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BioAssay: AID 1818

Summary of High-throughput multiplex screening for ABC transporter inhibitors

The three major types of multidrug resistance (MDR) proteins in humans include members of the ABCB, the ABCC and the ABCG subfamilies. These proteins influence oral absorption and disposition of a wide variety of drugs. As a result, their expression levels have important consequences for susceptibility to drug-induced side effects, interactions, and treatment efficacy. ..more
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AID: 1818
Data Source: NMMLSC (UNM_ABC_TRANSPORTERS_SUMMARY)
BioAssay Type: Summary, Candidate Probes/Leads with Supporting Evidence
Depositor Category: NIH Molecular Libraries Probe Production Network
BioAssay Version:
Deposit Date: 2009-06-17
Modify Date: 2012-10-10

Data Table ( Complete ):           View Active Data    View All Data
Targets
BioActive Compound: Chemical Probe: 1    Active: 1
Related Experiments
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AIDNameTypeComment
1325High-throughput multiplex screening for ABC transporter inhibitors: specifically ABCG2 screen, ABCB1 counter-screenScreeningdepositor-specified cross reference: HTS Multiplex Screen for ABC transporter inhibitors, specifically ABCG2 (ABCB1 counterscreen)
1326High-throughput multiplex screening for ABC transporter inhibitors: specifically ABCB1 screen, ABCG2 counter-screenScreeningdepositor-specified cross reference: HTS Multiplex Screen for ABC transporter inhibitors, specifically ABCB1 (ABCG2 counterscreen)
1451Single point confirmation for ABC transporter inhibitors: specifically ABCB1 screen, ABCG2 counter-screenScreeningdepositor-specified cross reference: Single point confirmation for ABC transporter inhibitors, specifically ABCB1 (ABCG2 counterscreen)
1453Single point confirmation screening for ABC transporter inhibitors: specifically ABCG2 screen, ABCB1 counter-screenScreeningdepositor-specified cross reference: HTS Multiplex Screen for ABC transporter inhibitors, specifically ABCG2 (ABCB1 counterscreen)
1480Profiling compound fluorescence in IgMXP3 at 488/530 nm; counter screen to single point confirmation of ABCG2 screenScreeningdepositor-specified cross reference: Profiling compound fluorescence in IgMXP3 at 488/530 nm; counter screen to single point confirmation
1483Profiling compound fluorescence in CCRF-Adr at 488/530 nm; counter screen to single point confirmation of ABCB1 screenScreeningdepositor-specified cross reference: Profiling compound fluorescence in CCRF-Adr at 488/530 nm; counter screen to single point confirmati
1689Dose Response for ABC transporter inhibitors: specifically ABCB1 screen, ABCG2 counter-screenConfirmatorydepositor-specified cross reference: Dose Response for ABC transporter inhibitors, specifically ABCB1 (ABCG2 counter-screen)
1690Dose Response for ABC transporter inhibitors: specifically ABCG2 screen, ABCB1 counter-screenConfirmatorydepositor-specified cross reference: Dose Response for ABC transporter inhibitors, specifically ABCB1 (ABCG2 counter-screen)
2830Chemo Reversal assay in ABCG2-expressing cells for one set of SAR compoundsOtherdepositor-specified cross reference: Chemo Reversal assay in ABCG2-expressing cells for one set of SAR compounds
2833Chemo Reversal assay in ABCB1-expressing cells for one set of SAR compoundsOtherdepositor-specified cross reference: Chemo Reversal assay in ABCB1-expressing cells for one set of SAR compounds
488973SAR hit validation chemo-reversal assay specifically in in ABCB1-expressing cells, ABCG2 counterscreenOtherdepositor-specified cross reference: SAR hit validation chemo-reversal assay specifically in in ABCB1-expressing cells, ABCG2 counterscre
488974SAR hit validation chemo-reversal assay specifically in in ABCG2-expressing cells, ABCB1 counterscreenOtherdepositor-specified cross reference: SAR hit validation chemo-reversal assay specifically in in ABCG2-expressing cells, ABCB1 counterscre
489002Dose Response for ABC transporter inhibitors: specifically ABCB1 screen, ABCG2 counter-screen. Follow-up assay.Confirmatorydepositor-specified cross reference: Dose Response for ABC transporter inhibitors: specifically ABCB1 screen, ABCG2 counter-screen. Follo
489003Dose Response for ABC transporter inhibitors: specifically ABCG2 screen, ABCB1 counter-screen. Follow-up assay.Confirmatorydepositor-specified cross reference: Dose Response for ABC transporter inhibitors: specifically ABCG2 screen, ABCB1 counter-screen. Follo
504476Chemo Reversal assay in ABCB1-expressing cells for set2 of SAR compoundsOtherdepositor-specified cross reference
504477Chemo Reversal assay in ABCG2-expressing cells for set 2 of SAR compoundsConfirmatorydepositor-specified cross reference
504566Dose Response for ABC transporter inhibitors: specifically ABCG2 screen, ABCB1 counter-screen. Follow-up assay on set 2Confirmatorydepositor-specified cross reference
504569Dose Response for ABC transporter inhibitors: specifically ABCB1 screen, ABCG2 counter-screen. Follow-up assay for Set 2Confirmatorydepositor-specified cross reference
Description:
University of New Mexico Assay Overview:
Assay Support: 1R03MH081228-01A1
High-throughput multiplex screening for ABC transporter inhibitors
Assay Provider: Richard Larson
Assay Development: Irena Ivnitski-Steele PhD, J. Jacob Strouse PhD
Assay Implementation: J. Jacob Strouse PhD,
Irena Ivnitski-Steele PhD, Terry Foutz, Anna Waller, Mark Carter
Screening Center PI: Larry Sklar

Assay Background and Significance:
The three major types of multidrug resistance (MDR) proteins in humans include members of the ABCB, the ABCC and the ABCG subfamilies. These proteins influence oral absorption and disposition of a wide variety of drugs. As a result, their expression levels have important consequences for susceptibility to drug-induced side effects, interactions, and treatment efficacy.

The use of low toxicity ABC transporter inhibitors is a common treatment strategy to circumvent MDR in cancers [Gillet et al. 2007, O'Connor 2007]. Although definitive results for any of the compounds concerning their efficacy for multiple drug resistance has not been forthcoming, it is not surprising, given their specificity for other molecular targets, that many problems such as significant side effects, dose effects, and changes in chemotherapy pharmacokinetics are of constant concern and provide ample justification for identifying new classes of modulators and exploring the biology around them.


A duplex assay was constructed in which ABCB1 and ABCG2 transporters were evaluated in parallel using fluorescent J-aggregate-forming lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide as substrate. ABCB1-expressing cells (CCRF-Adr) were color-coded to allow their distinction from ABCG2-expressing cells(IgMXP3).

This report summarizes the series of assays used to identify and characterize small molecule modulators of ABC transporters from the MLSMR library collection.

This project resulted in a probe compound specfic for ABCG2 transporter:
(SID / CID / ML#)
88095709 / 44640177 / ML230
Protocol
The screening assay was a high throughput no-wash procedure conducted in 384-well format micro plates in a total volume of 15.1 microliters. CCRF-Adr cells (ABCB1) were color-coded with FarRed DDAO CellTrace SE (Invitrogen) and combined with unlabeled IgMXP3 cells (ABCG2). The label binds covalently to amine groups in cells and is detected at red fluorescence emission wavelengths of 665 +/- 10 nm upon excitation at 635 nm. Final in-well concentration of test compound was 6.6 micromolar and of cells was 3 million cells/ml (1:1 ratio of the two cell types). Sample analysis was conducted with the HyperCyt(R) high throughput flow cytometry platform [Kuckuck, et al., 2001; Ramirez, et al., 2003]. The assay response range was defined by control wells containing Nicardipine (pump inhibitor, high fluorescence) or DMSO (no pump inhibition, low fluorescence).

Keywords:
NIH Roadmap, NMMLSC, high-throughput flow cytometry, drug-resistance transporters, ABCB1, ABCB2, multiplex cell-based screening

Summary and Sequence of Assays:

AID 1325: HTS Multiplex Screen for ABC transporter inhibitors, specifically ABCG2 (ABCB1 counterscreen)
Method: flow cytometry, multiplex cell-based, ligand binding competition assay
Test concentration: 6.6 microM
Activity criterion: >80% inhibition
# evaluated: 194,934
# active: 200

AID 1326: HTS Multiplex Screen for ABC transporter inhibitors, specifically ABCB1 (ABCB2 counterscreen)
Method: flow cytometry, multiplex cell-based, ligand binding competition assay
Test concentration: 6.6 microM
Activity criterion: >40% inhibition
# evaluated: 193,675
# active: 130

AID 1451: Single point confirmation for ABC transporter inhibitors, specifically ABCB1 (ABCG2 counterscreen)
Method: flow cytometry, multiplex cell-based, ligand binding competition assay
Test concentration: 6.6 microM
Activity criterion: >50% inhibition
# evaluated: 273
# active: 18

AID 1453: HTS Multiplex Screen for ABC transporter inhibitors, specifically ABCG2 (ABCB1 counterscreen)
Method: flow cytometry, multiplex cell-based, ligand binding competition assay
Test concentration: 6.6 microM
Activity criterion: >50% inhibition
# evaluated: 273
# active: 16

AID 1689: Dose Response for ABC transporter inhibitors, specifically ABCB1 (ABCG2 counter-screen)
Method: flow cytometry, multiplex cell-based, ligand binding competition assay
Test concentration: 9-point concentration curve from 0.15 microM to 66 microM
Activity criterion: EC50 < 10 microM
# evaluated: 40
# active: 9

AID 1690: Dose Response for ABC transporter inhibitors, specifically ABCB1 (ABCG2 counter-screen)
Method: flow cytometry, multiplex cell-based, ligand binding competition assay
Test concentration: 9-point concentration curve from 0.15 microM to 66 microM
Activity criterion: EC50 < 20 microM
# evaluated: 40
# active: 16

Additional SAR studies have been reported in AIDs currently OnHold: 2830, 2833, 488973 and 488974
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1ML#ML probe numberString
2EC50EC50 reported in probe report of target proteinFloatμM
Additional Information
Grant Number: 1R03MH081228-01A1

Data Table (Concise)
Data Table ( Complete ):     View Active Data    View All Data
Classification
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