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BioAssay: AID 588347

qHTS Assay for Inhibitors of GCN5L2: Hit confirmation

Histone acetyltransferase (HAT) enzymes transfer a methyl group from acetyl-coenzyme A to the epsilon-amino group of conserved lysine residues of N-terminal histone tails. Acetylation results in charge neutralization of this positively charged amino acid, decreasing interactions with the positively charged DNA backbone and making the DNA more accessible for transcription. HATs have been more ..
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 Tested Compounds
 Tested Compounds
All(145)
 
 
Active(96)
 
 
Inconclusive(49)
 
 
 Tested Substances
 Tested Substances
All(145)
 
 
Active(96)
 
 
Inconclusive(49)
 
 
AID: 588347
Data Source: NCGC (GCN5102)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-09-08

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 96
Related Experiments
AIDNameTypeComment
504398qHTS Assay for Inhibitors of GCN5L2: SummarySummarydepositor-specified cross reference: Summary AID of Project
504327qHTS Assay for Inhibitors of GCN5L2Confirmatorysame project related to Summary assay
Description:
Histone acetyltransferase (HAT) enzymes transfer a methyl group from acetyl-coenzyme A to the epsilon-amino group of conserved lysine residues of N-terminal histone tails. Acetylation results in charge neutralization of this positively charged amino acid, decreasing interactions with the positively charged DNA backbone and making the DNA more accessible for transcription. HATs have been recognized as an emerging drug target for treatment of cancer, diabetes, asthma, chronic obstructive pulmonary disease, cardiac disease, and viral infection [1]. Protein identified with HAT activity include the 5 yeast homolog like 2 (GCN5L2, a.ka. Histone acetyltransferase KAT2A) which acetylates histone H3 at Lys9, 14, 18, 13, and to lesser extent histones H4 and H2B. This activates transcription of the gene by modulating chromatin structure and installing the acetyllysine recognition mark for bromodomain-containing coactivator proteins.
A quantitative high throughput screen [2,3] was developed to identify small molecule inhibitors. The assay is based on the principle that it measures formation of reduced CoA with the pro-fluorescent maleimide derivative ThioGlo1 that becomes fluorescent upon reaction with the free thiol of CoA.

A large number of artifacts are expected in this primary screen, compounded by the assay format (fluorescent artifacts) and by the high top concentration tested for each substance. This BioAssay data deposition should be used with caution, and counterscreen and secondary assay validation is essential since very few actives from the primary screen are expected to be true inhibitors of GCN5L2.

[1] Esteller, M. Epigenetics in Cancer. N Engl J Med 358(11), 1148-1159. (2008). PMID: 18337604

[2] Yasgar, et al. Compound Management for Quantitative High-Throughput Screening. JALA. 2008 Apr;13(2):79-89. PMID: 18496600

[3] Inglese, et al. Quantitative high-throughput screening: a titration-based approach that efficiently identifies biological activities in large chemical libraries. Proc Natl Acad Sci. 1;103(31):11473-8. 2006. PMID: 16864780

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production Network [MLPCN]

MLCPN Grant: MH084681-02
Assay Submitter (PI): Structural Genomic Consortium - Toronto, Peter Brown
Protocol
To each well of a 1,536w plate, 3ul of GCN5L2 (final concentration 20nM, in 50mM HEPES, 0.1mM EDTA (pH 7.5), 0.01% Tween-20 buffer) are added using a Kalypsys dispenser. Only buffer is added to some wells as a control. A Kalypsys pin-tool transferred 23nl of library compound solution in DMSO to each well, as well as the control inhibit garcinol. . Following a 15 min incubation of enzyme with compounds at room temperature, 1ul mixture of ThioGlo1 (50uM final), H3(1-25) peptide (75uM final), and Acetyl-CoA (70uM final) is added. Plates are read immediately on the ViewLux (PerkinElmer) (Ex340nm/Em540nm) for a 0 min read, incubated for 20 min at room temperature, and read again on the ViewLux with the same settings. The initial read was carried out to identify compounds that are potentially fluorescent.
Comment
Compound Ranking:
1. Compounds are first classified as having full titration curves, partial modulation, partial curve (weaker actives), single point activity (at highest concentration only), or inactive. See data field "Curve Description". For this assay, apparent inhibitors are ranked higher than compounds that showed apparent activation.
2. For all inactive compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active compounds, a score range was given for each curve class type given above. Active compounds have PUBCHEM_ACTIVITY_SCORE between 40 and 100. Inconclusive compounds have PUBCHEM_ACTIVITY_SCORE between 1 and 39. Fit_LogAC50 was used for determining relative score and was scaled to each curve class' score range.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
Result Definitions
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TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PhenotypeIndicates type of activity observed: inhibitor, activator, fluorescent, cytotoxic, inactive, or inconclusive.String
2Potency*Concentration at which compound exhibits half-maximal efficacy, AC50. Extrapolated AC50s also include the highest efficacy observed and the concentration of compound at which it was observed.FloatμM
3EfficacyMaximal efficacy of compound, reported as a percentage of control. These values are estimated based on fits of the Hill equation to the dose-response curves.Float%
4Analysis CommentAnnotation/notes on a particular compound's data or its analysis.String
5Curve_DescriptionA description of dose-response curve quality. A complete curve has two observed asymptotes; a partial curve may not have attained its second asymptote at the highest concentration tested. High efficacy curves exhibit efficacy greater than 80% of control. Partial efficacies are statistically significant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the data to the Hill equation (calculated based on Molar Units).Float
7Fit_HillSlopeThe Hill slope from a fit of the data to the Hill equation.Float
8Fit_R2R^2 fit value of the curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the data to the Hill equation.Float%
11Fit_CurveClassNumerical encoding of curve description for the fitted Hill equation.Float
12Excluded_PointsWhich dose-response titration points were excluded from analysis based on outlier analysis. Each number represents whether a titration point was (1) or was not (0) excluded, for the titration series going from smallest to highest compound concentrations.String
13Max_ResponseMaximum activity observed for compound (usually at highest concentration tested).Float%
14Activity at 0.0003225714 uM (0.000322571μM**)% Activity at given concentration.Float%
15Activity at 0.0006451462 uM (0.000645146μM**)% Activity at given concentration.Float%
16Activity at 0.0009677143 uM (0.000967714μM**)% Activity at given concentration.Float%
17Activity at 0.00194 uM (0.00193544μM**)% Activity at given concentration.Float%
18Activity at 0.00290 uM (0.00290314μM**)% Activity at given concentration.Float%
19Activity at 0.00581 uM (0.00580632μM**)% Activity at given concentration.Float%
20Activity at 0.00871 uM (0.00870949μM**)% Activity at given concentration.Float%
21Activity at 0.017 uM (0.0174189μM**)% Activity at given concentration.Float%
22Activity at 0.026 uM (0.0261437μM**)% Activity at given concentration.Float%
23Activity at 0.052 uM (0.0522568μM**)% Activity at given concentration.Float%
24Activity at 0.078 uM (0.0781014μM**)% Activity at given concentration.Float%
25Activity at 0.112 uM (0.111607μM**)% Activity at given concentration.Float%
26Activity at 0.232 uM (0.231933μM**)% Activity at given concentration.Float%
27Activity at 0.464 uM (0.464466μM**)% Activity at given concentration.Float%
28Activity at 0.707 uM (0.707244μM**)% Activity at given concentration.Float%
29Activity at 1.493 uM (1.493μM**)% Activity at given concentration.Float%
30Activity at 2.116 uM (2.1164μM**)% Activity at given concentration.Float%
31Activity at 4.064 uM (4.06368μM**)% Activity at given concentration.Float%
32Activity at 6.357 uM (6.3572μM**)% Activity at given concentration.Float%
33Activity at 13.06 uM (13.0625μM**)% Activity at given concentration.Float%
34Activity at 19.05 uM (19.0476μM**)% Activity at given concentration.Float%
35Activity at 35.55 uM (35.5537μM**)% Activity at given concentration.Float%
36Activity at 57.14 uM (57.1429μM**)% Activity at given concentration.Float%
37Activity at 114.3 uM (114.286μM**)% Activity at given concentration.Float%
38Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH084681

Data Table (Concise)
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