STRUCTURE-GUIDED DESIGN OF CARBONIC ANHYDRASE IX SELECTIVE INHIBITORS AS A BREAST CANCER TREATMENT By CARRIE L. LOMELINO A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2018 © 2018 Carrie L. Lomelino To my parents and grandparents ACKNOWLEDGMENTS First and foremost, I would like to thank my mentor, Robert McKenna, for his expertise and guidance throughout the last four years. Not only has he prepared me for a career as an independent scientist in the field of structural biology, but he has also made my entire graduate school experience enjoyable and a time I will never forget. He has been patient with me at the times I am most stubborn and supportive when I am stressed and indecisive. I know I would not have survived graduate school without his constant sarcasm and humor. It would be remiss if I did not also thank Mavis Agbandje- McKenna as she has essentially been my unofficial co-mentor. She is a wonderful role model for a woman starting her career in science and I have always appreciated her advice. Most importantly, she has consistently reminded me to be confident in myself and my work. I would also like to acknowledge the members of my committee, Linda Bloom, Susan Frost, Nicole Horenstein, Robert Huigens, and David Ostrov. I greatly appreciate the advice you have all given over the last few years to improve my research. I would like to recognize all the members of the McKenna and Agbandje- McKenna labs for making the lab feel more like a family than a workplace. A special shout out to Brian Mahon for mentoring me when I joined the lab; Mam Mboge for helping me achieve my TL1 aims and being a fun travel buddy; Bri Murray for always listening to me vent and always making me feel self-assured; and Jacob Andring for keeping me sane and working through so many manuscripts and projects with me (team science!). Also, thank you to my IDP friends Andrea, Casey, Christina, Kevin, and Maggie who have been there from the beginning- we survived! 4 Finally, there are not enough words to express the gratitude I have for the constant support from my family. I know that I would not be where I am today without them. Thank you to my parents, Dale and Lori, for encouraging me to chase my dreams. They have each set examples of what it means to succeed- every day my father demonstrates the importance of diligence and dedication while my mother proves that you can always achieve your goals with determination and persistence. Thank you for raising me to be responsible, hard working, independent, and honest. Thank you to my sister, Jenny, for always reminding me to be myself and have fun; my Aunt, Cheri Cohen, for providing a home away from home; and my best friend, Brandi Cawthorn, for always being there no matter how far apart. One last special thank you to my grandfather, Carl Whitaker, for always believing in me and for telling me I couldn’t do something knowing full well I was capable and that it would push me to prove that I could- I hope my dissertation would make you proud. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 8 LIST OF FIGURES .......................................................................................................... 9 LIST OF ABBREVIATIONS ........................................................................................... 11 ABSTRACT ................................................................................................................... 13 CHAPTER 1 INTRODUCTION .................................................................................................... 15 CA IX Expression, Regulation, and Function .......................................................... 16 CA IX Structure ....................................................................................................... 18 CA Mechanism ....................................................................................................... 19 Inhibition of CA ....................................................................................................... 21 Structure-Guided Drug Design ................................................................................ 22 2 METHODS .............................................................................................................. 33 Protein Expression and Purification ........................................................................ 33 Stopped Flow CO2 Hydrase Assay ......................................................................... 34 Crystallization ......................................................................................................... 34 X-ray Crystallography ............................................................................................. 35 MTT Assay .............................................................................................................. 35 Migration and Invasion ............................................................................................ 36 3 SULFONAMIDE-BASED INHIBITION OF CA IX .................................................... 43 Benzene Sulfonamide Inhibitors ............................................................................. 44 Nitrogenous Base Inhibitors .................................................................................... 46 Multi-tail Inhibitors ................................................................................................... 50 Summary ................................................................................................................ 52 4 CARBOXYLIC ACID-BASED INHIBITION OF CA IX.............................................. 66 Heteroaryl-pyrazole Carboxylic Acids ..................................................................... 68 Nicotinic and Ferulic Acid ........................................................................................ 69 Summary ................................................................................................................ 70 5 SWEETENER-BASED INHIBITION OF CA IX ....................................................... 78 Acesulfame Potassium ........................................................................................... 78 6 Sucralose ................................................................................................................ 81 Summary ................................................................................................................ 83 6 FRAGMENT-BASED INHIBITION OF CA IX .......................................................... 92 Sulfamate ................................................................................................................ 92 Cyclic Sulfonamide ................................................................................................. 95 Summary ................................................................................................................ 98 7 CONCLUSION ...................................................................................................... 107 APPENDIX A MECHANISM OF WATER NETWORK REPLENISHMENT ................................. 115 Overview ............................................................................................................... 115 Methods ................................................................................................................ 120 Protein Expression and Purification ................................................................ 120 Crystallization ................................................................................................. 120 CO2 Entrapment ............................................................................................. 120 Data Collection ............................................................................................... 121 Structure Determination and Refinement ....................................................... 121 Results .................................................................................................................. 122 CO2 Binding Sites ........................................................................................... 122 H64 and Water Network ................................................................................. 123 Entrance Conduit Waters ............................................................................... 124 Restoration of Water Network ........................................................................ 125 Summary .............................................................................................................. 126 B XFELs ................................................................................................................... 133 Overview ............................................................................................................... 133 Methods ................................................................................................................ 135 Macromolecule Production ............................................................................. 135 Microbatch Mixing .......................................................................................... 135 Batch Crystallization