MLPCN PGC1a Modulators Measured in Cell-Based System Using Plate Reader - 2139-01_Inhibitor_SinglePoint_HTS_Activity
Keywords: GCN5,flag PGC1a adenovirus, acetyl transferase, SIRT1 deacetylase, Alphascreen, acetylation, inflammation, diabetes ..more
BioActive Compounds: 601
Depositor Specified Assays
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
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.
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 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: The plate pattern correction algorithm 'Runwise 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 -40.
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)