Development of a Novel Class of Multifunctional Virulence-Attenuating Antibiotics by Bryan Daniel Yestrepsky A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Medicinal Chemistry) in the University of Michigan 2013 Doctoral Committee: Research Professor Scott D. Larsen, Co-chair Assistant Professor Matthew B. Soellner, Co-chair Professor Henry I. Mosberg Professor Ronald W. Woodard Acknowledgement The preparation of this document caps an experience that has undoubtedly been the most challenging I’ve faced so far. Although the level of rigor and time commitment was high, everyone involved contributed to making the pursuit of my doctoral degree a pleasant and rewarding experience. First and foremost, I need to extend my deepest gratitude to my wife Michelle. On top of being a wonderful partner, you have been my best friend, training coach, therapist, and motivational speaker. I can say that without your companionship and eternal optimism over the last seven years, the course of my graduate work, and indeed my life, would unequivocally have been altered for the worse. I love you more than anything and I’m excited to see what the future holds for us. As a lifelong resident of southern Michigan, I have been blessed with the almost unfair advantage of being near my family and friends as I progressed through my graduate studies. My mother and father, Dan and Diane, continue to be endless sources of encouragement and kindness. Thank you for your unconditional endorsement of the choices I’ve made and every dream that I’ve pursued. Thank you also to my grandparents, Andrew and Margaret Yestrepsky and Albert and Judith Noel. I appreciate your pride in my accomplishments despite the fact I am 27 and just finishing school. Also worthy of a special mention is my brother Adam. Your continued successes have stoked a particularly high-achieving form of sibling rivalry that has contributed greatly to my own motivations. My family support group extends beyond these individuals, and includes my parents-in-law Pat and Jean Wicksall and sister-in-law Rachel; my uncles Paul, Jim, Al, Mitch, David Porteous, and their significant others; and all my cousins and extended family members. It is a true blessing to be surrounded by so many supportive and positive people that have given me nothing but encouragement. I also need to thank my friends and Med Chem colleagues. I owe a lot to my lab mates in the Larsen group: Dr. Jessica Bell, Janice Sindac, Helen Waldschmidt, and particularly Scott Barraza. We shared a lot, including stupid internet videos, constant jokes, a few beers, and occasional input on each other’s project difficulties. Scott, I’d like to say thank you for keeping ii me sane in lab, but I think everyone would agree that our part of the lab could not be defined as “sane.” Though I was technically the senior student in the lab, I think it’s up for debate who taught the other more chemistry over the last 4 years. Other Michigan students I need to acknowledge are Joel McDade, Kristoffer Brandvold, Douglas Hansen, Kyle Heslip, Ronak Shah, Corinne Weisheit, and Dr. Ronald Jenkins. I wish you all the best of luck and expect to meet up with you all at conventions and football games once in a while. I must also mention my friends from outside of the University, including Mike Wiese, Gary Peters, Andrew and Ashley Mirasol, Randy McGrail, Ben McArthur, and many more than I can list here. My road to a doctoral degree did not begin in the Fall of 2008, but rather years before, when my interest in science was stimulated by several excellent teachers. Thank you to my high school chemistry teachers, Dr. Claudia Heinbuck and the late Edward Cackowski, and my professors at Albion College, in particular Drs. Clifford Harris, Vanessa McCaffrey, Chris Rohlman, and Molly Scheel, for teaching me the scientific tools that I use every day. This acknowledgement would not be complete without thanking the people directly involved with me on my thesis project. In particular my deepest gratitude goes to Dr. Scott Larsen, who deserves most of the credit for my development into a capable synthetic organic chemist. Between your vast theoretical knowledge of medicinal and organic chemistry and Mr. Mike Wilson’s equally broad knowledge of bench chemistry, the Larsen group was the ideal place to learn the ropes of medicinal chemistry research. I must also thank Dr. Walajapet Rajeswaran, Roderick Sorenson, Jenny Ryu, and Meghan Breen for their contributions to the synthetic chemistry portion of this project, and the rest of the VMCC for assistance and advice. The biochemical work performed by my collaborators, led by Dr. Hongmin Sun and Dr. David Ginsburg, made this project possible. In particular, Dr. Yuanxi Xu, Dr. Yibao Ma, and Dr. Colin Kretz put significant portions of their time into this study for which I am very appreciative. Finally, I would like to extend my gratitude to the faculty that served on my thesis committee, including Dr. Ronald Woodard, Dr. Garry Dotson, Dr. Matt Soellner, Dr. Hank Mosberg, and especially Dr. Jason Gestwicki for all of your thoughtful contributions and critiques of my research plan over the years. The rest of the University of Michigan faculty and staff, including Cherie Dotson, Dr. Scott Woehler, and Sarah Lloyd also deserve mention here. iii Putting all of my acknowledgements into writing overwhelms me with thoughts of how much I have received from all of you over the years. In return, I will take it upon myself to achieve to the best of my ability and continue to make you all proud. -Bryan Yestrepsky iv Table of Contents Acknowledgement ............................................................................................................... ii List of Figures ................................................................................................................... vii List of Schemes ................................................................................................................. viii List of Tables ........................................................................................................................x Chapter 1: Introduction ................................................................................................................1 Bacterial Infection and Antibiotics ......................................................................................1 Anti-Virulence Antibiotics ....................................................................................................2 Group A Streptococcus ........................................................................................................2 Identifying GAS-SK Modulators ..........................................................................................4 Chapter 2: GAS-SK SAR Study ...................................................................................................9 SAR Logic.............................................................................................................................9 GAS-SK Assay ....................................................................................................................10 Synthesis .............................................................................................................................11 SAR Discussion ..................................................................................................................22 Microsomal Stability and Metabolite Identification ..........................................................30 Aqueous Solubility .............................................................................................................33 Mammalian Cytotoxicity and Scaffold Alteration..............................................................34 Follow-up HTS and Commercial SAR Campaign .............................................................35 Conclusions ........................................................................................................................38 Chapter 3: Target Identification Study .....................................................................................40 Phenotypic Screening and Target Identification ...............................................................40 v First Generation Probes: Biotinylated Ligands ................................................................43 Second Generation Probes: Photolabile Biotinylated Probes...........................................46 Tag-free Photoprobes ........................................................................................................50 SAR Analysis ......................................................................................................................55 Solvent Capture Study ........................................................................................................56 Target Identification Assays ..............................................................................................57 Conclusions ........................................................................................................................60 Chapter 4: Biofilm Inhibition SAR Study .................................................................................62 Biofilms, Virulence, and Clinical Relevance .....................................................................62 Biofilm Inhibition Assay .....................................................................................................64 Initial