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 ..
BioActive Compounds: 1627
Keywords:AF4, AF9, Alpha screen, inhibitor,peptide
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.
Inhibitor binding is detectable by a decrease in light emission
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.
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
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.
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.
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.
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
** Test Concentration.
Data Table (Concise)