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BioAssay: AID 651548

EZH2/PRC2 methyltransferase inhibitors Measured in Biochemical System Using Plate Reader - 2125-01_Inhibitor_SinglePoint_HTS_Activity

Keywords: Cancer, target-based, Polycomb Repressive complex 2, histone-modifying enzymes, histone methyltransferases ..more
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 Tested Compounds
 Tested Compounds
All(57013)
 
 
Active(201)
 
 
Inactive(56771)
 
 
Inconclusive(41)
 
 
 Tested Substances
 Tested Substances
All(57246)
 
 
Active(201)
 
 
Inactive(57004)
 
 
Inconclusive(41)
 
 
 Related BioAssays
 Related BioAssays
AID: 651548
Data Source: Broad Institute (2125-01_Inhibitor_SinglePoint_HTS_Activity)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: Other
Deposit Date: 2012-08-23

Data Table ( Complete ):           View Active Data    View All Data
Target
Sequence: histone-lysine N-methyltransferase EZH2 isoform a [Homo sapiens]
Description ..   
Protein Family: SET (Su(var)3-9, Enhancer-of-zeste, Trithorax) domain

Gene:EZH2     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 201
Description:
Keywords: Cancer, target-based, Polycomb Repressive complex 2, histone-modifying enzymes, histone methyltransferases

Assay Overview: The central goal of this project is to develop small-molecule probes that can be used to better understand the regulation of H3K27Me3 by EZH2 and associated proteins in PRC2, their connections in cancer development, and their roles in cancer treatment.
Protocol
Protocol:
PRC2 activity was measured using Dissociation-Enhanced Lanthanide Fluoro-ImmunoAssays (DELFIA) performed on 384-well, white, streptavidin-coated plates (PerkinElmer). In short, PRC2 was diluted to 30 ng and 3 ng per well in 20 uL 2X Enzyme Buffer (50mM Tris HCl pH 8.5, 10 mM DTT, 5 mM MgCl). Compounds were pinned at 100 nL per well and reactions were initiated with 20 uL of a 5 uM SAM (NEB) solution containing 600 nM H3[21-44]-GK-biotin (AnaSpec; PRC2).

Plates were incubated at room temperature for 1 hr and then washed three times with 100 uL of Wash Buffer (50mM Tris pH 7.4, 150 mM NaCl, 0.05% Tween 20, 0.2% BSA). 50 ul of Fluoroimmunoassay (FI) Buffer (50 mM Tris HCl pH 7.8, 150 mM NaCl, 0.05% Tween 40, 25 iM DTPA, 0.2% BSA, 0.05% BGG)containing a 1:4000 dilution of anti-H3K27me2 rabbit IgG (Cell Signaling, #9728) with 691 ng/mL Eu-N1-anti-rabbit IgG (PerkinElmer)was added to each well for PRC2 reactions.

Following 1 hr incubation at room temperature, the plates were washed three times with Wash Buffer and 50 uL of Enhancement Solution (PerkinElmer) was added to each well. Plates were incubated for 30 min at room temperature and time-resolved fluorescence (TRF) was measured on Wallac Envision 2104 Multilabel Reader (400 us window, 400 us delay, 320 excitation, 615 emission).
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 1 (inactive) :
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_12.5uM_(%) (12.5μM**)The calculated activity for the indicated sampleFloat%
4REPLICATE_B_ACTIVITY_SCORE_12.5uM_(%) (12.5μM**)The calculated activity for the indicated sampleFloat%

** Test Concentration.

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