Document Type
Dissertation - Open Access
Award Date
2018
Degree Name
Doctor of Philosophy (PhD)
Department / School
Chemistry and Biochemistry
First Advisor
Surtaj H. Iram
Keywords
ATP-binding cassette proteins, Biosensors, Fluorescence resonance energy transfer, Multidrug resistance, P-glycoprotein, Two-color MRP1
Abstract
ATP-binding cassette (ABC) transporter proteins are a vast, ubiquitous superfamily of proteins most of which unlock the energy from ATP-binding and hydrolysis to influx or efflux a wide spectrum of structurally dissimilar substrates across cell membranes. All eukaryotic ABC transporters including P-gp and MRP1 only exist as efflux pumps whose prominent expression in cells is associated with multidrug resistance of antiretrovirals, antiepileptics, antimalarials and anticancer agents. P-gp and MRP1 reduce the bioavailability and efficacy of chemotherapeutic drugs by actively pumping out these drugs, thereby, leading to poor clinical outcomes during chemotherapy. Due to their clinical importance, profiling of the interaction of MRP1 and P-gp with therapeutic drugs is pertinent. Here, we developed an innovative two-colorMRP1 and P-gp biosensors by fusing the transporter with green fluorescent (GFP) and red fluorescent (RFP) proteins. These biosensors exhibit large dynamic FRET changes in response to movements of the transporter upon substrate-binding and is useful for high throughput screening for potential modulators. Following a steady-state FRET screening, the two-colorMRP1 biosensor identified 10-anticancer drug-hits of 40, and the two-color P-gp detected 6 hits of 50 antitumor drugs, which are ligands and potential substrates of their respective transporters. In the absence of a single known high throughput screening for detection of drug-ABC transporter interaction, our coupled ensemble FRET model is transformative as it holds promise for fluorescent plate reader-based high throughput screening for substrates and ligands of these transporters. Future projects will focus on vesicular transport-coupled LC-MS/MS assay to verify the ABC transporter-substrate status of the anticancer drug-hits discovered in the present study.
Library of Congress Subject Headings
ATP-binding cassette transporters.
Biosensors.
Membrane proteins.
Description
Includes bibliographical references
Format
application/pdf
Number of Pages
152
Publisher
South Dakota State University
Recommended Citation
Osa-Andrews, Bremansu, "Engineering of Two-Color ATP-Binding Cassette Biosensor Proteins for Discovery of Novel Substrates and Modulators" (2018). Electronic Theses and Dissertations. 2955.
https://openprairie.sdstate.edu/etd/2955
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Rights Statement
