AID	Name	Type	Comment
1707	Counterscreen for APE1 Inhibitors: Fluorescent Dye Displacement Validation Assay	confirmatory	Primary qHTS assay for APE1 inhibitors
1708	Counterscreen for APE1 Inhibitors: qHTS Validation Assay for Inhibitors of Endonuclease IV	confirmatory	EndoIV counterscreen
1705	qHTS Validation Assay for Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)	confirmatory	DNA binding counterscreen
2573	qHTS FP-Based Assay for Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)	confirmatory	FP counterscreen
2324	Probe Development Summary of Inhibitors of the Human Apurinic/apyrimidinic Endonuclease 1 (APE1)	summary

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 bioassay provides profiling data on Caco-2 cell permeability of one of the lead compounds developed in the APE1 inhibitors project. This in vitro model is often predictive of in vivo absorption of the compound across the intestinal lining by using a Caco2-cell line and measuring the compound's rate of transport into the cytosol.

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]

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.

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 permeability*10 rounded to the closet whole number.

Grant Number: MH086444-01