Document Type

Dissertation - Open Access

Award Date

2021

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry and Biochemistry

First Advisor

Surtaj H. Iram

Keywords

ATP-binding cassette transporters, ELISA, Modulators, MRP1/ABCC1, MRP2/ABCC2, Multidrug resistance

Abstract

ATP-binding cassette (ABC) transporters are known to play a critical role in conferring multidrug resistance (MDR) in various cancers. Several retrospective analyses of chemotherapy results have reported high expression of Multidrug Resistance Protein 1 (MRP1) and Multidrug Resistance Protein 2 (MRP2) in tumor cells exhibiting the MDR phenotype. High MRP1 and MRP2 expression in cancer patients predict a higher risk of treatment failure, resulting in relapse and disease recurrence as well as shortened survival rates. The key role of MRP1 and MRP2 play in the development of MDR makes them important therapeutic targets that hold a great promise for addressing multidrug resistance in cancer cells. Since MRP1 and MRP2 play critical roles in the regulation of various cellular pathways by altering the levels of several key signaling molecules, finding ways of modulating the activities and expression of these transporters in cancer cells is of great clinical interest in oncology research. We identified four novel modulators of MRP1 from our initial screening of 30 therapeutic compounds using an In-Cell ELISA assay. Three of these compounds; Amuvatinib, SB743921 HCl, TG101348 (SAR302503), which were identified to be ATP competitive inhibitors based on their mode of action, decreased MRP1 expression whereas Felbamate (a recently approved FDA drug) increased MRP1 protein expression. Our findings revealed that these ATP competitive inhibitors decreased MRP1- mediated calcein accumulation. These compounds inhibited the growth of HEK293 MRP1-overexpressing cells at clinically achievable concentrations, and also reversed MRP1- mediated resistance in these cells. Since regulation of the activity of activators and effectors of specific biochemical pathways provide a means of regulating downstream signaling, we investigated the effect of a novel Tie2 kinase inhibitor and mTOR inhibitor, Everolimus, on MRP1 activity and expression. Tie2 is an activator of the PI3K/Akt pathway (a pathway known to modulate MRP1 activity and expression) whereas mTOR is a downstream effector of this pathway. We demonstrated using a flow cytometry-based calcein accumulation assay, and MTT based reversal resistance studies that Tie2 kinase inhibitor and Everolimus can decrease MRP1 mediated calcein efflux and reverse MRP1 mediated resistance towards vincristine in HEK293 MRP1-overexpressing cells. Lastly, we identified 49 modulators of MRP2 from our initial screening of 372 FDA-approved drugs from a recently approved FDA drug library representing 13.17% of total compounds screened. Thirty-nine (39) drugs increased MRP2 expression whereas 10 drugs lowered expression of MRP2 after drug treatment. Results from this screening reaffirm the promiscuous nature of the MRP2 transporter, and how important it is to investigate the interaction between both old and newly developed drugs with MRP2. The modulators identified from this study would be further characterized in future projects. Overall, our findings signify the importance of profiling drug interactions with these transporters, and the data obtained would provide essential information to improve combinatorial drug therapy and precision medicine as well as reduce drug toxicity of various cancer chemotherapies.

Number of Pages

179

Publisher

South Dakota State University

Rights

Copyright © the Author

Included in

Biochemistry Commons

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