ACT GAC TAC TGT CGT CGA TCA TCG TGC ATC ACA GTG/3BHQ_1/
AVM #1265, 60-mer: CAC TGT GAT GCA CGA TGA TTG ACG ACA GTA GTC AGT GCT TTT TTT TTT TTT TTT TTT TTT
AVM #265, 60-mer: CCT GCA TAC AGA TGT TGA CCC AGC ACT GAC TAC TGT CGT CAA TCA TCG TGC ATC ACA GTG
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 40.
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 0.5.
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
tSamples passing PAR_T only were assigned an outcome of 3 (inconclusive) :
PUBCHEM_ACTIVITY_SCORE < AT, and PCT_ACTIVE_REP >= PAR_T
Categorized Comment - additional comments and annotations
From PubChem:
Assay Format: Biochemical
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_10.41uM_(%) (10.41μM**)The calculated activity for the indicated sampleFloat%
4REPLICATE_B_ACTIVITY_SCORE_10.41uM_(%) (10.41μM**)The calculated activity for the indicated sampleFloat%

** Test Concentration.
Additional Information
Grant Number: R03 DA033981-01A1

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