University of Nebraska Medical Center DigitalCommons@UNMC Theses & Dissertations Graduate Studies Fall 12-16-2016 Target Validation and Pharmacological Characterization of Novel NMDAR Modulators Kiran Sapkota University of Nebraska Medical Center Follow this and additional works at: https://digitalcommons.unmc.edu/etd Part of the Behavioral Neurobiology Commons, Cellular and Molecular Physiology Commons, Mental Disorders Commons, Other Neuroscience and Neurobiology Commons, and the Pharmacology Commons Recommended Citation Sapkota, Kiran, "Target Validation and Pharmacological Characterization of Novel NMDAR Modulators" (2016). Theses & Dissertations. 177. https://digitalcommons.unmc.edu/etd/177 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@UNMC. It has been accepted for inclusion in Theses & Dissertations by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. TARGET VALIDATION AND PHARMACOLOGICAL CHARACTERIZATION OF NOVEL NMDAR MODULATORS by Kiran Sapkota A DISSERTATION Presented to the Faculty of the University of Nebraska Graduate College in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Pharmacology and Experimental Neuroscience Graduate Program Under the Supervision of Professor Daniel T. Monaghan University of Nebraska Medical Center Omaha, Nebraska December, 2016 Supervisory Committee: Wallace B. Thoreson, Ph.D. Huangui Xiong, M.D., Ph.D. Shelley D. Smith, Ph.D. Woo-Yang Kim, Ph.D. Shashank Dravid, Ph.D. i Acknowledgments First, I sincerely thank my advisor, Dr. Daniel T. Monaghan, for his continuous support, encouragement, and guidance. Thank you for believing in me and providing me an opportunity to work under your mentorship. I am very much thankful for your valuable time spent on me. During my graduate life under your supervision, I had the opportunity to learn about science as well as life. Your mentoring has changed my way of thinking about science, and definitely, your suggestions and instructions will remain valuable throughout my career in being a better scientist and, most importantly, a better person. I would also like to thank my graduate advisory committee members: Dr. Wallace B. Thoreson, Dr. Huangui Xiong, Dr. Shelley D. Smith, Dr. Woo-Yang Kim and Dr. Shashank Dravid, for their suggestions, guidance, and critical comments throughout the years of my graduate school, which always directed me to focus on goal-oriented research. I am also thankful to Dr. Keshore Bidasee and Dr. Myron Toews. You were always there to help when I had questions, whether it be about academic courses or about scientific research. I am grateful to my laboratory members: Hasaan Alsaad, Zhihao Mao, and Dr. Kang Tang for helping me in different ways. The time spent with each of you will be a happy memory. I appreciate the help from the administrative staff in the Department of Pharmacology and Experimental Neuroscience for their continuous support in various aspects during my graduate life, whether it be for arranging the room for a committee meeting or arranging a travel itinerary for scientific conferences. I am thankful to Dr. Howard fox and his laboratory staff for letting us use their NanoDrop and PCR machine. Finally, I would like to thank my parents, my wife, and my family for believing in me. Without your continuous support and encouragement, this process would not have been possible. ii TARGET VALIDATION AND PHARMACOLOGICAL CHARACTERIZATION OF NOVEL NMDAR MODULATORS Kiran Sapkota, Ph.D. University of Nebraska, 2016 Supervisor: Daniel T. Monaghan, Ph.D. Abstract The N-methyl-D aspartate receptors (NMDARs) are ligand-gated ion channels, which play important roles in learning and memory. Excessive activity of NMDARs is implicated in damage due to stroke and neurodegenerative diseases, whereas hypoactivity of NMDARs contributes to schizophrenia. The initial goal of my dissertation is to evaluate the potential role of the GluN2D-containing NMDARs in neuropathological, behavioral and cognitive alterations associated with schizophrenia and characterize the pharmacology and mechanisms of action of NMDAR modulators which could potentially be used to modulate these receptors in schizophrenia. A subanesthetic dose of the NMDAR antagonist ketamine elicits symptoms of schizophrenia. This property led to the well-supported theory of NMDARs-hypofunction in schizophrenia. We found that ketamine increases [14C]-2-deoxy-glucose uptake in the medial prefrontal cortex and entorhinal cortex in wild type (WT) but not in GluN2D-knock out (KO) mice. Ketamine also increases locomotor activity and gamma-band oscillatory power in WT but not in GluN2D-KO mice. These results suggest a critical role of GluN2D-containing NMDARs in ketamine-induced schizophrenia-like symptoms in mice. Also, consistent with a role for GluN2D in schizophrenia is that the GluN2D-KO mice displayed impaired spatial memory acquisition and reduced parvalbumin (PV)-immunopositive staining compared to control mice. iii To develop NMDAR modulator for treating schizophrenia and other neurological diseases, we characterized several different naphthalene and phenanthrene based compounds for their positive allosteric modulator (PAM), negative allosteric modulator (NAM) and competitive antagonistic activity at NMDARs. We discovered that UBP684 and UBP753 are general NMDAR PAMs, UBP792 is a GluN2C/GluN2D-preferring NAM and UBP791 is a GluN2C/GluN2D-preferring competitive antagonist. Subsequent studies identified the mechanisms of action of the new compounds. The general PAMs UBP684/UBP753 increase agonist efficacy. They increase the channel open probability (Popen), and prolong the deactivation time upon glutamate removal. They bind to both the inactive and active states of the receptor and stabilize the glutamate-bound state of the GluN2 ligand-binding domain (LBD). The GluN2C/GluN2D-selective NAM UBP792 is a non-competitive antagonist and acts in a voltage- independent manner. Like the PAMs UBP684 and UBP753, UBP792 also acts by stabilizing the GluN2 LBD. iv Table of Contents Acknowledgments ......................................................................................................................................... i Table of Contents ........................................................................................................................................ iv List of Tables ................................................................................................................................................ x List of Figures ............................................................................................................................................. xi List of Abbreviations ................................................................................................................................. xv Chapter 1 Introduction ............................................................................................................................... 1 1 NMDARs, schizophrenia, and NMDAR modulators ............................................................................... 2 1.1 N-methyl-D-aspartate receptors (NMDARs) ................................................................................... 2 1.1.1 Structure and pharmacology of NMDARs .......................................................................... 2 1.1.2 Developmental expression of NMDARs in brain ............................................................... 9 1.2 Schizophrenia ................................................................................................................................. 10 1.2.1 NMDAR hypofunction in schizophrenia ........................................................................... 11 1.2.2 Genes associated with schizophrenia ................................................................................ 13 1.2.3 NMDARs in parvalbumin cells and schizophrenia ........................................................... 14 1.2.4 Role of specific NMDAR subtypes in schizophrenia ........................................................ 16 1.2.5 Glutamatergic agents for schizophrenia treatment in future ............................................. 18 1.3 Allosteric modulators of NMDARs ................................................................................................ 20 1.3.1 Positive allosteric modulators (PAMs) of NMDARs ........................................................ 21 1.3.2 Negative allosteric modulators (NAMs) of NMDARs ...................................................... 24 1.3.3 Allosteric modulation of NMDARs by neurosteroids ....................................................... 26 1.3.4 Allosteric modulation of NMDARs by protons ................................................................ 28 1.4 Competitive antagonists of NMDARs ............................................................................................ 30 Chapter 2 Evaluation of GluN2D as a potential target for treating schizophrenia ............................. 33 2 GluN2D NMDAR subunit contribution to the stimulation of brain activity and gamma oscillations by ketamine; implications for schizophrenia .......................................................................................... 34 v 2.1 Abstract .......................................................................................................................................... 35 2.2 Introduction