Discovery and Characterization of Novel Inhibitors of Protein Kinase R
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Discovery and Characterization of Novel Inhibitors of Protein Kinase R by Unkyung Shin Department of Biochemistry McGill University, Montreal August 2013 A thesis submitted to McGill University in partial fulfillment of the requirements of the degree of Master of Science Unkyung Shin 2013 TABLE OF CONTENTS TABLE OF CONTENTS .................................................................................... iii LIST OF FIGURES .............................................................................................. v ABSTRACT .......................................................................................................... vi RÉ SUMÉ ............................................................................................................. vii ACKNOWLEDGEMENTS .............................................................................. viii PREFACE ............................................................................................................. ix CONTRIBUTION OF AUTHORS ..................................................................... x ORIGINAL CONTRIBUTION TO KNOWLEDGE ....................................... xi CHAPTER 1: INTRODUCTION ...................................................................... 12 1.1 Protein Synthesis ......................................................................................... 12 1.2 Eukaryotic Translation Initiation ................................................................ 13 1.2.1 Cap-dependent initiation....................................................................... 13 1.2.2 IRES-mediated initiation ...................................................................... 15 1.3 Regulation of Translation Initiation in Eukaryotes ..................................... 17 1.3.1 eIF4F complex formation and eIF4E availability ................................. 17 1.3.2 Ternary complex formation and eIF2 phosphorylation ........................ 19 1.4 Kinases Mediating eIF2 Phosphorylation ................................................... 20 1.4.1 Heme-regulated inhibitor (HRI or EIF2AK1) ...................................... 20 1.4.2 dsRNA-dependent protein kinase (PKR or EIF2AK2) ........................ 22 1.4.3 PKR-like endoplasmic reticulum kinase (PERK or EIF2AK3) ........... 25 1.4.4 General control non-depressible 2 (GCN2 or EIF2AK4) ..................... 26 1.5 Pathological Implications of eIF2α Kinases ............................................... 28 1.6 Small Molecule Inhibitors of eIF2α Kinases .............................................. 30 1.6.1 Quercetin – inhibitor of HRI ................................................................ 31 1.6.2 2-Aminopurine – inhibitor of PKR ....................................................... 31 1.6.3 C16 – inhibitor of PKR ......................................................................... 32 1.6.4 GSK PERK inhibitor – inhibitor of PERK ........................................... 32 1.6.5 Syk inhibitor – inhibitor of GCN2 ........................................................ 33 iii 1.7 Compound Screening Strategy .................................................................... 33 1.8 Overview and Rationale for the Thesis ....................................................... 37 CHAPTER 2: RESULTS ................................................................................... 38 2.1 Identification of Hymenialdisine and Isohymenialdisine as Stimulators of Translation ......................................................................................................... 38 2.2 Isohymenialdisine Inhibits PKR-mediated Phosphorylation of eIF2. ......... 49 CHAPTER 3: DISCUSSION ............................................................................. 55 CHAPTER 4: MATERIALS & METHODS .................................................... 61 4.1 Materials and General Methods .................................................................. 61 4.2 Isolation of Hymenialdisine and Its Derivatives from Stylissa massa ........ 61 4.3 In vitro Transcription and Translation Reactions. ....................................... 62 4.4 In vitro Kinase Assays................................................................................. 63 4.5 Computational Mapping of the PKR Structure ........................................... 64 4.6 Docking of Hymenialdisine and Isohymenialdisine ................................... 65 CHAPTER 5: CONCLUSION........................................................................... 66 ACKNOWLEDGEMENTS ............................................................................... 67 REFERENCES .................................................................................................... 68 iv LIST OF FIGURES CHAPTER 1 Figure 1.1 Overview of eukaryotic cap-dependent translation initiation. ............ 14 Figure 1.2 Key regulatory mechanisms of eukaryotic translation initiation. ........ 18 Figure 1.3 Overview of eIF2α phosphorylation. ................................................... 21 Figure 1.4 Chemical structures of eIF2α kinase inhibitors. .................................. 31 Figure 1.5 Outline of the compound screening strategy ....................................... 35 CHAPTER 2 Figure 2.1 Effects of Hymenialdisine, Debromohymenialdisine, Isohymeni- aldisine on translation reactions in rabbit reticulocyte lysates (RRL). ................. 39 Figure 2.2 Effects of Hymenialdisine Debromohymenialdisine, Isohymeni- aldisine on translation from the HCV and CrPV IRESes in RRL. ....................... 42 Figure 2.3 Effects of Cmpds 1 - 3 on translation in Krebs, Wheat Germ, and E. coli S30 extracts. ................................................................................................... 44 Figure 2.4 Effect of adding Cmpds 1 - 3 to actively translating extracts. ........ 47 Figure 2.5 Inhibitory effect of Isohymenialdisine on GST-PKR-KD-mediated eIF2α phosphorylation and GST-PKR-KD autophosphorylation. ........................ 51 v ABSTRACT Double-stranded RNA-dependent protein kinase (PKR) is an interferon- inducible serine/threonine kinase that plays a crucial role in innate immunity against viral infections. Activated PKR downregulates global protein synthesis through phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF2) and induces transcriptional output of cellular mRNAs implicated in limiting viral spread. Accumulating evidence suggests that PKR is involved in a multitude of other cellular processes including signal transduction, apoptosis, cell growth and differentiation, as well as the pathogenesis of several neurodegenerative diseases. In this context, small molecules targeting PKR would be useful as tools to help understand the intricacies of such biological pathways. In screening a library of natural and synthetic products for eukaryotic translation modulators, we identified two small molecules, isohymenialdisine and hymenialdisine, that exhibit stimulatory effects on translation. Characterization of the mode of action of isohymenialdisine revealed that it directly acts on PKR by inhibiting its autophosphorylation activity and perturbs the PKR-eIF2α phosphorylation axis. vi RÉ SUMÉ La protéine kinase dépendante de l’ARN double brin (PKR) est une kinase à sérine/threonine don’t l’activité est induite par l’interféron qui joue une rôle clé dans la défense immunitaire contre les infections virales. Lorsque PKR est active, elle régule négativement la synthèse protéique via la phosphorylation de la sous-unité α du facteur d’initiation de la traduction 2 (eIF2) et induit la transcription de certains ARN messagers cellulaires qui codent pour des gènes impliqués dans le contrôle de la propagation virale. PKR participe aussi à une multitude d’autres processus cellulaires qui incluent le contrôle de l’activité certaines voies de signalisation, l’apoptose, la croissance cellulaire, la différentiation ainsi que dans la pathogenèse de plusieurs maladies dégénératives. À cet égard, l’identification de molécules inhibitrices ciblant dette protéine pourrait faciliter son étude et la compréhension de ses fonctions biologiques. Lors d’un criblage d’une librairie de composés naturels pouvant moduler la synthèse protéique eucaryote, nous avons identifié deux molécules, l’isohymenialdisine et l’hymenialdisine, qui présentent un effet stimulateur sur la traduction. La caractérisation du mécanisme d’action de l’isohymenialdisine à démontré qu’elle agit directement sur PKR en bloquant son autophosphorylation et, par conséquent, celle d’eIF2α. vii ACKNOWLEDGEMENTS First and foremost, I would like to thank my supervisor Jerry Pelletier for his guidance, support, and insightful discussions throughout my graduate studies. His passion for science and contagious enthusiasm for new discoveries have been a constant source of inspiration. I am also grateful for the members of my Research Advisory Committee, Dr. David Y. Thomas and Dr. Martin Schmeing for their helpful comments and suggestions. My appreciation extends to the current and previous members of the lab (Aouod Agenor, Regina Cencic, Jennifer Chu, Gabriela Galicia-Vázquez, Alexandra Katigbak, Amina Kreps, Teresa Lee, Rayelle Maiga, Abba Malina, John Mills, Sophia Moraitis, Zeina Nasr, Francis Robert, Patrick Sénéchal and Tanner Shpiruk) for their help and friendship in addition to making the working environment stimulating and enjoyable. Special thanks go to Francis for translating the abstract in French and Jennifer for proofreading this thesis. I also wish