Identification and Characterization of Isoform-Selective Inhibitors
Total Page：16
File Type：pdf, Size：1020Kb
IDENTIFICATION AND CHARACTERIZATION OF ISOFORM-SELECTIVE INHIBITORS FOR PROTEIN ARGININE METHYLTRANSFERASES by KUN QIAN (Under the Direction of Yujun George Zheng) ABSTRACT In recent years, the field of epigenetic drug discovery has been rapidly growing and have resulted in several FDA-approved therapies. Epigenetic mechanisms predominantly consist of DNA methylation, histone modifications and non-coding RNA regulations. The nine isoforms of mammalian protein arginine methyltransferases (PRMT1 to PRMT9) are crucial histone modifying enzymes responsible for arginine methylation, which is an abundant posttranslational modification in eukaryotic cells. PRMTs are categorized into type I, II and III based on their catalytic products, which are asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA) and monomethylarginine (MMA), respectively. The nine PRMTs have different roles in cells, many of which are not well understood. Numerous studies have suggested that PRMTs are promising drug targets for cancers because of their crucial biological functions. Therefore, PRMT inhibitors are potential therapeutic agents for cancer treatment and useful chemical tools for cancer research. Despite the steady progress being made in PRMT inhibitor research, challenges remain in the discovery of potent, isoform-selective, cell- permeable and in vivo-active PRMT inhibitors. This work focuses on the identification and characterization of small molecule inhibitors for two PRMT isoforms, PRMT1 (type I) and PRMT5 (type II). Combinatorial inhibitor discovery approaches including virtual screening, target-based high throughput screening, chemical modification applied in this study have resulted in several novel chemical entities for potent and isoform-selective inhibition of PRMT1 or PRMT5 in vitro and in vivo. These compounds also showed significant anticancer efficacy in PRMT dysregulation-associated cancer models. Furthermore, an innovative stopped flow fluorescence assay was developed to measure PRMT enzymatic activity, representing the first continuous assay for PRMT inhibitor characterization that can determine potency and distinguish the mechanism of inhibition simultaneously. INDEX WORDS: Epigenetics, Drug Discovery, Protein Arginine Methyltransferases, Small Molecule Inhibitors, Diamidines, Anticancer, Stopped Flow IDENTIFICATION AND CHARACTERIZATION OF ISOFORM-SELECTIVE INHIBITORS FOR PROTEIN ARGININE METHYLTRANSFERASES by KUN QIAN B.S. Central China Normal University, China, 2012 A Dissertation Submitted to the Graduate Faculty of The University of Georgia in Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ATHENS, GEORGIA 2017 © 2017 Kun Qian All Rights Reserved IDENTIFICATION AND CHARACTERIZATION OF ISOFORM-SELECTIVE INHIBITORS FOR PROTEIN ARGININE METHYLTRANSFERASES by KUN QIAN Major Professor: Yujun George Zheng Committee: Brian S. Cummings Scott D. Pegan Jeffrey Urbauer Jason Zastre Electronic Version Approved: Suzanne Barbour Dean of the Graduate School The University of Georgia December 2017 DEDICATION I dedicate this dissertation work to my loving family, whose words of encouragement and push have made me who I am today. This dissertation is also dedicated to the most important person I met in Athens: Ruizhe Lin, I am truly grateful for having you in my life. I also dedicate this work to Emmiline K. Pritchett, who always has been my best friend for the past five and a half years. iv ACKNOWLEDGEMENTS I would like to express my gratefulness to my advisor, Dr. George Zheng for giving me the opportunity to join his research group. Dr. Zheng always have been supportive to me with innumerous guidance and hand-on skills since the earliest stage of my journey in science, which will continue to be valuable in my future career. I would like to acknowledge my committee, Dr. Brian Cummings, Dr. Scott Pegan, Dr. Jeffrey Urbauer and Dr. Jason Zastre for their great suggestions to research and academic study. I am also thankful to many faculty members who helped me in my doctoral program. I would like to thank all the laboratory members, who have also become my dearest friends, for not only providing their assistance and discussion in research, but also their selfless help in my daily life. I am so glad to be your companion for the past five years. I am indebted to Dr. Jing Zhang, Dr. Hao Hu and Dr. Maomao He, who have been my closest collaborators in the laboratory and my reliable consultants. I am obliged to Ms. Liza Ngo, Ms. Melody Fulton for their suggestions on my dissertation writing. Ms. Hui Xu is greatly acknowledged for her assistance in the stopped flow experiments and PRMT5:MEP50 complex protein purification. I would like to appreciate all the collaborators outside the lab, who have made important contributions to my research project: Dr. Chunli Yan and Dr. Ivaylo Ivanov for their work in virtual screening and other computational studies; Nhat Quach, Wided Missaoui, Dr. Brian Cummings and Dr. Tai Guo for their work of K280 and K280S studies in prostate cancer cells and mice models; Hairui Su and Dr. Xinyang Zhao for their work in cellular characterization of v the diamidine compounds; Sudeepti Kuppa, Lindsay Berman and Dr. Mandy Murph for the examination of K313 activity in melanoma cells. Lastly, I would like to thank all the other staffs, colleagues and graduate students I have met in the College of Pharmacy at the University of Georgia. I had wonderful experience studying here because of all of you. I would not become who I am today without your positive influence on me. vi TABLE OF CONTENTS Page ACKNOWLEDGEMENTS .............................................................................................................v LIST OF TABLES ...........................................................................................................................x LIST OF FIGURES ....................................................................................................................... xi LIST OF ABBREVIATIONS ...................................................................................................... xiv CHAPTER 1 INTRODUCTION .........................................................................................................1 1.1 Targeting Epigenetics in Drug Discovery ..........................................................1 1.2 Protein Arginine Methyltransferases .................................................................2 1.3 Catalytic Activity of PRMTs .............................................................................4 1.4 Pharmacological Significance ............................................................................6 1.5 Biochemical Assays for PRMT Inhibitor Screening .........................................8 1.6 PRMT Inhibitors ..............................................................................................10 1.7 Conclusions ......................................................................................................28 1.8 Rationale and Goal of This Work ....................................................................31 2 DISCOVERY OF DIAMIDINE COMPOUNDS AS ISOFORM-SELECTIVE INHIBITORS FOR PRMT1 ........................................................................................32 2.1 Introduction ......................................................................................................32 2.2 Materials and Methods .....................................................................................34 2.3 Furamidine is a Lead PRMT1-Selective Inhibitor ...........................................40 vii 2.4 Discovery of Decamidine as an Improved PRMT1 Inhibitor ..........................46 2.5 Discovery of K313 as a Potent PRMT1 Inhibitor ............................................49 2.6 Conclusions ......................................................................................................60 3 K280S IS A DUAL-TARGET INHIBITOR FOR PRMT5 AND MICROTUBULIN IN PROSTATE CANCER ...........................................................................................65 3.1 Introduction ......................................................................................................65 3.2 Materials and Methods .....................................................................................67 3.3 Identification of K280 as a Potent and Selective PRMT5 Inhibitor ................70 3.4 K280S Showed Improved Stability Compared to K280 ..................................74 3.5 Structure Activity Relationship of K280 Derivatives in PRMT Inhibition .....75 3.6 Mechanism of Action Studies of K280 ............................................................78 3.7 Target Validation and Anticancer Efficacy Assessment of K280S in Prostate Cancer Cells and TRAMP-C2 Induced Prostate Cancer Mice Model ...................81 3.8 Conclusions ......................................................................................................84 4 DETECTION OF PRMT INHIBITORS WITH STOPPED FLOW FLUORESCENCE .......................................................................................................86 4.1 Introduction ......................................................................................................86 4.2 Materials and Methods .....................................................................................88 4.3 Fluorescent Changes of Fluorescein-Labeled Histone H4 Peptide during PRMT1 Catalysis ...................................................................................................91