SAR hit validation chemo-reversal assay specifically in in ABCG2-expressing cells, ABCB1 counterscreen
Assay Implementation: Hadya Njus, Diane Jimenez-Stinson, J. Jacob Strouse, Anna Waller, Mark Carter, Annette Evangelisti ..more
BioActive Compounds: 23
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.
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.
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.
NIH Roadmap, UNM Center for Molecular Discovery (UNM CMD), high-throughput flow cytometry, drug-resistance transporters, ABCB1, ABCB2, multiplex cell-based screening
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Data Table (Concise)