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BioAssay: AID 488973

SAR hit validation chemo-reversal assay specifically in in ABCB1-expressing cells, ABCG2 counterscreen

Assay Implementation: Hadya Njus, Diane Jimenez-Stinson, J. Jacob Strouse, Anna Waller, Mark Carter, Annette Evangelisti ..more
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 Tested Compounds
 Tested Compounds
All(37)
 
 
Active(9)
 
 
Inactive(28)
 
 
 Tested Substances
 Tested Substances
All(37)
 
 
Active(9)
 
 
Inactive(28)
 
 
AID: 488973
Data Source: NMMLSC (UNMCMD_SAR_HIT_VALIDATION_CHEMOREVERSAL_SPECIFICALLY_ABCB1_..)
Depositor Category: NIH Molecular Libraries Probe Production Network
Deposit Date: 2010-10-29
Hold-until Date: 2011-10-28
Modify Date: 2011-10-30

Data Table ( Complete ):           View Active Data    View All Data
Target
BioActive Compounds: 9
Related Experiments
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AIDNameTypeProbeComment
1325High-throughput multiplex screening for ABC transporter inhibitors: specifically ABCG2 screen, ABCB1 counter-screenScreening depositor-specified cross reference: High-throughput multiplex screening for ABC transporter inhibitors: specifically ABCG2 screen, ABCB1
1326High-throughput multiplex screening for ABC transporter inhibitors: specifically ABCB1 screen, ABCG2 counter-screenScreening depositor-specified cross reference: High-throughput multiplex screening for ABC transporter inhibitors: specifically ABCB1 screen, ABCG2
1451Single point confirmation for ABC transporter inhibitors: specifically ABCB1 screen, ABCG2 counter-screenScreening depositor-specified cross reference: Single point confirmation for ABC transporter inhibitors: specifically ABCB1 screen, ABCG2 counter-s
1453Single point confirmation screening for ABC transporter inhibitors: specifically ABCG2 screen, ABCB1 counter-screenScreening depositor-specified cross reference: Single point confirmation screening for ABC transporter inhibitors: specifically ABCG2 screen, ABCB1
1689Dose Response for ABC transporter inhibitors: specifically ABCB1 screen, ABCG2 counter-screenConfirmatory depositor-specified cross reference: Dose Response for ABC transporter inhibitors: specifically ABCB1 screen, ABCG2 counter-screen
1690Dose Response for ABC transporter inhibitors: specifically ABCG2 screen, ABCB1 counter-screenConfirmatory depositor-specified cross reference: Dose Response for ABC transporter inhibitors: specifically ABCG2 screen, ABCB1 counter-screen
1818Summary of High-throughput multiplex screening for ABC transporter inhibitorsSummary1 depositor-specified cross reference: Summary of High-throughput multiplex screening for ABC transporter inhibitors.
2830Chemo Reversal assay in ABCG2-expressing cells for one set of SAR compoundsOther depositor-specified cross reference: on hold until 05/10/2011
2831A small molecule screen for inhibitors of the PhoP regulon in Salmonella Typhimurium (2) using purified and synthesized compoundsConfirmatory depositor-specified cross reference: on hold until 05/10/2011
1480Profiling compound fluorescence in IgMXP3 at 488/530 nm; counter screen to single point confirmation of ABCG2 screenScreening same project related to Summary assay
1483Profiling compound fluorescence in CCRF-Adr at 488/530 nm; counter screen to single point confirmation of ABCB1 screenScreening same project related to Summary assay
2833Chemo Reversal assay in ABCB1-expressing cells for one set of SAR compoundsOther same project related to Summary assay
488974SAR hit validation chemo-reversal assay specifically in in ABCG2-expressing cells, ABCB1 counterscreenOther same project related to Summary assay
489002Dose Response for ABC transporter inhibitors: specifically ABCB1 screen, ABCG2 counter-screen. Follow-up assay.Confirmatory same project related to Summary assay
489003Dose Response for ABC transporter inhibitors: specifically ABCG2 screen, ABCB1 counter-screen. Follow-up assay.Confirmatory same project related to Summary assay
504476Chemo Reversal assay in ABCB1-expressing cells for set2 of SAR compoundsOther same project related to Summary assay
504477Chemo Reversal assay in ABCG2-expressing cells for set 2 of SAR compoundsConfirmatory same project related to Summary assay
504566Dose Response for ABC transporter inhibitors: specifically ABCG2 screen, ABCB1 counter-screen. Follow-up assay on set 2Confirmatory same project related to Summary assay
504569Dose Response for ABC transporter inhibitors: specifically ABCB1 screen, ABCG2 counter-screen. Follow-up assay for Set 2Confirmatory same project related to Summary assay
Description:
University of New Mexico Assay Overview:
Assay Support: 1 R03 MH081228-01A1
High-throughput multiplex screen for ABC transporter inhibitors
Assay Provider: Richard Larson
Screening Center/ PI: UNM Center for Molecular Discovery (UNM CMD)Larry Sklar
Lead Biologist: J. Jacob Strouse
Assay Development: Hadya Njus, Richard Larson
Assay Implementation: Hadya Njus, Diane Jimenez-Stinson, J. Jacob Strouse, Anna Waller, Mark Carter, Annette Evangelisti
Chemistry Center / PI: University of Kansas Specialized Chemistry Center / Jeff Aube
Chemistry Center Manager: Jennifer Golden

Assay Background and Significance:
The three major subfamilies of human multidrug resistance (MDR) proteins (ABCB, ABCC, and ABCG) 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 [Franks, et al, 2003; Mercier et al, 2003]. Dual treatment with ABC transporter inhibitors in conjunction with chemotherapeutics is a common treatment strategy to circumvent MDR in cancers. However, manifestation of significant side effects, toxic 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. More than 48 members of the ABC transporter superfamily have been identified and three of these members, MDR1/Pgp (ABCB1), MRP1 (ABCC1), and BCRP (ABCG2), have unambiguously been shown to contribute to cancer multidrug resistance [Kubota et al, 2001].

The primary HTS associated with this project was a high-throughput flow cytometry-based [Ramirez et al, 2003, Kuckuck, 2001] duplex assay reported in PubChem AIDs 1325 and 1326. The ABCB1 and ABCG2 transporters were evaluated in parallel using the fluorescent J-aggregate-forming lipophilic cation 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1) as substrate. ABCB1-expressing cells (CCRF-Adr) were color-coded with FarRed DDAO CellTrace SE (Invitrogen) to allow their distinction from ABCG2-expressing cells (Ig-MXP3) via a red fluorescence emission wavelength of 665 +/- 10 nm (635 nm excitation). A total of 194394 compounds was tested with 200 and 130 actives noted in ABCB1 and ABCG2, respectively. A subsequent cherry pick resulted in single point confirmatory testing of 273 compounds (AIDs 1451 and 1453) revealed 18 and 16 actives in ABCB1 and ABCG2 respectively.

In the first set of chemosensitization assays (AIDs 2830 and 2833 for ABCG2 and ABCB1, respectively, a total of 15 compounds tested for each, release date 05/10/2011), five active compounds were reported for for ABCG2, and one for ABCB1. A detailed summary report (AID 1818) is accessible on PubChem.

In the assay reported here, these actives were validated in dose response over a compound concentration range of 0.1 microM to 100 microM. As in the single point analysis, this assay is an assessment of the viability of drug resistant cells in the presence of a known chemotherapeutic agent (Daunorubicin (DNR) or Mitoxantrone (Mtx)for ABCB1 and ABCG2-expressing cells, respectively. Compound effectiveness is evaluated over a concentration gradient for restoration of the killing effect of the drug.
Protocol
To quantify the effects of hit compounds in conferring sensitivity in drug-resistant cells, the ABCB1 or ABCG2-reversal agent (test compound) is evaluated over a concentration range to determine the dose-dependent effect of the compound on cell viability in 100,000 cells/mL maintained in a fixed concentration of selective agent. Cells (either Jurkat/DNR or Igrove for testing ABCB1 or ABCG2, respectively) are incubated with test compound (concentration range 0.1 microM -100 microM) over a 7-day period in the presence of selective agent: either DNR for ABCB1 transporter testing, or Mtx for ABCG2 transporter testing. Cell viability is evaluated by trypan blue staining and enumeration under light microscopy. A chemreversal index (Chem Reversal50) is determined from the viability assessment. Using a similar approach, an assessment is determined of the direct toxic effects of the transporter-reversal agents that resulted in cell death in resistant cells maintained in medium alone (Toxic Dose50; TD50). Results are compared with the survival of parental cells in the presence of drug (the positive control, resulting in 100% cell death) as well as the survival of drug-resistant cells in the presence of chemotherapeutic drug (the negative control yielding 100% cell viability). The differences between the Chem Reversal50 (CR50) and TD50 curves yields an approximation of an in vitro "therapeutic index" for each compound.

Calculations:
PUBCHEM_ACTIVITY_SCORE is based on Ratio of Chem Reversal50 to Toxic Dose50 for all compounds with Toxic Dose50 less than 15 microM. An Active test compound has a ratio greater than 10.

Keywords:
NIH Roadmap, UNM Center for Molecular Discovery (UNM CMD), high-throughput flow cytometry, drug-resistance transporters, ABCB1, ABCB2, multiplex cell-based screening
Categorized Comment - additional comments and annotations
From PubChem:
Assay Cell Type: Jurkat
Result Definitions
TIDNameDescriptionHistogramTypeUnit
OutcomeThe BioAssay activity outcomeOutcome
ScoreThe BioAssay activity ranking scoreInteger
1Chemo_Reversal50 (microM)Test compound concentration eliciting 50% cell viability in the presence of chemotheraputic agentFloatμM
2TD50_QualifierQualifier for the value of Toxicity Dose 50String
3TD50 (microM)Test compound concentration resulting in 50% cell viabilityFloatμM
4Ratio_TD50/CR50Ratio of TD50 to CR50Float
5TD50_AnnotationAnnotation of test compound being toxicString
Additional Information
Grant Number: 1 R03 MH081228-01A1

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