ABSTRACT CHARACTERIZATION OF POTENTIAL ANTI-INFECTIVE AGENTS OF Burkholderia pseudomallei TARGETING IspF Joy Michelle Blain, Ph.D. Department of Chemistry and Biochemistry Northern Illinois University, 2016 James R. Horn, Co-Director Timothy J. Hagen, Co-Director There is an urgent need for new anti-infective agents. Pathogenic organisms continue to develop resistance to modern-day antibiotics, while there has been a significant decline in the development of antibiotics over the last 30 years. One particular pathway of interest in the development of new antibiotics is the biosynthesis of isoprenoids. These compounds are essential chemical building blocks in pathogenic organisms such as Plasmodium falciparum, Mycobacterium tuberculosis, and Burkholderia pseudomallei. Notably, there are two distinct pathways in the biosynthesis of isoprenoids called the Mevalonate (MVA) pathway and the 2C-methyl-D-erythritol phosphate (MEP) pathway. The MEP pathway enzymes are attractive targets in developing novel anti-infective agents due to the fact that humans only use the MVA pathway to produce isoprenoids. Burkholderia pseudomallei is a Gram-negative bacterium found in untreated water and soil in Southeast Asia and northern Australia. The bacterium has been classified as a category B bioterrorism agent/disease by the category B bioterrorism agent/disease by the Centers for Disease Control and Prevention. Antibiotic resistance to traditional anti-microbial agents in such pathogenic organisms has increased due to the primary mechanisms of resistance, which are enzyme inactivation, drug efflux from the cell, and target deletion. Consequently, new anti- infective agents targeting B. pseudomallei are of great interest. The focus of this research project was to examine the inhibition of the fifth enzyme in the MEP pathway, 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF) using small molecules that were developed based on fragment hits and structure activity relationships (SAR). In this study, enzyme engineering was utilized for the development of a high throughput thermal-shift assay. The assay was used to assess the relative potency of compounds targeting IspF. The inhibitor binding affinities and thermodynamics were determined using Surface Plasmon Resonance (SPR) and Isothermal Titration Calorimetry (ITC). Enzyme inhibition values for the most potent inhibitors were then evaluated using a High Performance Liquid Chromatography (HPLC) activity assay. Finally, structural analysis for two compounds in complex with IspF was completed to assess the mode of binding in the active site, which will aid in future inhibitor design strategies. NORTHERN ILLINOIS UNIVERSITY DE KALB, ILLINOIS AUGUST 2016 CHARACTERIZATION OF POTENTIAL ANTI-INFECTIVE AGENTS OF Burkholderia pseudomallei TARGETING IspF BY JOY MICHELLE BLAIN © 2016 Joy Michelle Blain A DISSERTATION SUBMITTED TO THE GRADUATE SCHOOL IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY AND BIOCHEMISTRY Doctoral Director: James R. Horn, Co-Director Timothy J. Hagen, Co-Director ACKNOWLEDGEMENTS I would like to first thank my graduate co-advisor, Professor James Horn, for his continual support, guidance, and patience. He has constantly challenged me throughout the last five years and I have become a better scientist because of his encouragement. I would also like to thank my co-advisor, Professor Timothy Hagen, for his guidance and enthusiasm. It has been an honor to work with both Professors Horn and Hagen. It is with tremendous gratitude that I acknowledge Rick Walter and Gina Raneiri for their invaluable help at the Advanced Photon Source. They have not only helped me immeasurably with my research but have become more than just colleagues. I have enjoyed all of our conversations and appreciate all your encouragement over the last two years. I would also like to thank Matt Pokross for all the knowledge he has passed on to me to in determining the crystal structures. I am also thankful to my committee members Professor Gary Baker and Professor Oliver Hofstetter. They have continually supported and aided in my work through numerous valuable discussions. Many thanks to Dr. Heike Hofstetter for her help with the NMR assay and all our conversations. My family and friends have been an incredible support system throughout my graduate school career. Their support when I needed it the most has been amazing and this would not have been possible without you. To my parents, Leland and Soon Ik Blain thank you for continual love and support. You have always encouraged me in my academic interests. I would not be iii where I am now without your inspiration and willingness to help me. To my sisters, Jennifer and Jill, my best friends and always being there for me and supporting me. To my nephews and niece, Leonidas, Leandra, and Liev, you are the light in my life. Thanks to my paternal grandparents, Phillip and Nora Blain for your endless support. My maternal grandparents, Jin Darl Son and Do Hee Kim, have passed away, but the work ethic of my grandfather and kindness of my grandmother have guided me throughout my life. I wish to thank my aunt and uncle Eun Ik and Duk Yoon Song for their love and kindness and my cousins Eun Sun Song and Eun Hee Lee and her family, Young, Yu Jin, Yoori, and Yoo Hyun Lee for their love and support. I would also like to thank my aunt Connie Blain, and my uncle and aunt, Robert and Coleen Blain and their family, for their encouragement. I am also grateful to my maternal uncles and aunts living in Korea: Dea Ik Son, Dong Ik Son, Ik Soon Son, Kyong Ik Son, their spouses and all of my cousins. Although we live far apart and are not able to see each other you are always close in my thoughts. Several thanks go out to all members of the Horn and Hagen labs, past and present. You have provided valuable feedback throughout our conversations. The group members have become not only colleagues but also friends. I would like to acknowledge Northern Illinois University and the Department of Chemistry and Biochemistry for funding support. Additionally, I would like to thank the NIU Foundation, National Institute of Health (1R15AI113653-01), and National Science Foundation (MCB-0953323) for providing the funding and financial support I have received. DEDICATION To Leonidas “Leo” Lucien Blain Christen: March 23, 2010 – April 8, 2010 Always on my mind Forever in my heart TABLE OF CONTENTS Page LIST OF TABLES ........................................................................................................ x LIST OF FIGURES ...................................................................................................... xi LIST OF APPENDICES ................................................................................................ xv LIST OF ABBREVIATIONS ....................................................................................... xvi LIST OF SYMBOLS .................................................................................................... xx CHAPTER 1 INTRODUCTION Cellular Targets for Antibiotic Development................................................... 5 Inhibition of Cell Wall Biosynthesis...................................................... 7 Inhibition on DNA/RNA Synthesis ....................................................... 9 Inhibition of Protein Biosynthesis ......................................................... 11 Mechanisms of Antibiotic Resistance .............................................................. 13 Investigation of Novel Targets and Novel Classes of Antibiotics ................... 15 Known Targets of Antibiotics ................................................................ 15 Bacterial Genes which are Essential, Conserved ................................... 16 Biosynthesis of Isoprenoids as a Potential Anti-Infective Target .......... 17 Two Distinct Isoprenoid Biosynthetic Pathways Mevalonate and Non- mevalonate ....................................................................................................... 19 vi Mevalonate Isoprenoid Biosynthetic Pathway ................................................. 19 Discovery of the Non-mevalonate Isoprenoid Biosynthetic Pathway ... 21 Methylerythritol Phosphate (MEP) Pathway an Attractive Target for Developing Novel Antibiotics .......................................................................... 23 MEP pathway Regulation ...................................................................... 25 Known MEP Pathway Inhibitors ........................................................... 25 Targeting Cyclodiphosphate Synthase, IspF .................................................... 29 Active Site of Cyclodiphosphate synthase (IspF) .................................. 31 Cyclodiphosphate synthase (IspF) Hydrophobic Cavity ....................... 34 Targeting Burkholderia pseudomallei IspF ........................................... 34 2 DEVELOPMENT OF A HIGH-THROUGHPUT THERMAL-SHIFT ASSAY Introduction ...................................................................................................... 36 Methods ............................................................................................................ 39 Expression and Purification of Burkholderia pseudomallei IspF .......... 39 Circular Dichroism................................................................................. 40 Solvent Accessible Surface Area Calculations ...................................... 41 Computational Stability