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Published on: November 16, 2017
Identification of NanoLuciferase Substrates Transported by Human ABCB1 and ABCG2 and Their Zebrafish Homologs at the
John A Quinlan1, Sashank Sabbineni1, Robert W Robey1
1Fischell Department of Bioengineering, University of Maryland, College Park, Maryland (J.A.Q., C.T.I., H.-C.H.); Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland (J.A.Q., S.S., R.W.R., C.C.L., C.T.I., J.R.T., M.M.G.); and Promega Corporation, San Luis Obispo, California (J.R.W., W.Z.).
Abstract:
ATP-binding cassette (ABC) transporters expressed at the blood-brain barrier (BBB) impede delivery of therapeutic agents to the brain, including agents to treat neurodegenerative diseases and primary and metastatic brain cancers. Two transporters, ABCB1 and ABCG2, are highly expressed at the BBB and are responsible for the efflux of numerous clinically useful chemotherapeutic agents, including irinotecan, paclitaxel, and doxorubicin. Based on a previous mouse model, we have generated transgenic zebrafish in which expression of NanoLuciferase (NanoLuc) is controlled by the promoter of glial fibrillary acidic protein, leading to expression in zebrafish glia. To identify agents that disrupt the BBB, including inhibitors of ABCB1 and ABCG2, we identified NanoLuc substrates that are also transported by ABCB1, ABCG2, and their zebrafish homologs. These substrates will elevate the amount of bioluminescent light produced in the transgenic zebrafish with BBB disruption. We transfected HEK293 cells with NanoLuc and either human ABCB1, ABCG2, or their zebrafish homologs Abcb4 or Abcg2a, respectively, that are expressed at the zebrafish BBB. We evaluated the luminescence and transporter substrate status of 16 NanoLuc substrates. We identified eight substrates that were efficiently pumped out by ABCB1, six by Abcb4, seven by ABCG2, and seven by Abcg2a. These data will aid in the development of a transgenic zebrafish model of the BBB to identify novel BBB disruptors and should prove useful in the development of other animal models that use NanoLuc as a reporter. SIGNIFICANCE STATEMENT: The ATP-binding cassette (ABC) transporters ABCB1 and ABCG2 at the blood-brain barrier (BBB) hinder pharmacological treatment of brain-related diseases. Consequently, there is a need for tools to identify BBB disruptors. This study screened 16 NanoLuciferase substrates, identifying the brightest and those that were transported by human and zebrafish ABC transporters at the BBB. This work supports and complements development of a transgenic zebrafish model, in which NanoLuciferase is expressed within glial cells, enabling detection of BBB disruption.
Insights
This study identified NanoLuciferase substrates that are transported by ATP-binding cassette (ABC) transporters at the blood-brain barrier (BBB). These findings support the development of a transgenic zebrafish model for discovering novel BBB-disrupting agents to improve drug delivery.
Area of Science:
- Neuroscience
- Pharmacology
- Genetics
Background:
- ATP-binding cassette (ABC) transporters, specifically ABCB1 and ABCG2, are highly expressed at the blood-brain barrier (BBB).
- These transporters efflux therapeutic agents, hindering drug delivery for brain diseases like neurodegenerative disorders and cancers.
- There is a critical need for tools to identify agents that can disrupt BBB function.
Purpose of the Study:
- To identify NanoLuciferase (NanoLuc) substrates that are efficiently transported by human and zebrafish ATP-binding cassette (ABC) transporters at the blood-brain barrier (BBB).
- To support the development of a transgenic zebrafish model for detecting BBB disruption and identifying novel therapeutic agents.
Main Methods:
- Generated transgenic zebrafish expressing NanoLuc under the glial fibrillary acidic protein promoter.
- Transfected HEK293 cells with NanoLuc and human/zebrafish ABC transporters (ABCB1, ABCG2, Abcb4, Abcg2a).
- Evaluated luminescence and transporter substrate status of 16 NanoLuc substrates for human and zebrafish ABC transporters.
Main Results:
- Identified eight NanoLuc substrates efficiently transported by human ABCB1.
- Identified seven NanoLuc substrates efficiently transported by human ABCG2.
- Identified six and seven NanoLuc substrates transported by zebrafish Abcb4 and Abcg2a, respectively.
Conclusions:
- The identified NanoLuc substrates can be used to detect BBB disruption in the developed transgenic zebrafish model.
- This research aids in the development of novel BBB-disrupting agents for treating brain diseases.
- The findings are valuable for creating other animal models utilizing NanoLuc as a reporter system.
