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BioAssay: AID 2712

Counterscreen for BLMA Inhibitors: ADP Fluorescence Polarization Displacement Assay

In order to gain further insight into the mode of action of the BLMAscreening hits, we have profiled them in a set of miniaturized fluorescence polarization assays designed to report on compounds which competitively displace either co-substrate (ATP or DNA). The appropriate fluorescently-labeled probe was used: BODIPY Texas Red-labeled ADP was expected to be competed off by ATP-competitive inhibitors, while single-TAMRA labeled forked-duplex or short single-stranded DNA molecules served as probes for DNA-competitive compounds. ..more
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 Tested Compounds
 Tested Compounds
All(529)
 
 
Active(139)
 
 
Inactive(282)
 
 
Inconclusive(109)
 
 
 Tested Substances
 Tested Substances
All(534)
 
 
Active(143)
 
 
Inactive(282)
 
 
Inconclusive(109)
 
 
 Related BioAssays
 Related BioAssays
AID: 2712
Data Source: NCGC (BLMA419)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-03-26

Data Table ( Complete ):           View Active Data    View All Data
BioActive Compounds: 139
Related Experiments
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2528qHTS Assay for Inhibitors of Bloom's syndrome helicase (BLM)Confirmatorydepositor-specified cross reference: qHTS Assay for Inhibitors of Bloom's syndrome helicase (BLM)
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504662Inhibitors of BLM Helicase: DNA Unwinding Measured by Gel Electrophoresis - BLM Helicase ActivityConfirmatorydepositor-specified cross reference
504663Inhibitors of BLM Helicase: DNA Unwinding Measured by Gel Electrophoresis - RecQL1 Helicase CounterscreenConfirmatorydepositor-specified cross reference
504736Inhibitors of Bloom's syndrome helicase: Efflux Ratio Profiling AssayOthersame project related to Summary assay
504737Inhibitors of Bloom's syndrome helicase: Caco-2 Permeability Profiling AssayOthersame project related to Summary assay
504738Inhibitors of Bloom's syndrome helicase: Aqueous Profiling AssayOthersame project related to Summary assay
504739Inhibitors of Bloom's syndrome helicase: Metabolic Stability ProfilingOthersame project related to Summary assay
504740Inhibitors of Bloom's syndrome helicase: Mouse Plasma Stability ProfilingOthersame project related to Summary assay
504741Inhibitors of Bloom's syndrome helicase: PBS Stability Profiling AssayOthersame project related to Summary assay
720549qHTS for Inhibitors of Bloom's syndrome helicase (BLM): Helicase ATPase Orthogonal Confirmatory Assay for SAROthersame project related to Summary assay
720550qHTS for Inhibitors of Bloom's syndrome helicase (BLM): Thiazole Orange DNA Binding Counterscreen for SAR.Othersame project related to Summary assay
720555qHTS for Inhibitors of Bloom's syndrome helicase (BLM): Helicase DNA unwinding fluorescent orthogonal confirmatory assay for SAROthersame project related to Summary assay
Description:
In order to gain further insight into the mode of action of the BLMAscreening hits, we have profiled them in a set of miniaturized fluorescence polarization assays designed to report on compounds which competitively displace either co-substrate (ATP or DNA). The appropriate fluorescently-labeled probe was used: BODIPY Texas Red-labeled ADP was expected to be competed off by ATP-competitive inhibitors, while single-TAMRA labeled forked-duplex or short single-stranded DNA molecules served as probes for DNA-competitive compounds.

Assay Providers:
Ian Hickson, University of Oxford
Opher Gileadi, Structural Genomics Consortium, University of Oxford

Screening Center PI: Austin, C.P.
Screening Center: NIH Chemical Genomics Center [NCGC]
Protocol
Three uL of reaction buffer (25 mM Tris-HCl (pH 8.0), 5 mM NaCl, 2 mM MgCl2, 1mM DTT, 0.01% Tween- 20, and 2.5 ug/ml poly(dI-dC), containing 300 nM BLMAand 5 nM TAMRA-labeled forked duplex (upper strand 5#-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCGTACCCGATGTGTTCGTTC-TAMRA-3#, lower strand 5#-GAACGAACACATCGGGTACGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3#) were dispensed into a 1536-well Greiner black assay plate via solenoid-valve based nanoliter dispensers; 5 nM TAMRA-labeled forked duplex without BLMAprotein served as a fully-displaced control. Compounds (23 nl each in columns 5-48) were transferred via a Kalypsys pintool equipped with a 1536-pin array. The plates were incubated for 15 min at room temperature and were then transferred into a ViewLux high-throughput CCD imager, where the endpoint fluorescence polarization was measured using 525 nm excitation and 598 nm emission protocol.
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.
Categorized Comment - additional comments and annotations
From ChEMBL:
Assay Type: Functional
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 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 0.0000272478 uM (2.72478e-05μM**)% Activity at given concentration.Float%
15Activity at 0.0000544957 uM (5.44957e-05μM**)% Activity at given concentration.Float%
16Activity at 0.0001089913 uM (0.000108991μM**)% Activity at given concentration.Float%
17Activity at 0.0002179827 uM (0.000217983μM**)% Activity at given concentration.Float%
18Activity at 0.0006220067 uM (0.000622007μM**)% Activity at given concentration.Float%
19Activity at 0.00126 uM (0.00125691μM**)% Activity at given concentration.Float%
20Activity at 0.00191 uM (0.00191282μM**)% Activity at given concentration.Float%
21Activity at 0.00388 uM (0.00387979μM**)% Activity at given concentration.Float%
22Activity at 0.00590 uM (0.00590446μM**)% Activity at given concentration.Float%
23Activity at 0.014 uM (0.0138825μM**)% Activity at given concentration.Float%
24Activity at 0.034 uM (0.0341502μM**)% Activity at given concentration.Float%
25Activity at 0.052 uM (0.052368μM**)% Activity at given concentration.Float%
26Activity at 0.106 uM (0.106015μM**)% Activity at given concentration.Float%
27Activity at 0.162 uM (0.16174μM**)% Activity at given concentration.Float%
28Activity at 0.378 uM (0.377959μM**)% Activity at given concentration.Float%
29Activity at 0.936 uM (0.936155μM**)% Activity at given concentration.Float%
30Activity at 1.441 uM (1.4409μM**)% Activity at given concentration.Float%
31Activity at 3.380 uM (3.38018μM**)% Activity at given concentration.Float%
32Activity at 7.083 uM (7.08302μM**)% Activity at given concentration.Float%
33Activity at 10.25 uM (10.2527μM**)% Activity at given concentration.Float%
34Activity at 25.62 uM (25.6173μM**)% Activity at given concentration.Float%
35Activity at 39.25 uM (39.2511μM**)% Activity at given concentration.Float%
36Activity at 91.86 uM (91.8599μM**)% Activity at given concentration.Float%
37Activity at 228.3 uM (228.277μM**)% Activity at given concentration.Float%
38Activity at 457.1 uM (457.143μM**)% Activity at given concentration.Float%
39Compound QCSource of compound QCString

* Activity Concentration. ** Test Concentration.
Additional Information
Grant Number: MH087284-01

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