qHTS Assay for Inhibitors of Histone Lysine Methyltransferase G9a: Hit Confirmation
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 ..
BioActive Compounds: 327
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) . 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 . 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.
 Quinn et al. A chemiluminescence-based method for identification of histone lysine methyltransferase inhibitors. Mol Biosyst 6(5): 782-8. 2010. PMID: 20567762
 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 Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production Network [MLPCN]
MLPCN Grant: MH084681-02
Assay Submitter (PI): Structural Genomic Consortium - Toronto, Peter Brown
To each well, 2 uL of HMT enzyme (final 20 nM) will be added using a nanoliter dispenser. The G9a- and GLP-inhibitor BIX-01294 will be used as an intraplate control, with a 16-point titration in duplicate. A Kalypsys pin-tool will be employed to transfer 23 nL of library compound solution in DMSO to each well. Following a 15 min incubation of enzyme with compounds at room temperature, 1 uL mixture of b-H3K9 peptide substrate (final 500 nM) and SAM cofactor (final 20 uM) will be added. Reactions are allowed to proceed at room temperature for 2 hours. Methylated b-H3K9 peptide product is 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 will be added in a 1 uL dispense, for a final volume of 4 uL, and plates will be incubated protected from the light for 10 min at room temperature. Microplates will then be read on an EnVision multilabel plate reader using the 1,536 plate HTS AlphaScreen aperture (excitation time 80 ms, measurement time 240 ms).
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
* Activity Concentration. ** Test Concentration.
Data Table (Concise)