qHTS Assay for Inhibitors of BAZ2B: Hit Validation in AlphaScreen Counterscreen.
BAZ2B (bromodomain adjacent to zinc finger domain, 2B) belongs to a family of ubiquitously expressed bromodomain containing proteins, which biological function has not yet been elucidated. However, it is suggested that BAZ2B has a similar function as the Drosophila Acf1 protein which regulates nucleosome mobilization through the ATP-dependent chromatin remodelling factor ISWI , resulting in alteration in the translational position of the histone and hence transcriptional regulation. The interaction of BAZ2B with ISWI mediated by the BAZ1 motif has recently been described . ..more
BioActive Compounds: 2
BAZ2B (bromodomain adjacent to zinc finger domain, 2B) belongs to a family of ubiquitously expressed bromodomain containing proteins, which biological function has not yet been elucidated. However, it is suggested that BAZ2B has a similar function as the Drosophila Acf1 protein which regulates nucleosome mobilization through the ATP-dependent chromatin remodelling factor ISWI , resulting in alteration in the translational position of the histone and hence transcriptional regulation. The interaction of BAZ2B with ISWI mediated by the BAZ1 motif has recently been described .
There is a need for chemical probes to allow the elucidation of the roles of this protein in health and disease and hence a quantitative high-throughput screen [3,4] was developed. The protocol is based on the AlphaScreen (PerkinElmer) displacement assay developed by NCGC , but uses the peptide ligand Biot-H3K14Ac, a high affinity binding partner for BAZ2B, in an AlphaScreen format.
Although AlphaScreen has significant advantages with its utility in a variety of epigenetic target assays, the primary screening data (linked to this assay) should be used with caution due to the prevalence of screening artifacts . An AlphaScreen counterscreen was developed and ran against any putative actives to eliminate non-specific artifacts. This alpha-screen uses AlphaScreen technology but does not include the target protein, Baz2B. Compounds that were active in the primary screen were run in this this counterscreen. Compounds that are active in this screen are compounds that are artifacts in the primary screen. Compounds that are inactive in this screen are compounds that are were genuine hits in the primary screen
 Eberharter, A. et al. ACF1 improves the effectiveness of nucleosome mobilization by ISWI through PHD-histone contacts. The EMBO journal 23, 4029-4039. 2004. PMID: 15457208
 Jones, M.H. et al. A novel family of bromodomain genes. Genomics 63, 40-45. 2000. PMID: 10662543
 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
 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 Molecular Libraries Probe Production Network [MLPCN]
NIH Chemical Genomics Center [NCGC]
Structural Genomics Consortium (SGC)
NIH Grant: 5U54 MH084681-02
Biotinylated-hexahistidine (PerkinElmer, Waltham, MA) will be added to 1,536-well plates in a 3microl dispense (15nM final concentration). Compounds will be added in a 23nl pin-transfer step, and plates will be incubated at room temperature for 15 min. Streptavidin-coated donor and nickel chelate acceptor AlphaScreen beads will be added (1microl) for final concentrations of 10 microg/ml each bead, and plates read as described above (qHTS assay) after a 30 min incubation at room temperature in the dark.
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".
2. For all inactive (genuine hits) compounds, PUBCHEM_ACTIVITY_SCORE is 0. For all active (artifacts) compounds, a score range was given for each curve class type given above. Active (artifacts) 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.
* Activity Concentration. ** Test Concentration.
Data Table (Concise)