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BioAssay: AID 504618

Inhibitors of APE1: Efflux Ratio Profiling

The apurinic/apyrimidinic endonuclease APE1 is the primary mammalian enzyme responsible for the removal of abasic (or AP) sites in DNA and functions centrally in the base excision DNA repair (BER) pathway. Recent studies suggested a link between an overexpression of APE1 in many cancers and resistance of these tumor cells to radio- and chemotherapy. Thus, targeting APE1 could improve the efficacy of current treatment paradigms by promoting selective sensitization or protection of diseased and normal cells, respectively. ..more
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 Tested Compounds
 Tested Compounds
All(1)
 
 
Active(1)
 
 
 Tested Substances
 Tested Substances
All(1)
 
 
Active(1)
 
 
AID: 504618
Data Source: NCGC (APE1803)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2011-03-29
Hold-until Date: 2011-05-01
Modify Date: 2011-05-03

Data Table ( Complete ):           Active    All
Target
BioActive Compound: 1
Depositor Specified Assays
AIDNameTypeComment
2324Probe Development Summary of Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)summarySummary
2573qHTS FP-Based Assay for Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)confirmatoryFP counterscreen
1705qHTS Validation Assay for Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)confirmatoryDNA binding counterscreen
1708Counterscreen for APE1 Inhibitors: qHTS Validation Assay for Inhibitors of Endonuclease IVconfirmatoryEndoIV counterscreen
1707Counterscreen for APE1 Inhibitors: Fluorescent Dye Displacement Validation AssayconfirmatoryPrimary qHTS assay for APE1 inhibitors
Description:
The apurinic/apyrimidinic endonuclease APE1 is the primary mammalian enzyme responsible for the removal of abasic (or AP) sites in DNA and functions centrally in the base excision DNA repair (BER) pathway. Recent studies suggested a link between an overexpression of APE1 in many cancers and resistance of these tumor cells to radio- and chemotherapy. Thus, targeting APE1 could improve the efficacy of current treatment paradigms by promoting selective sensitization or protection of diseased and normal cells, respectively.

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.

APE1 Project Principal Investigator: David M. Wilson, III, National Institute on Aging, NIH
Screening Center PI: Austin, C.P.
Screening Center: NIH Chemical Genomics Center [NCGC]
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 * 100.
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: MH086444-01

Data Table (Concise)
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