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Southern Methodist University SMU Scholar Biological Sciences Theses and Dissertations Biological Sciences Spring 5-18-2019 Reversal of P-Glycoprotein and Breast Cancer Resistance Protein Mediated Multidrug Resistance In Vitro Using In Silico Identified Novel Compounds Amila Nanayakkara Southern Methodist University, [email protected] Follow this and additional works at: https://scholar.smu.edu/hum_sci_biologicalsciences_etds Part of the Cancer Biology Commons, Cell Biology Commons, Other Chemicals and Drugs Commons, Pharmaceutical Preparations Commons, and the Pharmacology Commons Recommended Citation Nanayakkara, Amila, "Reversal of P-Glycoprotein and Breast Cancer Resistance Protein Mediated Multidrug Resistance In Vitro Using In Silico Identified Novel Compounds" (2019). Biological Sciences Theses and Dissertations. 3. https://scholar.smu.edu/hum_sci_biologicalsciences_etds/3 This Dissertation is brought to you for free and open access by the Biological Sciences at SMU Scholar. It has been accepted for inclusion in Biological Sciences Theses and Dissertations by an authorized administrator of SMU Scholar. For more information, please visit http://digitalrepository.smu.edu. REVERSAL OF P-GLYCOPROTEIN AND BREAST CANCER RESISTANCE PROTEIN MEDIATED MULTIDRUG RESISTANCE IN VITRO USING IN SILICO IDENTIFIED NOVEL COMPOUNDS. Approved by, __________________________ Prof. John Wise Associate Professor of Biology __________________________ Prof. Pia Vogel Professor of Biology __________________________ Prof. Steven Vik Professor of Biology __________________________ Prof. Alex Lippert Associate Professor of Chemistry REVERSAL OF P-GLYCOPROTEIN AND BREAST CANCER RESISTANCE PROTEIN MEDIATED MULTIDRUG RESISTANCE IN VITRO USING IN SILICO IDENTIFIED NOVEL COMPOUNDS A Dissertation Presented to the Graduate Faculty of Dedman College Southern Methodist University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy with a Major in Molecular and Cell Biology by Amila K. Nanayakkara B.S., Molecular Biology and Biochemistry, University of Colombo, 2011 May 18, 2019 Copyright (2019) Amila Nanayakkara All Rights Reserved ACKNOWLEDGMENTS First, I would like to thank Dr. Pia Vogel and Dr. John Wise for giving me this wonderful opportunity to pursue my Ph.D. in their lab. This work would have been impossible to accomplish without their unlimited support. Especially I would like to thank Dr. Vogel for responding to my email 7 years ago and arranging for me to shadow senior graduate students in the lab initially. I think that gave me an opportunity to get exposure I needed very much. Both Drs. Vogel and Wise you provided me the guidance exactly I needed to fulfill this task. I would like to thank my committee members Dr. Steven Vik and Dr. Alex Lippert for their guidance throughout the past 5 years. Also, I would like to acknowledge collaborators of our lab, Dr. Noelle Williams from UTSW and Dr. Ashlee Moses from OHSU for their support. I was blessed to have received their expertise that heavily benefitted my research. My present and past lab colleagues have been very supportive of my work. Specially, I am grateful for the guidance and mentorship by Dr. Courtney Follit. Further, I would like to thank Dr. McCormick, Maisa, Gang, Collette, Maha and Lauren. Also, I would like to thank two undergraduates who worked with me, Kasi and Beau, who were wonderfully supportive. Further, I would like to acknowledge both academic and nonacademic staff in SMU Biology department who were supportive to me in many ways. Outside the laboratory, I had the opportunity of getting to know wonderful new friends in Dallas who were supportive in various ways to my life and special thank goes to Aditi, Elnaz, Shruthy, Tetiena and Lena. iv Last but not least my main gratitude goes to my family. My parents always believed in my skills and gave me the freedom to follow my dreams. Finally, I am so grateful to my loving wife, Nipunika Somatilaka. She has shared all the good and bad times with me and supported me throughout my studies. v Amila K. Nanayakkara B.Sc. Molecular Biology and Biochemistry, University of Colombo, Sri Lanka REVERSAL OF P-GLYCOPROTEIN AND BREAST CANCER RESISTANCE PROTEIN MEDIATED MULTIDRUG RESISTANCE IN VITRO USING IN SILICO IDENTIFIED NOVEL COMPOUNDS. Advisor: John G. Wise, Ph.D. Doctor of Philosophy conferred May 18, 2019 Dissertation completed April 15, 2019 Multidrug resistance (MDR) is a major cause of chemotherapy failure. Overexpression of ATP-binding cassette (ABC) transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are two well-studied drug transporters which are associated with MDR. These two transporters also act as a major functional unit of the blood brain barrier to protect the brain from xenobiotics and toxins. Lack of clinically approved P-gp and BCRP inhibitors renders chemotherapy treatments of many MDR cancers ineffective and obstructs drug uptake into the brain. Using computational methods, we have identified new compounds that inhibit P-gp (Brewer et al., Mol. Pharmacol. 2014). Several of these compounds show successful MDR reversal in the drug resistant DU145TXR prostate cancer cell line (Follit et al., Pharmacol. Res. Perspect. 2015). Here, we further analyze these P-gp inhibitors 29, 34 and 45 in a P-gp over- expressing ovarian cancer cell line, A2780ADR, and a P-gp over-expressing prostate cancer cell line, DU145TXR. Treatment of chemotherapeutics with the in silico identified inhibitors led to a higher mortality in MDR cancer cells in tests conducted with both conventional 2D and 3D- spheroid cell-based assays. Using a novel assay based on cellular accumulation of compounds in a P-gp overexpressing cell line, we report that 29, 34 and 45 do not function as P-gp transport substrates. It was found that the efficacy of chemotherapy can be enhanced by initial cotreatment vi of chemotherapeutics with P-gp inhibitor 29 followed by an extended treatment using only inhibitor 29 without having chemotherapeutics in the media. Further, we describe the effects of chemical variants of the P-gp inhibitor 29 in experiments aimed at improving the pharmacological characteristics of the inhibitor. The variants were generated using computational approaches or by structure-based design in attempts to improve P-gp inhibition. Multiple variants showed an improved efficacy in reversing paclitaxel resistance in the P-gp over-expressing DU145 TXR prostate cancer cell line. We achieved a 100% success rate using computational lead optimization in obtaining variants of P-gp inhibitor 29 which do not function as transport substrates of P-gp. In related work, we developed a breast cancer cell line that overexpresses the BCRP transporter for the purpose of identifying inhibitors of the BCRP drug pump. Three of the variants of inhibitor 29 could reverse the BCRP-mediated MDR in this BCRP overexpressing breast cancer cell line. Further, we identified several other compounds that interfered with BCRP mediated transport in vitro screened from an inhouse library of small molecules. The experimental inhibitors of P-gp and BCRP in this study appear to be promising candidates for further development into co-therapeutics to treat MDR cancers and modulate the blood brain barrier. vii TABLE OF CONTENTS LIST OF FIGURES ..................................................................................................................... xiv LIST OF TABLES ...................................................................................................................... xvii CHAPTER 1: BACKGROUND ..................................................................................................... 1 1.1 Multi Drug Resistance and ABC transporters ........................................................... 1 1.2 P-glycoprotein (P-gp)................................................................................................. 2 1.2.1 P-gp structure and function ........................................................................................ 2 1.2.2 Role of P-glycoprotein in MDR cancers .................................................................... 5 1.3 Breast cancer resistance protein (BCRP) ................................................................... 6 1.3.1 BCRP structure and function ..................................................................................... 6 1.3.2 Role of BCRP in MDR cancers ................................................................................. 9 1.4 Physiological role of P-gp and BCRP in blood brain barrier ................................... 10 1.5 Inhibitors of P-gp and BCRP ................................................................................... 12 1.5.1 P-gp inhibitors .......................................................................................................... 12 1.5.2 BCRP inhibitors ....................................................................................................... 14 1.5.3 Clinical trials of P-gp and BCRP inhibitors ............................................................. 14 1.6 Rationale .................................................................................................................. 16 CHAPTER 2: TARGETED INHIBITORS OF P- GLYCOPROTEIN 29, 34 AND 45 INCREASE CHEMOTHERAPEUTIC-INDUCED MORTALITY OF MULTIDRUG RESISTANT TUMOR CELLS ................................................................................................. 18 2.1 Introduction .............................................................................................................

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