Bookmark and Share
BioAssay: AID 504332

qHTS Assay for Inhibitors of Histone Lysine Methyltransferase G9a

Methylated lysines, on the N-terminal tails of histone proteins serve as epigenetic markers to recruit factors that can then modify local chromatin structure to lead to functional consequences. G9a and other members of the SUV39 family of the SET domain-containing superfamily of histone lysine methyltransferases (HMT) have been identified to specifically methylate Lys9 of histone H3 (H3K9). G9a more ..
_
   
 Tested Compounds
 Tested Compounds
All(348343)
 
 
Active(30875)
 
 
Inactive(267011)
 
 
Inconclusive(51899)
 
 
 Tested Substances
 Tested Substances
All(353740)
 
 
Active(31109)
 
 
Inactive(270505)
 
 
Inconclusive(52126)
 
 
AID: 504332
Data Source: NCGC (G9A502)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-02-22

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 30875
Depositor Specified Assays
AIDNameTypeComment
504404qHTS Assay for Inhibitors of Histone Lysine Methyltransferase G9a: Summarysummary
Description:
Methylated lysines, on the N-terminal tails of histone proteins serve as epigenetic markers to recruit factors that can then modify local chromatin structure to lead to functional consequences. G9a and other members of the SUV39 family of the SET domain-containing superfamily of histone lysine methyltransferases (HMT) have been identified to specifically methylate Lys9 of histone H3 (H3K9). G9a catalyzes the mono- and di-methylation of H3K9 in mammalian euchromatic regions, where the resulting H3K9me2 is indicative of transcriptional repression. Hence, G9a has been recognized as a potential drug target for several human diseases, including cancer. The inhibition of G9a will result in transcriptional activation and work synergistically with DNA methyltransferase and histone deacetylase inhibitors, to kill cancer cells.

This qHTS assay for identification of G9a inhibitors is a chemiluminescence based AlphaScreen (PerkinElmer) [1]. Methylation of biotinylated-histone peptide is measured through specific antibody-based detection, in conjunction with streptavidin-coated donor and anti-IgG antibody-coated acceptor beads. The method is particularly well suited for detection of inhibitors acting by the desired histone peptide competitive mechanism and is applicable to testing other HMTs.

Although AlphaScreen has significant advantages with its utility in a variety of epigenetic target assays, the primary screening data provided in this deposition should be used with caution due to the prevalence of screening artifacts [2]. An AlphaScreen counterscreen is highly recommended to be run against any putative actives to eliminate non specific artifacts. Alternatively, one can use other AlphaScreen assays in PubChem to filter out promiscuous hits.

[1] Quinn et al. A chemiluminescence-based method for identification of histone lysine methyltransferase inhibitors. Mol Biosyst 6(5): 782-8. 2010. PMID: 20567762

[2] Baell,Holloway. New substructure filters for removal of pan assay interference compounds (PAINS) from screening libraries and for their exclusion in bioassays. J Med Chem. 53(7):2719-40. 2010. PMID: 20131845

NIH Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
Structural Genomics Consortium (SGC)
NIH Grant: 5U54 MH084681-02
Protocol
To each well of a 1,536w plate, 2ul of HMT enzyme (final 20nM) were added, using a nanoliter dispenser. The G9a- and GLP-inhibitor BIX-01294 (CID: 25150857) are used as an intraplate control, with a 16-point titration in duplicate. A Kalypsys pin-tool transferred 23nl of library compound solution in DMSO to each well. Following a 15 min incubation of enzyme with compounds at room temperature, 1ul mixture of b-H3K9 peptide substrate (final 500nM) and SAM cofactor (final 20uM) were added. Reactions were allowed to proceed at room temperature for 2 hours. Methylated b-H3K9 peptide product was detected with 0.5 ug/mL rabbit polyclonal anti-monomethyl histone H3K9 antibody (Abcam Inc., Cambridge, MA) and 20 ug/mL each streptavidin-coated donor and anti-rabbit IgG acceptor AlphaScreen beads. Antibody and beads were added in a 1ul dispense, for a final volume of 4ul, and plates were incubated, protected from the light, for 10 min at room temperature. Microplates were then read on an EnVision multilabel plate reader using the 1,536 plate HTS AlphaScreen aperture (excitation time 80 ms, measurement time 240 ms).
Comment
Keywords:
G9a, EHMT2, euchromatic histone-lysine N-methyltransferase 2, histone-lysine N-methyltransferase, H3 lysine-9 specific 3

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.
Result Definitions
Show more
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 0.00366 uM (0.00366μM**)% Activity at given concentration.Float%
15Activity at 0.00865 uM (0.00864669μM**)% Activity at given concentration.Float%
16Activity at 0.018 uM (0.0183μM**)% Activity at given concentration.Float%
17Activity at 0.041 uM (0.0409μM**)% Activity at given concentration.Float%
18Activity at 0.092 uM (0.0924575μM**)% Activity at given concentration.Float%
19Activity at 0.133 uM (0.132655μM**)% Activity at given concentration.Float%
20Activity at 0.202 uM (0.202001μM**)% Activity at given concentration.Float%
21Activity at 0.415 uM (0.414951μM**)% Activity at given concentration.Float%
22Activity at 0.501 uM (0.501175μM**)% Activity at given concentration.Float%
23Activity at 0.843 uM (0.842891μM**)% Activity at given concentration.Float%
24Activity at 1.264 uM (1.26401μM**)% Activity at given concentration.Float%
25Activity at 2.335 uM (2.33479μM**)% Activity at given concentration.Float%
26Activity at 3.417 uM (3.41653μM**)% Activity at given concentration.Float%
27Activity at 5.146 uM (5.14644μM**)% Activity at given concentration.Float%
28Activity at 10.61 uM (10.6103μM**)% Activity at given concentration.Float%
29Activity at 12.70 uM (12.6983μM**)% Activity at given concentration.Float%
30Activity at 21.52 uM (21.5185μM**)% Activity at given concentration.Float%
31Activity at 31.98 uM (31.977μM**)% Activity at given concentration.Float%
32Activity at 58.95 uM (58.9519μM**)% Activity at given concentration.Float%
33Activity at 101.6 uM (101.564μM**)% Activity at given concentration.Float%
34Activity at 134.7 uM (134.697μM**)% Activity at given concentration.Float%
35Activity at 186.0 uM (186μM**)% Activity at given concentration.Float%
36Compound QCSource of Compound QCString

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH084681-02

Data Table (Concise)
PageFrom: