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BioAssay: AID 651723

MLPCN PGC1a Modulators Measured in Cell-Based System Using Plate Reader - 2139-01_Activator_SinglePoint_HTS_Activity

Keywords: GCN5,flag PGC1a adenovirus, acetyl transferase, SIRT1 deacetylase, Alphascreen, acetylation, inflammation, diabetes ..more
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 Tested Compounds
 Tested Compounds
All(345860)
 
 
Active(740)
 
 
Inactive(344764)
 
 
Inconclusive(388)
 
 
 Tested Substances
 Tested Substances
All(349095)
 
 
Active(741)
 
 
Inactive(347966)
 
 
Inconclusive(388)
 
 
 Related BioAssays
 Related BioAssays
AID: 651723
Data Source: Broad Institute (2139-01_Activator_SinglePoint_HTS_Activity)
BioAssay Type: Primary, Primary Screening, Single Concentration Activity Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2012-10-28

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 740
Related Experiments
AIDNameTypeComment
651721Broad Institute HTS of Compounds Modulating PGC-1a Acetylation and Oxidative Metabolic Function Activator Probe ProjectSummarydepositor-specified cross reference: Summary
720513MLPCN PGC1a Modulators Measured in Cell-Based System Using Plate Reader - 2139-01_Activator_Dose_CherryPick_Activity_Set6Confirmatorysame project related to Summary assay
743463PGC-1a: Effect of activators of PGC-1a acetylation on target gene expression G6PC in hepatocyte, using qPCR Measured in Cell-Based System Using RT-PCR - 2139-05_Activator_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
743465PGC-1a: Effect of activators of PGC-1a acetylation of target gene expression PCK1 in hepatocytes, using qPCR Measured in Cell-Based System Using RT-PCR - 2139-04_Activator_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
743467PGC-1a: Effect of activators of PGC-1a acetylation on target gene expression PGC-1a in hepatocyte, using qPCR Measured in Cell-Based System Using RT-PCR - 2139-03_Activator_Dose_CherryPick_ActivityConfirmatorysame project related to Summary assay
Description:
Keywords: GCN5,flag PGC1a adenovirus, acetyl transferase, SIRT1 deacetylase, Alphascreen, acetylation, inflammation, diabetes

Assay Overview: The fact that the discovery and identification of chemical compounds that through deacetylation of PGC-
1a might mimic the "healthy" conditions of exercise and calorie restriction makes the outcomes of this
work of high biological and therapeutic significance. The close relationship between PGC-1a acetylation
and metabolic diseases suggests the possibility that the discovery of chemical probes to manipulate the
acetylation level would introduce new therapeutic applications in those diseases. Toward this end, our primary goal is to discover
chemical compounds that can be applied in biological/therapeutic research to protect against age-associated diseases including metabolic disorders.

Expected Outcome: The identification of chemical probes capable of mimicking calorie restriction and exercise by reducing PGC-1a acetylation, with the long-term goal of developing treatments for diseases in which PGC-1a is dysregulated, including diabetes, othre metabolic disorders, neuromuscular diseases and sarcopenia
Protocol
1.Thaw 6 vials U2OS cells into 4 triple flasks with growth media. Grow for 48hrs. Harvest and seed 4 Hyper flasks @ 15-20M cells ea. Grow 4-5 days until near/at confluence.
2.Change to assay media and infect with flag PGC1a adenovirus and GCN5 viruses . Incubate 4hrs. Replace media with Phenol Red free medium and incubate overnight.
3.Harvest , count, and plate cells @7000 cell/well in 50 uL assay media in 384 well plates.
4.Incubate @ 37 degrees c 4-8 hrs for cells to adhere.
5.Pin 100nl compound, continue incubation overnight.
6.Aspirate media and add 6ul of Lysis buffer,* incubate 1hr @ r.t.
7.Add 6ul of anti-acetyl Lysine Ab, incubate 1hr @ r.t.
8.Add 6ul of Alpha Acceptor beads, incubate 1hr @ r.t.
9.Add 6ul of Alpha Donor beads, incubate 1hr @ r.t
10.Read on Envision using ALPHA protocol.
Comment
PRESENCE OF CONTROLS: Neutral control wells (NC) and positive control wells (PC) were included on every plate.

EXPECTED OUTCOME: Active compounds result in increasing 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.

PATTERN CORRECTION: The plate pattern correction algorithm 'Runwise 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 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 1 (inactive) :
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
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_15.56uM_(%) (15.56μM**)The calculated activity for the indicated sampleFloat%
4REPLICATE_B_ACTIVITY_SCORE_15.56uM_(%) (15.56μM**)The calculated activity for the indicated sampleFloat%

** Test Concentration.
Additional Information
Grant Number: RO3 DA032468-01A1

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