Document Type

Dissertation - Open Access

Award Date

2019

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry and Biochemistry

First Advisor

Surtaj H. Iram

Keywords

ABCC1, drug absorption and disposition, high content screening, MRP1, MRP1 inhibitors, multidrug resistance

Abstract

Multidrug resistance protein 1 (MRP1/ABCC1) actively transports a variety of drugs, toxic molecules and important physiological substrates across the plasma membrane. It can confer broad-spectrum drug resistance and can decrease the bioavailability of many important drugs such as anti-cancer agents, antibiotics, antivirals, antidepressants and antiinflammatory drugs. Calcein-AM, a fluorescent reporter commonly used for studying compound interactions with MRP1 was recently used in the development of a high content imaging-based assay by our group. This assay was robust and had better sensitivity than fluorescent plate readouts. The assay identified 12 MRP1 inhibitors after screening an anticancer library of 386 compounds. Due to the multiple distinct substrate binding sites of MRP1, we sought to use different fluorescent probes to identify substrate selective inhibitors which were likely missed by the calcein-AM screening. The high content imaging-based uptake assay was modified using doxorubicin (anticancer drug) and CRO- 9 (dye) as fluorescent reporters which vary in structure and function. The doxorubicin assay, after screening the same 386 compound library identified a total of 28 MRP1 inhibitors including 16 inhibitors that have not been previously reported as inhibitors of MRP1. The CRO-9 assay identified a total of 50 MRP1 inhibitors including 19 additional inhibitors that have never been reported as inhibitors of MRP1. These 50 MRP1 inhibitors included 10 out of 12 hits identified using calcein-AM and 27 out of 28 inhibitors discovered through the doxorubicin assay. MRP1 inhibition was confirmed using flow cytometry, confocal microscopy and membrane-based transport assays. Selected drugs were evaluated for their ability to reverse resistance of MRP1-overexpressing H69AR lung cancer cells against various substrates. From the doxorubicin screening, mifepristone and doramapimod were the most effective in reversing MRP1 mediated resistance whiles celecoxib exhibited selective MRP1 inhibition. From the hits identified through the CRO- 9-based screening, LY2603618 and ZSTK474 were the most effective in reversing MRP1 mediated resistance in H69AR cells. Together, our findings signify the effectiveness and value of doxorubicin and CRO-9 based high content screening approach. Anti-cancer agents that exhibit MRP1 inhibition may be used to reverse multidrug resistance or to improve the efficacy and reduce the toxicity of various cancer chemotherapies.

Format

application/pdf

Number of Pages

170

Publisher

South Dakota State University

Rights

In Copyright - Educational Use Permitted
http://rightsstatements.org/vocab/InC-EDU/1.0/

Included in

Biochemistry Commons

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