Rockefeller University Digital Commons @ RU Student Theses and Dissertations 2020 Diverse Kappa Opioid Receptor Agonists: Relationships Between Signaling and Behavior Amelia Dunn Follow this and additional works at: https://digitalcommons.rockefeller.edu/ student_theses_and_dissertations Part of the Life Sciences Commons DIVERSE KAPPA OPIOID RECEPTOR AGONISTS: RELATIONSHIPS BETWEEN SIGNALING AND BEHAVIOR A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy by Amelia Dunn June 2020 © Copyright by Amelia Dunn 2020 Diverse Kappa Opioid Receptor Agonists: Relationships Between Signaling and Behavior Amelia Dunn, Ph.D. The Rockefeller University 2020 The opioid system, comprised mainly of the three opioid receptors (kappa, mu and delta) and their endogenous neuropeptide ligands (dynorphin, endorphin and enkephalin, respectively), mediates mood and reward. Activation of the mu opioid receptor is associated with positive reward and euphoria, while activation of the kappa opioid receptor (KOR) has the opposite effect. Activation of the KOR causes a decrease in dopamine levels in reward-related regions of the brain, and can block the rewarding effects of various drugs of abuse, making it a potential drug target for addictive diseases. KOR agonists are of particular interest for the treatment of cocaine and other psychostimulant addictions, because there are currently no available medications for these diseases. Studies in humans and animals, however, have shown that activation of the KOR also causes negative side effects such as hallucinations, aversion and sedation. Several strategies are currently being employed to develop KOR agonists that block the rewarding effects of drugs of abuse with fewer side effects, including KOR agonists with unique pharmacology. The goal of the research presented here was to profile the signaling pathways activated by KOR agonists and to investigate relationships between unique pharmacology and animal models of KOR-mediated behaviors, in order to better understand how to target the KOR for therapeutic use. First, we quantified the effects of KOR agonists at both G-protein and b-arrestin-2 signaling pathways to compare to a variety of downstream effects, including sedation behavior. We found that b-arrestin-2, but not G-protein, efficacy strongly correlated with sedation in mice. We found that there was no apparent relationship between either G-protein or b-arrestin-2 signaling pathways with other investigated downstream signaling pathways, such as ERK1/2 and mTOR, however. We also investigated the effects of KOR activation on protein-receptor interactions, to identify other potential mediators of KOR effects. Finally, we compared the effects of several unique KOR agonists on addiction-related behaviors in mice. We found that nalfurafine, which is approved for human use in Japan, very potent KOR agonist, was able to modulate the rewarding effects of cocaine at very low doses that did not cause sedation or aversion. We also found that the commonly-used, full KOR agonist U50,488 had a similar effect, suggesting that this “therapeutic window” could be a property of the KOR system in general. Overall, this work suggests that KOR agonists at very low doses, that show very little b-arrestin-2 signaling activity, may be able to modulate the rewarding effects of cocaine while causing fewer negative side effects. ACKNOWLEDGMENTS First and foremost, I want to thank my advisor Dr. Mary Jeanne Kreek. Her science has helped so many people, and I feel incredibly lucky for the opportunity to learn from her and her example of both scientific rigor and passion for her work. I’m so grateful for the support and guidance I’ve received during my time here. Dr. Kreek – thank you for everything. This work, of course, also belongs to many members of the Kreek lab. I’m forever grateful for the mentorship of Dr. Brian Reed, who was there for every single step of my PhD. Brian, I’ll strive to follow your example of going where the data leads, while always keeping the bigger picture, and the people you’re trying to help, in mind. Thank you for teaching me so much, and for the opportunity to contribute to this project. I’m lucky to have had so many wonderful mentors in the Kreek lab, including Drs. Yong Zhang and Eduardo Butelman. Eduardo, thank you for so many helpful suggestions and encouragement. Yong, thank you your many kind words and telling me to go home on the weekends. I’m incredibly grateful to Dr. Kyle Windisch for being a mentor, friend, and role model. Thank you for showing me how to be a careful and critical scientist, as well as a thoughtful and generous lab citizen. And of course, for the endless hours teaching and helping with surgeries. Finally, I want to thank the students and research assistants that I’ve been lucky enough to work with over the years, in particular, Josh, Catherine, Ali, Jose, Ariel and Phil. Thank you for making the endless hours in the CBC and the GTPgS room not only bearable, but sometimes even fun. I’m so lucky to have been a part of Team Kappa with you. I’ve received a great deal of support and mentoring from my thesis committee, Drs. Vanessa Ruta, Tom Sakmar and Brian Chait, as well. Your encouragement and thoughtful contributions to my project, both during and outside of my committee meetings, were incredibly helpful and iii essential for this work. Thank you to the Sakmar lab in particular for crucial technical help early on in my project, as well as welcoming me into their scientific community. And of course, the Chait lab was where I started my Rockefeller career in 2012. I’m grateful to Brian, and entire Chait lab, both for that opportunity and for the continued help, guidance and collaboration in the years since. In particular, I’d like to thank Dr. Wenzhu Zhang for so much patient technical help preparing and running the mass spectrometry samples, and Dr. Paul Olinares for always lending a helpful hand and kind ear. It has been such a privilege to be part of the Rockefeller community. I’m incredibly grateful to the Dean’s Office – Sid Strickland, Emily Harms, Cris Rosario, Stephanie Fernandez, Kristen Cullen, Andrea Morris, Marta Delgado – for their support through the graduate program, and their commitment to providing opportunities for students to get involved in research. Participating in the SURF program started me on this path, and teaching for the Summer Neuroscience Program here has been a highlight of my grad school career. A huge thank you to my fellow SNP directors, and especially to all of the incredible high school students, who made the last two weeks of August the craziest and most fun time of the year. Thank you to all of my friends that have made the past five years, the best five years. Your encouragement and support have meant the world to me, and you’ve made NYC home. And finally, I’m forever grateful for the endless support of my family and Ananth – I couldn’t have done it without you. iv TABLE OF CONTENTS ACKNOWLEDGMENTS…………………………………………………………….....iii TABLE OF CONTENTS ……………………………………………………………......v LIST OF FIGURES……………………………………………………………….….....vii LIST OF TABLES…………………………………………………………………..…...ix LIST OF ABBREVIATIONS……………………………………………………………x CHAPTER 1. Background and Introduction…………………………………………..1 1.1 Opioid receptors mediate mood and reward, including addiction ........................ 1 1.2 The KOR system negatively regulates reward .................................................... 3 1.3 Many KOR agonists have negative side effects in humans ................................. 4 1.4 KOR agonists activate multiple signaling pathways ........................................... 6 1.5 Additional signaling pathways may be involved in opioid receptor signaling ... 10 1.6 Research goals ................................................................................................. 11 CHAPTER 2. Materials and Methods…………………………………………………13 2.1 Chemical compounds ....................................................................................... 13 2.2 In vitro signaling studies .................................................................................. 14 2.2.1 b-arrestin-2 enzyme fragment complementation ...................................... 14 2.2.2 [35S]GTPgS Binding with U2OS cell membranes .................................... 14 2.2.3 [35S]GTPgS Binding with mouse striatum tissue ...................................... 15 2.2.4 In-cell western blots for ERK1/2 phosphorylation ................................... 16 2.2.5 Western blots for Rps6 phosphorylation .................................................. 16 2.2.6 LogRAi bias calculations ........................................................................ 17 2.3 Biochemical studies and imaging ..................................................................... 19 2.3.1 Stable-isotope labeling in cell culture (SILAC) and immunoprecipitation 19 2.3.2 Mass spectrometry analysis ..................................................................... 19 2.3.3 Immunofluorescent sample preparation and imaging ............................... 20 2.4 Animal model studies ...................................................................................... 20 2.4.1 Animals .................................................................................................. 20 2.4.2 Prolactin serum ....................................................................................... 21 2.4.3 Rotarod assay .........................................................................................