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BioAssay: AID 504662

Inhibitors of BLM Helicase: DNA Unwinding Measured by Gel Electrophoresis - BLM Helicase Activity

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(1)
 
 
Active(1)
 
 
 Tested Substances
 Tested Substances
All(1)
 
 
Active(1)
 
 
AID: 504662
Data Source: NCGC (BLMA603)
BioAssay Type: Confirmatory, Concentration-Response Relationship Observed
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-04-13
Hold-until Date: 2012-03-31
Modify Date: 2012-03-31

Data Table ( Complete ):           Active    All
Target
BioActive Compound: 1
Depositor Specified Assays
AIDNameTypeComment
2386Probe Development Summary for Inhibitors of Bloom's syndrome helicase (BLM)summaryProbe Development Summary for Inhibitors of Bloom's syndrome helicase (BLM)
2585qHTS Confirmation Assay for Inhibitors of Bloom's syndrome helicase (BLM)confirmatoryqHTS Confirmation Assay for Inhibitors of Bloom's syndrome helicase (BLM)
2528qHTS Assay for Inhibitors of Bloom's syndrome helicase (BLM)confirmatoryqHTS Assay for Inhibitors of Bloom's syndrome helicase (BLM)
2364qHTS Validation Assay for Inhibitors of Bloom's syndrome helicase (BLM)confirmatoryqHTS Validation Assay for Inhibitors of Bloom's syndrome helicase (BLM)
2712Counterscreen for BLMA Inhibitors: ADP Fluorescence Polarization Displacement AssayconfirmatoryCounterscreen for BLMA Inhibitors: ADP Fluorescence Polarization Displacement 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 DNA unwinding assay measured by gel electrophoresis (32P-labeled substrate) 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.

Initial screening hits were validated by the standard assay for measuring DNA helicase activity, which utilizes gel electrophoresis to monitor the helicase-mediated unwinding of a radiolabeled DNA substrate.
Protocol
Unwinding assays using BLM helicase was carried out in a reaction buffer containing 50 mM Tris-HCl (pH 7.5), 50 mM NaCl, 2 mM MgCl2, 2 mM ATP, 1 mM DTT, and 0.1 mg/ml BSA. Optimal substrate and enzyme concentrations as well as reaction times were determined experimentally. All reactions were performed at 37 degrees C and terminated by the addition of loading buffer containing 20 mM EDTA and 2.5% bromophenol blue. Reaction products were separated on 15% nondenaturing polyacrylamide gels in 1x TBE at 30 mA for 1 hr at 4 degrees C. The gels were then dried and visualized by autoradiography (Kodak BioMax MR-1).
Comment
Keywords, BLMA, RecQ Helicase, RecQ-like type 2, Bloom syndrome protein

Compounds that were inactive were given a PUBCHEM_ACTIVITY_SCORE of 0. Active compounds got a PUBCHEM_ACTIVITY_SCORE of -10*Log_IC50 rounded to the closest whole number.
Result Definitions
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, IC50.FloatμM
3Log_IC50The logarithm of the Potency (calculated based on Molar Units).Float

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

Data Table (Concise)
Classification
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