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BioAssay: AID 504736

Inhibitors of Bloom's syndrome helicase: Efflux Ratio Profiling Assay

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 more ..
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 Tested Compounds
 Tested Compounds
All(1)
 
 
Inconclusive(1)
 
 
 Tested Substances
 Tested Substances
All(1)
 
 
Inconclusive(1)
 
 
 Related BioAssays
 Related BioAssays
AID: 504736
Data Source: NCGC (BLMA602)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-05-09
Hold-until Date: 2012-05-06
Modify Date: 2012-05-08

Data Table ( Complete ):           All
Tested Compound:
Depositor Specified Assays
AIDNameTypeComment
2386Probe Development Summary for Inhibitors of Bloom's syndrome helicase (BLM)summarySummary AID
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. Bloom syndrome helicase (BLM), 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.

This bioassays provides profiling data on Caco-2 cell permeability of leads compounds developed for the HPGD project. This in vitro model is often predictive of in vivo efflux of the compound by measuring the ratio of transport in and out the plasma membrane of the Caco2-cell line.

NIH Chemical Genomics Center [NCGC]
NIH Molecular Libraries Probe Production Centers Network [MLPCN]

MLSCN Grant: MH087284
PI Name: Dr. Opher Gileadi, Structural Genomics Consortium, University of Oxford, UK
Protocol
1. Buffer Preparation:
Prepare 1M HEPES: add 23.83g HEPES to 100ml of 0.85% NaCl
Prepare HBSS+ buffer: add 12.8ml of 1M HEPES in 0.85% NaCl to 500ml HBSS buffer.

2. Take out the compounds from -80 degrees C, ultrasonicate or pre-warm for a few minutes to thaw the compounds.

3. Solution preparation

Prepare donor buffer:
For A-to-B direction:
HBSS buffer with 0.3% DMSO and 5uM Lucifer Yellow (LY): add 150mul DMSO and 50mul LY (5mM) into 50ml HBSS buffer (pH 7.4).
HBSS buffer with 0.1% DMSO and 5muM Lucifer Yellow (LY): add 50mul DMSO and 50mul LY (5mM) into 50ml HBSS buffer (pH 7.4).
HBSS buffer with 5uM Lucifer Yellow (LY): add 50ul LY (5mM) into 50ml HBSS buffer (pH 7.4).
For B-to-A direction:
HSS buffer with 0.3% DMSO: add 150mul DMSO into 50ml HBSS buffer (pH 7.4).
HBSS buffer with 0.1% DMSO: add 50mul DMSO into 50ml HBSS buffer (pH 7.4).
HBSS buffer without DMSO.

Prepare receiver buffer:
For A-to-B direction: Prepare HBSS buffer with 0.4% DMSO: add 200mul DMSO into 50ml HBSS buffer (pH 7.4)
For B-to-A direction: Prepare HBSS buffer with 0.4% DMSO and 5muM LY: add 200microl DMSO and 50mul LY (5 mM) into 50ml HBSS buffer (pH 7.4)

4. Centrifuge the diluted solutions at 4000 rpm, 5 min. Supernatants are collected for compound dosing.

5. TEER measurement.

6. Add donor and receiver solutions to corresponding wells:
A-B (Donor): 600ul A to B dosing solution (100mul for LY, 100mul for T0 sample);
A-B (Receiver): 800ul 0.4% DMSO HBSS;
B-A (Donor): 900ul B to A dosing solution (100ul for T0 sample);
B-A (Receiver): 500ul 0.4% DMSO HBSS+LY (100ul for LY).

7. Pre-warm assay plate at 37 degrees C for 5 min, take out 100ul from donor solution as T0 sample (A-B D0, B-A D0). And take another 100ul from donor solution (A-B) or receiver solution (B-A) to opaque plate as T0, LY for Lucifer Yellow determination. Then start incubation.

8. Lucifer Yellow measurement: at 90 min, take 100mul from basolateral side (receiver of A-B and donor of B-A) as T90, LY to opaque plate. The T0, LY and T90, LY plates are read by FlexStation 3 at excitation of 485 nm and emission of 535 nm to determine LY permeability.

9. Sample preparation:
For receiver solution: 60ul of sample + 60ul ACN with IS (200ng/ml Osalmid)
For donor solution: 6ul of sample + 54ul 0.4% DMSO/HBSS + 60ul ACN with IS (200 ng/ml Osalmid)

10. Samples are submitted for LC-MS/MS analysis.
Comment
Compounds are "active" if efflux is less than 1.5; "inconclusive" if efflux is between 1.5 and 2.5; "inactive" if efflux is greater than 2.5.

PUBCHEM_ACTIVITY_SCORE is the efflux ratio * 10.
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Efflux Ratio(B-->A)/(A-->B)Float
2Compound QCSource of compound QCString
Additional Information
Grant Number: MH087284

Data Table (Concise)
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