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BioAssay: AID 2353

qHTS Validation Assay for Inhibitors of RecQ-Like Dna Helicase 1 (RECQ1)

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(1272)
 
 
Active(23)
 
 
Inactive(1220)
 
 
Inconclusive(29)
 
 
 Tested Substances
 Tested Substances
All(1279)
 
 
Active(23)
 
 
Inactive(1227)
 
 
Inconclusive(29)
 
 
AID: 2353
Data Source: NCGC (RECQ201)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2010-02-12
Modify Date: 2010-03-11

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 23
Related Experiments
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
2367Probe Development Summary for Inhibitors of RecQ-Like Dna Helicase 1 (RECQ1)Summarydepositor-specified cross reference
2549qHTS Assay for Inhibitors of RecQ-Like Dna Helicase 1 (RECQ1)Confirmatorydepositor-specified cross reference
2708Confirmation qHTS Assay for Inhibitors of RecQ-Like Dna Helicase 1 (RECQ1)Confirmatorydepositor-specified cross reference
2711Counterscreen for RECQ1 Inhibitors: ADP Fluorescence Polarization Displacement AssayConfirmatorydepositor-specified cross reference
504841Confirmation qHTS Assay for Inhibitors of RecQ-Like Dna Helicase 1 (RECQ1) - Round 2 Cherry PicksConfirmatorysame 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 RECQ1, which is important in resolving abnormal DNA structures formed during replication or homologous recombination. Shutting down the expression of RECQ1 leads to chromosomal instability and higher radiation sensitivity in cultured cells. Remarkably, some cancer cell lines, but not normal cells, exhibit reduced growth and an increase in cell death when RECQ1 expression is inhibited by RNAi.

The validation assay was a fluorescence quenching based kinetic qHTS for RECQ1 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:
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 RECQ1T1), 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, RECQL, RecQ protein-like (DNA helicase Q1-like), RECQL1, Member of the RecQ DNA helicase family
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 0.204 uM (0.204486μM**)% Activity at given concentration.Float%
15Activity at 1.022 uM (1.02234μM**)% Activity at given concentration.Float%
16Activity at 5.111 uM (5.11149μM**)% Activity at given concentration.Float%
17Activity at 25.56 uM (25.556μM**)% Activity at given concentration.Float%
18Compound QCSource of compound QCString

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

Data Table (Concise)
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