qHTS Assay for Inhibitors of BAZ2B: Hit Validation
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: 23
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 provided in this deposition should be used with caution due to the prevalence of screening artifacts . The top compound concentration tested was 110uM in this assay, which increases number of actives and potential 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.
 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
To each well of a 1,536w plate, 2ul of BAZ2B protein (final 50nM) and 1ul biotinylated histone peptide acetylated at Lys14 [b-H3(5-23)K14Ac] (final 50nM) were added in 1x PBS buffer, pH 7.4, containing 0.01% Tween-20, using a nanoliter dispenser. Formation of a protein-peptide complex proceeded at room temperature for 30 min. A Kalypsys pin-tool was employed to transfer 23 nL of library compound solution in DMSO to each well. Following a 30 min incubation of the protein-peptide complex with compounds at room temperature, a mixture of 40 ug/mL each streptavidin-coated donor and nickel chelate acceptor AlphaScreen beads (final 10 ug/mL) were added in a 1ul dispense for a final volume of 4ul. A brief centrifugation followed (1000rpm, 1 min) and were then incubated protected from the light for 20 min at room temperature. Plates were 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)