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Discovery and Characterization of Novel Substrate Selective Inhibitors of Human MRP1 (ABCC1)

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Discovery and Characterization of Novel Substrate Selective Inhibitors of Human MRP1 (ABCC1) South Dakota State University Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange Electronic Theses and Dissertations 2019 Discovery and Characterization of Novel Substrate Selective Inhibitors of Human MRP1 (ABCC1) Angelina Sampson South Dakota State University Follow this and additional works at: https://openprairie.sdstate.edu/etd Part of the Biochemistry Commons Recommended Citation Sampson, Angelina, "Discovery and Characterization of Novel Substrate Selective Inhibitors of Human MRP1 (ABCC1)" (2019). Electronic Theses and Dissertations. 3660. https://openprairie.sdstate.edu/etd/3660 This Dissertation - Open Access is brought to you for free and open access by Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Open PRAIRIE: Open Public Research Access Institutional Repository and Information Exchange. For more information, please contact [email protected]. 2 -i DISCOVERY AND CHARACTERIZATION OF NOVEL SUBSTRATE SELECTIVE INHIBITORS OF HUMAN MRP1 (ABCC1) BY ANGELINA SAMPSON A dissertation submitted in partial fulfillment of the requirements for the Doctor of Philosophy Major in Biochemistry South Dakota State University 2019 iii I dedicate this dissertation to my husband, Dr. Emmanuel Blay whose support and advice has helped me tremendously. iv ACKNOWLEDGEMENTS I am grateful to the Lord Almighty for this accomplishment. I am also grateful to my husband and family for their unflinching support and motivation whenever the journey got tough. I thank my academic advisor, Dr Surtaj H. Iram for his training, guidance, mentorship and opportunities throughout the course of my doctoral studies. To my advisory committee members, Prof. Fathi Halaweish, Dr. Gregory Michna, Dr. Severine Van Slambrouck (former committee member), I am grateful for your useful reviews, feedback and overall assistance during my studies. I am also thankful to Dr. Rachel Willand Charnley (committee member) for accepting to be a part of my academic advisory committee when I needed it the most. I am also grateful to Dr. Kee Wee Tan (former post-doctoral research associate in Iram lab) for training me in most of the techniques used in this study. I thank Brian Peterson (former undergraduate student in Iram lab) for helping execute some of the assays in this study. I am thankful to all Iram Lab members for their support, feedback and questions during my research work in the lab. I am also grateful to the Department of Chemistry and Biochemistry of the South Dakota State University for giving me the opportunity to study and supporting me financially throughout my studies. Last but not least, I would like to thank the Holy Life Tabernacle Church, Brookings for being a great supportive system in my stay here in Brookings, South Dakota. ALL GLORY BE TO GOD. v TABLE OF CONTENTS LIST OF TABLES ......................................................................................................... xi LIST OF FIGURES ....................................................................................................... xii LIST OF ABBREVIATIONS ...................................................................................... xiv ............................................................................................................................ 1 1.0 Scope of the Study ..................................................................................................... 1 1.1 Introduction to ABC transporters .............................................................................. 1 1.2 Structure and function of ABC transporters .............................................................. 4 1.3 Multidrug resistance in cancer .................................................................................. 7 1.4 Modulators of ABC transporters ............................................................................. 12 1.5 In vitro screening methods for evaluating ABC transporter interaction ................. 13 1.5.1 Fluorescence accumulation and efflux assay .................................................... 14 1.6 Traditional membrane-based assays ........................................................................ 17 1.6.1 Inside-out Membrane Vesicles ......................................................................... 17 1.6.2 Membrane vesicular transport .......................................................................... 19 1.7 Current/alternate methods for assessment of compound interactions ..................... 21 1.7.1 High Content Imaging ...................................................................................... 22 1.8 Rationale of Study ................................................................................................... 24 REFERENCES .............................................................................................................. 26 vi .......................................................................................................................... 35 Doxorubicin as a Fluorescent Reporter Identifies Novel MRP1 (ABCC1) Inhibitors Missed by Calcein-Based High Content Screening of Anticancer Agents ................... 35 Abstract ......................................................................................................................... 35 1.0 Introduction ............................................................................................................. 38 2.0 Materials and methods ............................................................................................ 41 2.1 Chemicals ................................................................................................................ 41 2.2 Cell lines and cell culture ........................................................................................ 41 2.3 MRP1 inhibition screening with automated image acquisition and analysis .......... 42 2.4 Flow cytometry-based doxorubicin accumulation assay ........................................ 43 2.5 Doxorubicin accumulation assay using confocal microscopy ................................ 44 2.6 Membrane vesicle preparation ................................................................................ 44 2.7 Membrane vesicular transport assay ....................................................................... 45 2.8 Drug sensitivity assay .............................................................................................. 46 2.9 High content screening data analysis ...................................................................... 46 2.10 Statistical analysis ................................................................................................. 47 3.0 Results ..................................................................................................................... 48 3.1 Assay development and optimization ..................................................................... 48 3.2 Screening of anti-cancer compound library for MRP1 inhibitors ........................... 51 vii 3.3 Validation of identified MRP1 inhibitors ................................................................ 57 3.4 Membrane Transport assay for MRP1 inhibitors .................................................... 60 3.5 Resistance reversal by selected MRP1 inhibitors ................................................... 63 3.6 Effects of selected hit compounds on drug sensitivity of HEK293/BCRP and HEK293/P-gp cells ........................................................................................................ 67 4.0 Discussion ............................................................................................................... 70 REFERENCES .............................................................................................................. 76 .......................................................................................................................... 80 CRO-9 screening of unique anticancer library identifies novel inhibitors of human MRP1 (ABCC1). ........................................................................................................... 80 Abstract ......................................................................................................................... 80 1.0 Introduction ............................................................................................................. 82 2.0 Materials and methods ............................................................................................ 86 2.1 Chemicals ................................................................................................................ 86 2.2 Cell lines and cell culture ........................................................................................ 86 2.3 Screening for MRP1 inhibition via automated image acquisition and analysis ...... 86 2.4 Doxorubicin accumulation assay using confocal microscopy ................................ 88 2.5 Flow cytometry-based CRO-9 accumulation assay ................................................ 88 2.6 Membrane vesicle preparation ................................................................................ 89 2.7 Membrane vesicular transport assay ....................................................................... 90 viii 2.8 Drug sensitivity Assay ...........................................................................................
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