Development of a selective and stable Reactive Oxygen Species-activated anti-Acute Myeloid Leukemia agent and localizing DNA Aptamer a dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements of the degree of DOCTOR OF PHILOSOPHY (Ph.D.) In the Department of Chemistry of McMicken College of Arts and Sciences by Kaylin Grace Earnest Bachelor of Science (B.S.), Chemical Science and Biochemistry, Xavier University, Cincinnati, OH Dissertation Advisor: Edward J. Merino, PhD Abstract Anticancer agents that modify DNA are a mainstay of chemotherapy regimens, but development of new classes of these agents has slowed because of the modifications of DNA in non-cancerous cells. This is what gives rise to serious side effects via poor selectivity. The Merino Lab has developed a pro-drug strategy to achieve specificity by translating the finding that levels of reactive oxygen species (ROS) are elevated in cancers, such as Acute Myeloid Leukemia (AML). This pro-drug approach allows cellular ROS to oxidize the pro-drug into its active form to achieve selective cytotoxicity. Our current lead agent (A100) is shown to have 10-fold selectivity between AML cells over normal CD34+ blood cells in vitro and showed some efficacy in the in vivo AML mouse model; however, it did not perform as highly as expected. It was hypothesized that the poor in vivo results were due to poor solubility and susceptibility to metabolic enzymes. This work started with computational analysis to determine what parts of the molecule were a target for metabolic enzymes. The first step taken to improve the molecule was to add polyethylene glycol (PEG) to the free phenol, increasing both its solubility and metabolic stability. To prove metabolic stability, binding assays against CYP1A2 (Cytochrome P450, Isoform 1A2) were done, as CYP1A2 is known to attack alcohols. The synthetic addition of the PEG increased stability against CYP1A2 by almost 50%. To prove stability in a more complex matrix, total stability was measured via half-life in pooled human liver microsomes. The PEGylated compound (A100-PEG) showed i a 7-fold increase in the half-life of A100, as compared to A100 alone. A pharmacokinetic method was developed and optimized to test the stability of A100- PEG in a mouse model, using only 30μLs of blood. A100 alone was not detectable in mouse blood samples after 15 minutes; however, in all three mouse models, A100-PEG was detectable even after 100 minutes and was calculated to have a 6-fold increase in half-life, as compared to A100. Though stable, A100-PEG only showed similar efficacy in the in vivo AML mouse model; however, an IV injection was used instead of the previous IP injection. Having improved its solubility and metabolic stability, the next step was to improve agent delivery. This was done by developing an aptamer to selectively “shuttle” the molecule to the cancer cells. Aptamers are selective binding nucleic acid macromolecules that are evolved in vitro to bind a specific target, in this case, whole AML cells, through a process called SELEX, systematic evolution of ligands by exponential enrichment. Twelve rounds of SELEX, including two counter selections against fibroblast cells, were completed. Aptamer pools were sequenced, and 3 main candidate sequences were identified. These sequences consisted of two 23 bases primers and a 30-base sequence in between. Binding studies were done using flow cytometry, and the lead sequence was found to have a binding constant of 38+/-2.5nM to AML cells, while having no binding to fibroblast and umbilical cord blood cells at 200nM. A truncation study of the lead sequence was done using 9 shortened sequences, which proved that the 5’ primer was not important for binding. The binding of the lead sequence was tested against 7 ii primary AML patient samples, and 5 of the 7 cell lines showed binding at 200nM. In conclusion, an anti-Acute Myeloid Leukemia agent was metabolically stabilized using PEG and its stability was proven in an in vivo mouse model. Also, a localizing nucleic acid aptamer specific to AML cells was developed, sequenced, and characterized for future drug-aptamer conjugates. iii iv Acknowledgements It is with great humility that I have the chance to say to thank the people who have made this accomplishment possible. I first want to give gratitude to my God, who gave me the life to pursue this passion and the persistence to complete this dream. I want to thank my parents, Tom and Anita, for all of their love and support. So many times throughout this journey I called them wanting to give up, and they always gave me the sane advice I needed to hear. Without them, I don’t see how I could have done this. I also want to thank my siblings Alicia (and Sean), Matthew (and Morgan), and Melinda. I had to sacrifice so much time in this endeavor, that I felt I neglected them and the love they showed me. Without their support, both near and far, this would not have been possible. I cannot wait to be able to spend more time with my nephew Walter, and all the nephews and nieces to come. To my extended family – thank you for understanding when I couldn’t come for holidays or spend that extra time with you. I cannot wait to thank you and to make up for the time I lost in the pursuit of this dream, especially my godson Joseph. In high school, I lost a dear friend to the very disease I ended up researching, acute myeloid leukemia. Tony – thank you for your beautiful life, and I hope you know that I put the pressure on myself to succeed in this because of you. I want to also pay tribute to my cousin, Jennifer Scoles, who passed too soon from this world because of breast cancer in my first year of graduate school. Jenni – your passing lit a fire within me that constantly reminded me that the mountain I v was trying to conquer was well worth the time and energy. To all of the surviors of cancer, whether I know them personally or not, you supplied me with the happiness and drive I needed to carry on. I owe a great deal of thanks to those who have been a part of my education. First to the teachers and faculty at Bishop Dwenger High School, you all not only allowed me to grow as a young adult, but you also showed me that eduation and science could be fun. I’d like to specifically note Lara Fairchild, Maryanne Spohn, Carrie Bleeke, John Bennet, and Lisa Polhamus for their guidance and dedication to my education, especially when I didn’t realize how impactful it would be untll now. To Xavier University – where do I begin? I owe the Xavier Chemistry Department so much gratitude – Dr. Barbara Hopkins, Dr. Adam Banage, Dr. Mary Stroud, Dr. Roger Parker, and Dr. Justin Link (even if he’s technically Physics). If sophomore me could see me today, she wouldn’t recognize me. Thank you Dr. Dan McLoughlin, my academic advisor, for believing in me, even when I didn’t believe in myself. To Dr. Rick Mullins, my research advisor – who saw more potential in me than I ever thought I had. Thank you allowing me to study under you and sparking my interest in scientific research. You helped me get into graduate school and continued to support me during the last 5 years. To my Xavier friends – thank you understanding my chaotic graduate school schedule and life. Your support, even when I couldn’t see or hangout with you, helped me through this degree. vi I also want to thank my advisor Dr. Edward Merino and the University of Cincinnati Chemistry Department for giving me the opportunity to study the subject that I love. I want to thank my committee: Dr. Patrick Limbach, Dr. Laura, Sagle, and the late Dr. Joe Caruso. Thank you for your support and guidance throughout this journey. I also want to thank my collabortors at CCHMC Dr. Jim Mulloy and Mark Wunderlich – without you this wouldn’t have been possible. To Dr. Maria DeRosa, Dr. Erin McConnell, and Dr. Eman Hassan and all my friends at Carleton University in Ottawa, Ontario Canada – thank you for giving me such a warm home in Ottawa. Your guidance and patience did not go unnoticed. To my lab mates past and present – thank you. Thank you for putting up with my dancing and singing in lab. I will miss you all dearly. To my classmates – this journey has been unforgettable. I’d like to acknowledge my funding sources – the Univeristy of Cincinnati Chemistry Department, the R.I.T.E. Program, the NIH, the DOD. Obviously none of this would have been possible without that support. And lastly I’d like to thank my dog, Bruce. Every presentation or report I had to give – he listened, even though he had no idea what I was talking about. You make me smile every day, which something I definitely took for granted. I’d like to conclude my acknowledgment by thankfully remembering each and every one who helped me in so many countless ways along this journey. All my love, Kaylin Grace vii viii Table of Contents Chapter 1: Introduction to Cancer, Reactive Oxygen Species, A100, and Aptamers .............................................................................................................. 1 1.1 Reactive Oxygen Species: Production and Depletion ................................. 2 1.2 High levels of ROS leads to DNA Damage and Disease ............................. 3 1.3 Cancer Statistics .......................................................................................... 4 1.4. Current Chemotherapeutic Approaches ..................................................... 5 1.5 ROS-Activated Anti-Cancer Agents ............................................................