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 ..
BioActive Compounds: 96
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 . 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.
 Esteller, M. Epigenetics in Cancer. N Engl J Med 358(11), 1148-1159. (2008). PMID: 18337604
 Yasgar, et al. Compound Management for Quantitative High-Throughput Screening. JALA. 2008 Apr;13(2):79-89. PMID: 18496600
 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
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.
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)