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BioAssay: AID 651704

Inhibition of the MLL-AF4-AF9 Interaction in Pediatric Leukemia Measured in Biochemical System Using Plate Reader - 2160-01_Inhibitor_SinglePoint_HTS_Activity

This assay screens for inhibitors of the interaction of AF4 and AF9 . This is a homogeneous AlphaScreen assay that measures binding of a biotinylated AF4 peptide to FLAG-tagged AF9. The assay configuration comprises a 27-amino acid peptide biotinylated on the N-terminus. The selected peptide encompasses the portion of AF4 shown to be essential for AF9 binding, and an additional N-terminal more ..
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 Tested Compounds
 Tested Compounds
All(344456)
 
 
Active(1627)
 
 
Inactive(341368)
 
 
Inconclusive(1491)
 
 
 Tested Substances
 Tested Substances
All(348218)
 
 
Active(1633)
 
 
Inactive(345094)
 
 
Inconclusive(1491)
 
 
AID: 651704
Data Source: Broad Institute (2160-01_Inhibitor_SinglePoint_HTS_Activity)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2012-10-26
Modify Date: 2013-02-21

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 1627
Related Experiments
AIDNameTypeComment
651715Broad Institute Inhibition of the MLL-AF4-AF9 Interaction in Pediatric Leukemia Inhibitor Probe ProjectSummarydepositor-specified cross reference
720494Alphascreen Interference Assay Measured in Biochemical System Using Plate Reader - 2160-02_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
720495Inhibition of the MLL-AF4-AF9 Interaction in Pediatric Leukemia Measured in Biochemical System Using Plate Reader - 2160-01_Inhibitor_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
Description:
Keywords:AF4, AF9, Alpha screen, inhibitor,peptide


Assay Overview:
This assay screens for inhibitors of the interaction of AF4 and AF9 . This is a homogeneous AlphaScreen assay that measures binding of a biotinylated AF4 peptide to FLAG-tagged AF9. The assay configuration comprises a 27-amino acid peptide biotinylated on the N-terminus. The selected peptide encompasses the portion of AF4 shown to be essential for AF9 binding, and an additional N-terminal sequence to provide a spacer between the binding site and the streptavidin-coated AlphaScreen bead. Binding of biotinylated AF4 peptide to FLAG-tagged AF9 protein is detected by streptavidin-coated donor beads and anti-FLAG-coated acceptor beads. If peptide and protein bind, the beads are brought in proximity; laser excitation of the donor beads results in singlet oxygen (1O2) transfer to the acceptor beads and light emission. If an inhibitor disrupts the peptide-protein interaction by competing with the biotinylated AF4 peptide for binding to AF9, singlet oxygen transfer fails to occur due to the increased distance between donor and acceptor beads.

Expected Outcome:
Inhibitor binding is detectable by a decrease in light emission
Protocol
1. Compounds are delivered into Assay read plates (ARPs) including non-biotinylated AF4 peptide which will serve as the positive control in this screen
2. The plates used here are 384 proxiplates from PE.
3. A 2.5 X solution (5nM) of AF9-flag is prepared in 1x buffer and refreshed every 3hours.
4. A 2.5X solution (5nM) of Biotinylated AF4 (stock is 307uM) is prepared in two steps: step1- make intermediate stock of 3.07uM by diluting peptide in Citrate pH3 4uL into 396ul, first 4 ul peptide into 20ul citrate then bring up to 400ul in citrate. Step 2 make a 5nM stock in 1x buffer .
5. Make up a 5x stock (40ug/ml each bead) of alphalisa beads (biotin and antiflag beads).
6. Add 4ul of 2.5S AF9-Flag using a Combi dispenser.
7. Add 4ul of 2.5X Biotin AF4 using a Combi dispenser.
8. Incubate at RT for 1.5 hours.
9. Add 2ul of 5X beads using a Combi dispenser
10. Incubate for 1 hour at RT
11. read using Envision Readear.
Buffer notes-
1. 1x assay buffer is 1xPBS, 0.1% BSA, 0.01% Tween 20 made up fresh daily
2. citrate buffer used to make intermediate stock of bio-AF4 is 0.5M trisodium citrate dihydrate pH3.
Other assay notes-
All reagents are kept on ice during the course of the screen
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' method in Genedata Assay Analyzer (v7.0.3):
The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(NC).
A normalized activity value of -50 is defined as (0.5)(NC).
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: The plate pattern correction algorithm 'Assay Pattern (multiplicative)' in Genedata (v7.0.3) was applied to the normalized plate data.
PUBCHEM_ACTIVITY_SCORE:
This was set as equal to the mean of the normalized and corrected 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 -25%.
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
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_10uM_(%) (1μM**)The calculated activity for the indicated sampleFloat%
4REPLICATE_B_ACTIVITY_SCORE_10uM_(%) (1μM**)The calculated activity for the indicated sampleFloat%

** Test Concentration.
Additional Information
Grant Number: 1 R21 NS073046-01

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