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BioAssay: AID 720514

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

Keywords: GCN5,flag PGC1a adenovirus, acetyl transferase, SIRT1 deacetylase, Alphascreen, acetylation, inflammation, diabetes ..more
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 Tested Compounds
 Tested Compounds
All(2098)
 
 
Active(297)
 
 
Inactive(1768)
 
 
Inconclusive(33)
 
 
 Tested Substances
 Tested Substances
All(2103)
 
 
Active(300)
 
 
Inactive(1770)
 
 
Inconclusive(33)
 
 
 Related BioAssays
 Related BioAssays
AID: 720514
Data Source: Broad Institute (2139-01_Inhibitor_Dose_CherryPick_Activity)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2013-07-16

Data Table ( Complete ):           Active    All
BioActive Compounds: 297
Depositor Specified Assays
AIDNameTypeComment
651714Broad Institute HTS of Compounds Modulating PGC-1a Acetylation and Oxidative Metabolic Function Inhibitor Probe ProjectsummarySummary assay
Description:
Keywords: GCN5,flag PGC1a adenovirus, acetyl transferase, SIRT1 deacetylase, Alphascreen, acetylation, inflammation, diabetes

Assay Overview: The close relationship between PGC-1a acetylation and metabolic diseases suggests the possibility the discovery of chemical probes that manipulate the acetylation level would introduce new therapeutic applications in those diseases. Our primary goal is to discover chemical compounds that can modulate PGC1a acetylation levels to protect against age-associated diseases including metabolic disorders (Inhibitors) and hyperglycemia associated with Type 2 diabetes (Activators). In this project we use a homogeneous, whole cell lysate, AlphaLISA assay to measure PGC1a acetylation levels. The cell system is infected with flag PGC1a adenovirus and GCN5 acetyl transferase virus. The acetylation state of PGC1a is measured using a rabbit anti-acetylated lysine antibody, anti-rabbit Alpha-LISA donor bead and anti-flag Alpha-LISA acceptor bead. This assay is capable of measuring both increases and decreases in the PGC1a acetylation levels. Decreases in PGC1a acetylation can be the result of inhibiting GCN5 acetyl transferase or stimulating de-acetylation through deacetylases such as SirT1. This mimics the effects of exercise and caloric restriction. These compounds are found in the PGC1a inhibitor screen. Increases in hepatic PGC1a acetylation levels can suppress glucose production and ameliorate the hyperglycemia associated with Type 2 diabetes. These compounds are found in the PGC1a activator screen.

Expected Outcome: The identification of chemical probes capable of mimicking calorie restriction and exercise by reducing PGC-1a acetylation (Inhibitors), with the long-term goal of developing treatments for diseases in which PGC-1a is dysregulated, including diabetes, other metabolic disorders, neuromuscular diseases and sarcopenia. Identification of chemical probes that increase PGC1a acetylation levels (Activators) are expected to be long-term treatments for improving hyperglycemia associated with type 2 diabetes. An increase in signal with this assay represents a PGC1a activator which results in increased acetylation. A decrease in signal with this assay represents an inhibitor of PGC1a acetylation. Additional counter screens will be performed to remove Alpha-LISA artifacts and cytotoxic compounds.
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 decreasing readout signal.
The compounds were assayed in multiple independent instances using an identical protocol; each instance is called a 'test'. For each test, the following analysis was applied:

ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).

NORMALIZATION:
The raw signals of the plate wells were normalized using the 'Neutral Controls Minus Inhibitors' method in Genedata Assay Analyzer (v10.0.2):
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: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.

MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): AC50
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)

PUBCHEM_ACTIVITY_OUTCOME:
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.

PUBCHEM_ACTIVITY_SCORE:
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.

DATA AGGREGATION:
Once the data for each test was processed, the test number was appended to all column headers in that test's data set. The individual test results were then aggregated as follows:

1. The final PUBCHEM_ACTIVITY_OUTCOME was set to:
a. '2' (active) when all test outcomes were '2' (active), or
b. '1' (inactive) when all test outcomes were '1' (inactive), or
c. '3' (inconclusive) when the test outcomes were mixed.

2. The final ACTIVE_CONCENTRATION (AC) was set as follows:
a. If the final PUBCHEM_ACTIVITY_OUTCOME = 2, AC was set as the mean of the constituent test active concentrations;
b. If the final PUBCHEM_ACTIVITY_OUTCOME = 1 or 3, AC was left empty.

3. The final PUBCHEM_ACTIVITY_SCORE was calculated based on the aggregated ACTIVE_CONCENTRATION, using the same logic described above for individual test scores.

Note:
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1AC50_Qualifier'>','=', or '<'String
2AC50_uM*The concentration at which activity reaches 50% of the maximumFloatμM
3pAC50_MEqual to -1*log10(AC50)String
4ACTIVITY_OUTCOME_TEST1The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
5ACTIVITY_SCORE_TEST1The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
6ASSAYDATA_COMMENT_TEST1Comments relevant to the subset of data included in the indicated Test number.String
7AC50_Qualifier_TEST1'>','=', or '<'String
8AC50_uM_TEST1The concentration at which activity reaches 50% of the maximumFloatμM
9pAC50_M_TEST1Equal to -1*log10(AC50)String
10Hill_Slope_TEST1The slope at AC50Float
11S0_(%)_TEST1The fitted activity value at zero concentrationFloat%
12Sinf_(%)_TEST1The fitted activity value at infinite concentrationFloat%
13Num_Points_TEST1The number of data points used to generate the plotInteger
14Max_Activity_(%)_TEST1The maximum activity value observed, based on mean of replicates per concentrationFloat%
15Max_Activity_Conc_uM_TEST1The concentration at which the maximum activity is observedFloatμM
16Max_Concentration_uM_TEST1Maximum valid test concentrationFloatμM
17Activity_at_2.35uM_(%)_TEST1 (2.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
18Activity_at_2.6uM_(%)_TEST1 (2.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
19Activity_at_2.85uM_(%)_TEST1 (2.85μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
20Activity_at_4.6uM_(%)_TEST1 (4.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
21Activity_at_5uM_(%)_TEST1 (5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
22Activity_at_5.6uM_(%)_TEST1 (5.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
23Activity_at_9uM_(%)_TEST1 (9μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
24Activity_at_10uM_(%)_TEST1 (10μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
25Activity_at_11uM_(%)_TEST1 (11μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
26Activity_at_18uM_(%)_TEST1 (18μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
27Activity_at_19.5uM_(%)_TEST1 (19.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
28Activity_at_21uM_(%)_TEST1 (21μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
29ACTIVITY_OUTCOME_TEST2The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
30ACTIVITY_SCORE_TEST2The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
31ASSAYDATA_COMMENT_TEST2Comments relevant to the subset of data included in the indicated Test number.String
32AC50_Qualifier_TEST2'>','=', or '<'String
33AC50_uM_TEST2The concentration at which activity reaches 50% of the maximumFloatμM
34pAC50_M_TEST2Equal to -1*log10(AC50)String
35Hill_Slope_TEST2The slope at AC50Float
36S0_(%)_TEST2The fitted activity value at zero concentrationFloat%
37Sinf_(%)_TEST2The fitted activity value at infinite concentrationFloat%
38Num_Points_TEST2The number of data points used to generate the plotInteger
39Max_Activity_(%)_TEST2The maximum activity value observed, based on mean of replicates per concentrationFloat%
40Max_Activity_Conc_uM_TEST2The concentration at which the maximum activity is observedFloatμM
41Max_Concentration_uM_TEST2Maximum valid test concentrationFloatμM
42Activity_at_0.16uM_(%)_TEST2 (0.16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
43Activity_at_0.3uM_(%)_TEST2 (0.3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
44Activity_at_0.6uM_(%)_TEST2 (0.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
45Activity_at_1.2uM_(%)_TEST2 (1.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
46Activity_at_2.6uM_(%)_TEST2 (2.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
47Activity_at_5uM_(%)_TEST2 (5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
48Activity_at_10uM_(%)_TEST2 (10μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
49Activity_at_19.5uM_(%)_TEST2 (19.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
50ACTIVITY_OUTCOME_TEST3The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
51ACTIVITY_SCORE_TEST3The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
52ASSAYDATA_COMMENT_TEST3Comments relevant to the subset of data included in the indicated Test number.String
53AC50_Qualifier_TEST3'>','=', or '<'String
54AC50_uM_TEST3The concentration at which activity reaches 50% of the maximumFloatμM
55pAC50_M_TEST3Equal to -1*log10(AC50)String
56Hill_Slope_TEST3The slope at AC50Float
57S0_(%)_TEST3The fitted activity value at zero concentrationFloat%
58Sinf_(%)_TEST3The fitted activity value at infinite concentrationFloat%
59Num_Points_TEST3The number of data points used to generate the plotInteger
60Max_Activity_(%)_TEST3The maximum activity value observed, based on mean of replicates per concentrationFloat%
61Max_Activity_Conc_uM_TEST3The concentration at which the maximum activity is observedFloatμM
62Max_Concentration_uM_TEST3Maximum valid test concentrationFloatμM
63Activity_at_0.16uM_(%)_TEST3 (0.16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
64Activity_at_0.3uM_(%)_TEST3 (0.3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
65Activity_at_0.6uM_(%)_TEST3 (0.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
66Activity_at_1.2uM_(%)_TEST3 (1.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
67Activity_at_2.6uM_(%)_TEST3 (2.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
68Activity_at_5uM_(%)_TEST3 (5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
69Activity_at_10uM_(%)_TEST3 (10μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
70Activity_at_19.5uM_(%)_TEST3 (19.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
71ACTIVITY_OUTCOME_TEST4The PubChem Activity Outcome for the subset of data included in the indicated Test number.Integer
72ACTIVITY_SCORE_TEST4The PubChem Activity Score for the subset of data included in the indicated Test number.Integer
73ASSAYDATA_COMMENT_TEST4Comments relevant to the subset of data included in the indicated Test number.String
74AC50_Qualifier_TEST4'>','=', or '<'String
75AC50_uM_TEST4The concentration at which activity reaches 50% of the maximumFloatμM
76pAC50_M_TEST4Equal to -1*log10(AC50)String
77Hill_Slope_TEST4The slope at AC50Float
78S0_(%)_TEST4The fitted activity value at zero concentrationFloat%
79Sinf_(%)_TEST4The fitted activity value at infinite concentrationFloat%
80Num_Points_TEST4The number of data points used to generate the plotInteger
81Max_Activity_(%)_TEST4The maximum activity value observed, based on mean of replicates per concentrationFloat%
82Max_Activity_Conc_uM_TEST4The concentration at which the maximum activity is observedFloatμM
83Max_Concentration_uM_TEST4Maximum valid test concentrationFloatμM
84Activity_at_0.12uM_(%)_TEST4 (0.12μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
85Activity_at_0.15uM_(%)_TEST4 (0.15μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
86Activity_at_0.16uM_(%)_TEST4 (0.16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
87Activity_at_0.26uM_(%)_TEST4 (0.26μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
88Activity_at_0.285uM_(%)_TEST4 (0.285μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
89Activity_at_0.3uM_(%)_TEST4 (0.3μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
90Activity_at_0.35uM_(%)_TEST4 (0.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
91Activity_at_0.5uM_(%)_TEST4 (0.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
92Activity_at_0.56uM_(%)_TEST4 (0.56μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
93Activity_at_0.6uM_(%)_TEST4 (0.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
94Activity_at_0.68uM_(%)_TEST4 (0.68μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
95Activity_at_1uM_(%)_TEST4 (1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
96Activity_at_1.1uM_(%)_TEST4 (1.1μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
97Activity_at_1.2uM_(%)_TEST4 (1.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
98Activity_at_1.35uM_(%)_TEST4 (1.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
99Activity_at_1.95uM_(%)_TEST4 (1.95μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
100Activity_at_2.35uM_(%)_TEST4 (2.35μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
101Activity_at_2.6uM_(%)_TEST4 (2.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
102Activity_at_4.2uM_(%)_TEST4 (4.2μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
103Activity_at_4.6uM_(%)_TEST4 (4.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
104Activity_at_5uM_(%)_TEST4 (5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
105Activity_at_5.6uM_(%)_TEST4 (5.6μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
106Activity_at_8uM_(%)_TEST4 (8μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
107Activity_at_9uM_(%)_TEST4 (9μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
108Activity_at_10uM_(%)_TEST4 (10μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
109Activity_at_11uM_(%)_TEST4 (11μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
110Activity_at_16uM_(%)_TEST4 (16μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
111Activity_at_18uM_(%)_TEST4 (18μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
112Activity_at_19.5uM_(%)_TEST4 (19.5μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%
113Activity_at_21uM_(%)_TEST4 (21μM**)The average measured activity of all accepted replicates at the specified concentrationFloat%

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

Data Table (Concise)
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