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Updated: Jun 22, 2026
A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans
Published on: October 16, 2015
Identification of inhibitors of ABCG2 by a bioluminescence imaging-based high-throughput assay
Yimao Zhang1, Youngjoo Byun, Yunzhao R Ren
1Russell H. Morgan Department of Radiology and Radiological Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland, USA.
Abstract:
ABCG2 is a member of the ATP-binding cassette (ABC) family of transporters, the overexpression of which is associated with tumor resistance to a variety of chemotherapeutic agents. Accordingly, combining ABCG2 inhibitor(s) with chemotherapy has the potential to improve treatment outcome. To search for clinically useful ABCG2 inhibitors, a bioluminescence imaging (BLI)-based assay was developed to allow high-throughput compound screening. This assay exploits our finding that d-luciferin, the substrate of firefly luciferase (fLuc), is a specific substrate of ABCG2, and ABCG2 inhibitors block the export of d-luciferin and enhance bioluminescence signal by increasing intracellular d-luciferin concentrations. HEK293 cells, engineered to express ABCG2 and fLuc, were used to screen the Hopkins Drug Library that includes drugs approved by the Food and Drug Administration (FDA) as well as drug candidates that have entered phase II clinical trials. Forty-seven compounds showed BLI enhancement, a measure of anti-ABCG2 activity, of > or =5-fold, the majority of which were not previously known as ABCG2 inhibitors. The assay was validated by its identification of known ABCG2 inhibitors and by confirming previously unknown ABCG2 inhibitors using established in vitro assays (e.g., mitoxantrone resensitization and BODIPY-prazosin assays). Glafenine, a potent new inhibitor, also inhibited ABCG2 activity in vivo. The BLI-based assay is an efficient method to identify new inhibitors of ABCG2. As they were derived from a FDA-approved compound library, many of the inhibitors uncovered in this study are ready for clinical testing.
Insights
Researchers developed a bioluminescence imaging assay to find new ABCG2 inhibitors. This assay identified 47 compounds, many ready for clinical testing, to combat chemotherapy resistance in tumors.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- ABCG2 transporter overexpression is linked to chemotherapy resistance in tumors.
- Targeting ABCG2 with inhibitors could enhance cancer treatment outcomes.
- Developing efficient screening methods for ABCG2 inhibitors is crucial for drug discovery.
Purpose of the Study:
- To develop and validate a high-throughput bioluminescence imaging (BLI)-based assay for identifying novel ABCG2 inhibitors.
- To screen the Hopkins Drug Library for compounds that inhibit ABCG2 activity.
- To identify clinically relevant ABCG2 inhibitors for potential combination cancer therapy.
Main Methods:
- Engineered HEK293 cells expressing ABCG2 and firefly luciferase (fLuc).
- Utilized d-luciferin as a substrate for fLuc, which is exported by ABCG2.
- Developed a BLI assay where ABCG2 inhibition increases intracellular d-luciferin and enhances luminescence.
- Screened the Hopkins Drug Library, comprising FDA-approved drugs and clinical candidates.
Main Results:
- Identified 47 compounds exhibiting a > or =5-fold BLI enhancement, indicating anti-ABCG2 activity.
- The majority of identified compounds were previously unknown as ABCG2 inhibitors.
- Validated the assay by detecting known ABCG2 inhibitors and confirming novel inhibitors via established in vitro assays.
- Discovered Glafenine as a potent new ABCG2 inhibitor with in vivo activity.
Conclusions:
- The BLI-based assay is an efficient and effective method for high-throughput screening of ABCG2 inhibitors.
- The study identified numerous novel ABCG2 inhibitors, many from an FDA-approved library, suitable for clinical development.
- This approach holds significant potential for improving cancer chemotherapy by overcoming ABCG2-mediated drug resistance.
