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BioAssay: AID 892

qHTS Assay for Inhibitors of BRCT-Phosphoprotein Interaction (Red Fluorophore)

Inhibitors of the BRCT:pBACH1 interaction should prove useful in studies of BRCA1's tumor suppression role and to potentially sensitive tumors to chemotherapeutic agents. A complex of BRCT (C-terminal portion of BRCA1, MW ~35 kDa, His-tagged) and fluorescently labeled pBACH1 phosphorylated 10 aa peptide fragment of BACH1, a helicase implicated in DNA repair, is incubated with library members. more ..
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 Tested Compounds
 Tested Compounds
All(73910)
 
 
Active(20)
 
 
Inactive(73847)
 
 
Inconclusive(45)
 
 
 Tested Substances
 Tested Substances
All(75028)
 
 
Active(20)
 
 
Inactive(74962)
 
 
Inconclusive(46)
 
 
AID: 892
Data Source: NCGC (BRCA738)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Screening Center Network
BioAssay Version:
Deposit Date: 2007-12-17
Modify Date: 2010-07-12

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 20
Related Experiments
AIDNameTypeComment
875qHTS Assay for Inhibitors of BRCT-Phosphoprotein Interaction (Green Fluorophore)Confirmatorydepositor-specified cross reference
Description:
Assay Overview:

NIH Molecular Libraries Screening Centers Network [MLSCN]
NIH Chemical Genomics Center [NCGC]
MLSCN Grant: MH081227-01
PI Name: Natarajan, Amarnath; University of Texas

Inhibitors of the BRCT:pBACH1 interaction should prove useful in studies of BRCA1's tumor suppression role and to potentially sensitive tumors to chemotherapeutic agents. A complex of BRCT (C-terminal portion of BRCA1, MW ~35 kDa, His-tagged) and fluorescently labeled pBACH1 phosphorylated 10 aa peptide fragment of BACH1, a helicase implicated in DNA repair, is incubated with library members. Inhibitors of BRCT-pBACH1 binding are detected by a decrease in the fluorescence polarization (FP) of the fluorophore. In order to minimize the effect of fluorescence artifacts and to increase the overall robustness of the screen, the compound library members were incubated in separate reactions with rhodamine-labeled probe-protein complexes. Protein and labeled probes were supplied by Dr. Amarnath Natarajan, Department of Pharmacology and Toxicology, University of Texas Medical Branch Galveston, TX.
Protocol
Reagents/Controls:

Assay Buffer: 16mM Na2HPO4, 4 mM NaH2PO4, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.02% NaN3, and 0.01% Tween 20 at pH 7.3.

Assay Controls: 100 nM TAMRA labeled decapeptide dispensed into columns 3 and 4 to generate negative control (full inhibition of
binding, -100 % activity). 100 nM labeled decapeptide/100 nM protein in columns 1, 2, 5 - 48. Titration of unlabeled decapeptide control (from 10 mM, then 1:2 dilution in duplicate) pin-transferred to column 2, rows 1 to 16. Column 1 is neutral.

Assay Steps:

Three uL of reagents were dispensed to 1536-well Greiner black solid-bottom plates. Compounds and controls (23 nL) were transferred via Kalypsys PinTool. The plates were incubated for 12 min at room temperature, and then read on ViewLux (Perkin-Elmer) High-throughput CCD imager using BODIPY for the rhodamine-labeled decapeptide probe polarization filter sets. During dispense, reagent bottles were kept submerged into 4 deg C water bath and all liquid lines were covered with aluminum foil to minimize probe and protein degradation. All screening operations were performed on a Kalypsys robotic system (Kalypsys, Inc., San Diego, CA) containing one RX-130 and two RX-90 anthropomorphic robotic arms. The timing and order of assay plates passing through the screening system were adjusted such that each compound library plate was assayed against the fluorescein- and rhodamine-labeled assay systems at immediately adjacent time points.
Comment
Keywords: NIH Roadmap, MLSCN, MLI, MLSMR, SGC, qHTS, NCGC, brct, brca1

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
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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 signficant, but below 80% of control.String
6Fit_LogAC50The logarithm of the AC50 from a fit of the ratio data to the Hill equation.Float
7Fit_HillSlopeThe Hill slope from a fit of the ratio data to the Hill equation.Float
8Fit_R2R^2 fit value of ratio curve. Closer to 1.0 equates to better Hill equation fit.Float
9Fit_InfiniteActivityThe asymptotic efficacy from a fit of the ratio data to the Hill equation.Float%
10Fit_ZeroActivityEfficacy at zero concentration of compound from a fit of the ratio 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 0.0009850345 uM (0.000985035μM**)% Activity at given concentration.Float%
15Activity at 0.00220 uM (0.00220254μM**)% Activity at given concentration.Float%
16Activity at 0.00492 uM (0.00492487μM**)% Activity at given concentration.Float%
17Activity at 0.011 uM (0.011012μM**)% Activity at given concentration.Float%
18Activity at 0.025 uM (0.0246229μM**)% Activity at given concentration.Float%
19Activity at 0.055 uM (0.0550567μM**)% Activity at given concentration.Float%
20Activity at 0.123 uM (0.123107μM**)% Activity at given concentration.Float%
21Activity at 0.275 uM (0.275267μM**)% Activity at given concentration.Float%
22Activity at 0.615 uM (0.615497μM**)% Activity at given concentration.Float%
23Activity at 1.376 uM (1.37625μM**)% Activity at given concentration.Float%
24Activity at 3.077 uM (3.0773μM**)% Activity at given concentration.Float%
25Activity at 6.881 uM (6.88084μM**)% Activity at given concentration.Float%
26Activity at 15.39 uM (15.3856μM**)% Activity at given concentration.Float%
27Activity at 34.40 uM (34.4021μM**)% Activity at given concentration.Float%
28Activity at 76.92 uM (76.9231μM**)% Activity at given concentration.Float%
29Compound QCNCGC designation for data stage: 'qHTS', 'qHTS Verification', 'Secondary Profiling'String

* Activity Concentration. ** Test Concentration.

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
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