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BioAssay: AID 2718

Fluorescence Cell-Free Homogeneous Primary HTS to Identify Inhibitors of Histone Deacetylase 3

Assay Overview: HDAC3 is incubated with compounds for 10 minutes before addition of a coumarin-linked tripeptide substrate containing a terminal acetylated lysine, and trypsin. If HDAC3 is not inhibited it will deacetylate the substrate, which renders the peptide a substrate for trypsin. Trypsin can then liberate the coumarin, increasing fluorescence ..more
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 Tested Compounds
 Tested Compounds
All(318295)
 
 
Active(483)
 
 
Inactive(313816)
 
 
Inconclusive(4001)
 
 
 Tested Substances
 Tested Substances
All(318388)
 
 
Active(485)
 
 
Inactive(313902)
 
 
Inconclusive(4001)
 
 
AID: 2718
Data Source: Broad Institute (2062-01_INHIBITORS_SINGLE-POINT_MLPCN-HTS)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: Other
Deposit Date: 2010-03-29

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 483
Related Experiments
AIDNameTypeComment
2722Summary of Broad Institute Histone Deacetylase 3 ProjectSummarydepositor-specified cross reference
Description:
Keywords: histone deacetylase, HDAC3, epigenetics

Assay Overview: HDAC3 is incubated with compounds for 10 minutes before addition of a coumarin-linked tripeptide substrate containing a terminal acetylated lysine, and trypsin. If HDAC3 is not inhibited it will deacetylate the substrate, which renders the peptide a substrate for trypsin. Trypsin can then liberate the coumarin, increasing fluorescence


Expected Outcome: HDAC3 inhibitors will prevent deacetylation of the peptide substrate, rendering it an unsuitable substrate for trypsin. This will prevent the liberation of the coumarin fluorophore and fluorescence levels will be reduced compared to noninhibited (DMSO) wells.
Protocol
1) Assay runs were 100 1536 plates each (Aurora 00029844). Assay ready plates (ARPs) with 7.5 nL compound per plate (10 mM stock) were stored vacuum sealed at -20 C until 12 hours before use when they were unsealed and thawed at RT.
2) Positive control suberoylanilide hydroxamic acid (SAHA 1.4 mM stock concentration, 7.5 mM final concentration, synthesized in house) was applied to 112 wells/plate (50 nL/well) using a MultiDrop Combi nL liquid dispenser (Thermo Scientific, model: 838).

3) For each run of 100 plates, 1.5L of HDAC assay buffer was utilized (HAB; 100 mM KCl (EMD cat# 7300, lot# 0827B018), 50 mM HEPES pH 7.4 (GIBCO cat# 15630-114, lot# 697278), 0.05% BSA (CalBiochem cat# 126626, lot# D00085439), and 0.001% Tween-20 (Zymed cat# 00-3005, lot# D00007104)).
4) For 100 plates, 149.25 ul HDAC3 enzyme stock (BPS Biosciences cat# 50003, lot# 91014, 0.67 mg/mL) was diluted into 1 L of HAB. Six microliters of this HDAC3 solution was added to each well (HDAC3 final concentration in each well = 0.78 nM) using a MultiDrop Combi nL liquid dispenser (Thermo Scientific, model: 838).

5) Each plate was then incubated at RT for 10 minutes before addition of 3 uL substrate and trypsin solution addition (S+T) (For 500 mL S+T stock; 141.05 uL 100 mM MAZ1600 substrate (final concentration 9.4 uM), 4.95 mL of 45 uM Trypsin stock (45 uM stock made from lyophilized powder, Worthington cat# 3744 lot# 57E965B) (final concentration in well 150 nM), 494.9 ml of R.T. HAB), using a second MultiDrop Combi nL liquid dispenser (Thermo Scientific, model: 838).

6) After incubation at RT for 45 minutes, plates were spun for 1 minute @ 1000 rpm. Following centrifugation, plates were read using the Viewlux (Perkin Elmer model 1430-002) reader @ 355/460nm.
Comment
HTS Data Analysis
Neutral control wells (NC) and positive controls wells (PC) were included on every plate.
NC: DMSO
PC: SAHA, 7.6uM
Active compounds result in decreased readout signal.
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 NC wells was set to a normalized activity value of 0.
The median raw signal of the intraplate PC wells was set to a normalized activity value of -100.
Experimental wells were scaled to this range, giving an activity score as percent change in signal relative to the intraplate controls.
For one run, the positive control did not work, so the -100 full inhibition value was set to (0.33)(median of NC), and the compound wells scaled accordingly.
The plate pattern correction algorithm 'Runwise Pattern (Additive)' in Genedata applied to the normalized plate data.
The replicate activity scores were multiplied by -1 to convert Genedata negative percent inhibition values to Pubchem positive percent activity values.
The final PUBCHEM_ACTIVITY_SCORE was set as equal to the mean of the valid replicates.
The PUBCHEM_ACTIVITY_OUTCOME class was assigned as described below, based on an inhibition threshold of 25% inhibition:
Activity_Outcome = 1 (inactive)
Percent of active replicates is < 50%
Activity_Outcome = 2 (active)
Percent of active replicates = 100% AND the count of active replicates > 1
Activity_Outcome = 3 (inconclusive)
Percent of active replicates = 100% AND the count of replicates = 1
or
Percent of active replicates is < 100% AND >=50%
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 InvalidFloat
2BROAD_SCREENING_RUNIDSThis is a comma separated list of unique IDs given to each screening run at the Broad Institute.String
3REPLICATE_A_ACTIVITY_SCORE (8.3μM**)The calculated activity score for the indicated sample, a NULL here denotes data was not used or was not producedFloat%
4REPLICATE_B_ACTIVITY_SCORE (8.3μM**)The calculated activity score for the indicated sample, a NULL here denotes data was not used or was not producedFloat%
5REPLICATE_C_ACTIVITY_SCORE (8.3μM**)The calculated activity score for the indicated sample, a NULL here denotes data was not used or was not producedFloat%
6REPLICATE_D_ACTIVITY_SCORE (8.3μM**)The calculated activity score for the indicated sample, a NULL here denotes data was not used or was not producedFloat%
7DATE_REPORTEDThe date the data was internally reportedString

** Test Concentration.

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