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BioAssay: AID 588809

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 [1], 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 [2]. ..more
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 Tested Compounds
 Tested Compounds
All(25)
 
 
Active(23)
 
 
Inconclusive(2)
 
 
 Tested Substances
 Tested Substances
All(25)
 
 
Active(23)
 
 
Inconclusive(2)
 
 
AID: 588809
Data Source: NCGC (BAZ2724)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-11-17

Data Table ( Complete ):           Active    All
Target
BioActive Compounds: 23
Depositor Specified Assays
AIDNameTypeComment
504391qHTS Assay for Inhibitors of BAZ2B: SummarysummarySummary
Description:
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 [1], 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 [2].

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 [5], 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 [6]. 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.

[1] 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

[2] Jones, M.H. et al. A novel family of bromodomain genes. Genomics 63, 40-45. 2000. PMID: 10662543

[3] Yasgar, et al. Compound Management for Quantitative High-Throughput Screening. JALA. 2008 Apr;13(2):79-89. PMID: 18496600

[4] 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

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

[6] 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 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).
Comment
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 0.00465 uM (0.00464783μM**)% Activity at given concentration.Float%
15Activity at 0.019 uM (0.0186005μM**)% Activity at given concentration.Float%
16Activity at 0.026 uM (0.0261284μM**)% Activity at given concentration.Float%
17Activity at 0.077 uM (0.0770059μM**)% Activity at given concentration.Float%
18Activity at 0.109 uM (0.109157μM**)% Activity at given concentration.Float%
19Activity at 0.255 uM (0.254601μM**)% Activity at given concentration.Float%
20Activity at 0.448 uM (0.447591μM**)% Activity at given concentration.Float%
21Activity at 0.705 uM (0.705467μM**)% Activity at given concentration.Float%
22Activity at 0.893 uM (0.892857μM**)% Activity at given concentration.Float%
23Activity at 1.190 uM (1.19048μM**)% Activity at given concentration.Float%
24Activity at 2.113 uM (2.11333μM**)% Activity at given concentration.Float%
25Activity at 3.571 uM (3.57143μM**)% Activity at given concentration.Float%
26Activity at 5.757 uM (5.75651μM**)% Activity at given concentration.Float%
27Activity at 7.143 uM (7.14286μM**)% Activity at given concentration.Float%
28Activity at 13.93 uM (13.932μM**)% Activity at given concentration.Float%
29Activity at 19.05 uM (19.0476μM**)% Activity at given concentration.Float%
30Activity at 28.57 uM (28.5714μM**)% Activity at given concentration.Float%
31Activity at 57.14 uM (57.1428μM**)% Activity at given concentration.Float%
32Activity at 114.3 uM (114.286μM**)% Activity at given concentration.Float%
33Activity at 228.6 uM (228.571μM**)% Activity at given concentration.Float%
34Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

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

Data Table (Concise)
Classification
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