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BioAssay: AID 2364

qHTS Validation Assay for Inhibitors of Bloom's syndrome helicase (BLM)

Survival of cells and the faithful propagation of the genome depend on elaborate mechanisms of detecting and repairing DNA damage. Treatment of advanced cancer relies on radiation therapy or chemotherapy, which kill cancer cells by causing extensive DNA damage. It is often found, that cancer cells develop resistance to therapy through enhanced activity of DNA repair functions; this has led to an increased interest in developing drugs that interfere with DNA repair, which could sensitize cancer cells to conventional therapy. ..more
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 Tested Compounds
 Tested Compounds
All(1273)
 
 
Active(30)
 
 
Inactive(522)
 
 
Inconclusive(725)
 
 
 Tested Substances
 Tested Substances
All(1280)
 
 
Active(30)
 
 
Inactive(522)
 
 
Inconclusive(728)
 
 
AID: 2364
Data Source: NCGC (BLMA201)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-02-16
Modify Date: 2010-03-11

Data Table ( Complete ):           View Active Data    View All Data
Target
Sequence: Bloom syndrome protein [Homo sapiens]
Description ..   
Protein Family: HELICc

Gene:BLM     Related Protein 3D Structures     More BioActivity Data..
BioActive Compounds: 30
Related Experiments
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AIDNameTypeComment
593qHTS Assay for Spectroscopic Profiling in Fluorescein Spectral RegionOtherdepositor-specified cross reference: Fluorescein region spectral profiling screen
594qHTS Assay for Spectroscopic Profiling in Rhodamine Spectral RegionConfirmatorydepositor-specified cross reference: Rhodamine region spectral profiling screen
2386Probe Development Summary for Inhibitors of Bloom's syndrome helicase (BLM)Summarydepositor-specified cross reference
2528qHTS Assay for Inhibitors of Bloom's syndrome helicase (BLM)Confirmatorydepositor-specified cross reference
2585qHTS Confirmation Assay for Inhibitors of Bloom's syndrome helicase (BLM)Confirmatorydepositor-specified cross reference
2712Counterscreen for BLMA Inhibitors: ADP Fluorescence Polarization Displacement AssayConfirmatorydepositor-specified cross reference
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:
Survival of cells and the faithful propagation of the genome depend on elaborate mechanisms of detecting and repairing DNA damage. Treatment of advanced cancer relies on radiation therapy or chemotherapy, which kill cancer cells by causing extensive DNA damage. It is often found, that cancer cells develop resistance to therapy through enhanced activity of DNA repair functions; this has led to an increased interest in developing drugs that interfere with DNA repair, which could sensitize cancer cells to conventional therapy.

This validation qHTS assay pertains to human BLM, which is important in resolving abnormal DNA structures formed during replication or homologous recombination. Shutting down the expression of BLM leads to chromosomal instability and higher radiation sensitivity in cultured cells.

The validation assay was a fluorescence quenching based kinetic qHTS for BLM Helicase DNA unwinding. The activity was measured as ATP-dependent separation of a 20-bp DNA duplex extended by 30-nt single-stranded tails (forked duplex). The forked DNA substrate was tagged with rhodamine fluorophore (carboxytetramethyl rhodamine, TAMRA) and BHQ-2 (Black Hole Quencher 2) dark quencher. Strand separation results in an increase in the fluorescence of TAMRA (excitation 525 nm, emission 598 nm). This substrate construct operates in a red-shifted region where very few compound library members have been noted to fluoresce (PubChem AIDs 593 and 594). An additional feature of the assay is the inclusion of 2.5 ug/ml poly (dIdC), to reduce interference by compounds such as DNA intercalators, a major source of false inhibitors.

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), including enzyme (50 nM BLM), were dispensed into a 1536-well Greiner black assay plate via solenoid-valve based nanoliter dispensers. Compounds (23 nl each in columns 5-48) and control (23 nl each in column 2 as dose-response) were transferred via a Kalypsys pintool equipped with a 1536-pin array. The plates were incubated for 15 min at room temperature, and then 1 ul of substrates (200 nM DNA and 2 mM ATP, final concentrations) was added to start the reaction. The plate was transferred into a ViewLux high-throughput CCD imager where the reaction progress was measured in a discontinuous kinetic mode (two reads spaced 2 hours apart, with intermediate room temperature incubation of the covered assay plate) by using standard red-fluorescence optics (excitation filter 525 nm, emission filter 598 nm). Enzyme-free and vehicle (DMSO) controls were included for signal normalization.
Comment
Keywords, BLMA, RecQ Helicase, RecQ-like type 2, Bloom syndrome protein
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 0.00366 uM (0.00366μM**)% Activity at given concentration.Float%
15Activity at 0.018 uM (0.0183μM**)% Activity at given concentration.Float%
16Activity at 0.091 uM (0.0914μM**)% Activity at given concentration.Float%
17Activity at 0.457 uM (0.457μM**)% Activity at given concentration.Float%
18Activity at 2.290 uM (2.29μM**)% Activity at given concentration.Float%
19Activity at 11.40 uM (11.4μM**)% Activity at given concentration.Float%
20Activity at 57.10 uM (57.1μM**)% Activity at given concentration.Float%
21Compound 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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