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BioAssay: AID 504737

Inhibitors of Bloom's syndrome helicase: Caco-2 Permeability 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)
 
 
Active(1)
 
 
 Tested Substances
 Tested Substances
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Active(1)
 
 
 Related BioAssays
 Related BioAssays
AID: 504737
Data Source: NCGC (BLMA601)
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 ):           Active    All
BioActive Compound: 1
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 permeability assay predicts the in vivo absorption of the lead across the intestinal lining by using a Caco-2 cell line and measuring the lead's rate of transport into the cytosol. This result helps with a wide range of drug discovery investigations.

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 150ul DMSO and 50ul LY (5mM) into 50ml HBSS buffer (pH 7.4).

- HBSS buffer with 0.1% DMSO and 5uM Lucifer Yellow (LY): add 50ul DMSO and 50ul 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 150ul DMSO into 50ml HBSS buffer (pH 7.4).

- HBSS buffer with 0.1% DMSO: add 50ul 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 200ul DMSO into 50ml HBSS buffer (pH 7.4)

- For B-to-A direction: Prepare HBSS buffer with 0.4% DMSO and 5uM LY: add 200microl DMSO and 50ul LY (5mM) 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 (100ul for LY, 100ul 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 100ul 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 permeability is greater than 1; "inconclusive" if permeability is between 0.15 and 0.99; "inactive" if permeability is less than 0.15.

PUBCHEM_ACTIVITY_SCORE is the closets whole number of (permeability * 10^-6).
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1PermeabilityB->A Papp Papp 10^-6 m/s (pH 7.4)Float
2Compound QCSource of compound QCString
Additional Information
Grant Number: MH087284

Data Table (Concise)
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