Design and Synthesis of Inhibitors of dTDP-D-glucose 4,6~ dehydratase (RmlB), an Enzyme Required for dTDP-L-Rhamnose Production in M.Tuberculosis by Neena Sujata Kadaba S.B. Chemistry Massachusetts Institute of Technology, 2002 SUBMITFED TO THE BIOLOGICAL ENGINEERING DIVISION IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN MOLECULAR AND SYSTEMS TOXICOLOGY AT THE MASSACHUSETTS INSTITUTE OF TECHNOLOGY JUNE 2003 © 2003 Neena S. Kadaba. All rights reserved. The author hereby grants to MIT permission to reproduce and to distribute publicly paper and electronic copies of this thesis document in whole or in part. Signature of A uthor............................................................................. V .................................... Neena S. Kadaba Biological Engineering Division May 9, 2003 Certified By................................................... ............... ........................................ ** \ jjJohn M. Essigmann Professor of Chemistry and Toxicology Thesis Advisor A ccep ted By ...................................................................... 2 ................................................... Ram Sasisekharan Chair, Biological Engineering Graduate Committee Design and Synthesis of Inhibitors of dTDP-D-glucose 4,6- dehydratase (RmlB), an Enzyme Required for dTDP-L-Rhamnose Production in M.Tuberculosis by Neena Sujata Kadaba Submitted to the Biological Engineering Division on May 9, 2003 in Partial Fulfillment of the Requirements for the Degree of Master of Science in Molecular and Systems Toxicology Abstract The purpose of this work is to probe the dTDP-L-rhamnose pathway in an effort to develop small molecule inhibitors that could act as therapeutics for Mycobacterium tuberculosis. The necessity for newer, more effective treatments for tuberculosis is growing, as the bacteria evolve resistance to traditional treatments. In an effort to develop more effective and perhaps more abbreviated courses of treatment, a plan was developed to investigate a pathway involved in cell wall biosynthesis as a promising target: the dTDP-L-rhamnose pathway. This pathway plays an essential role in linking the peptidoglycan and arabinogalactan portions of the mycolic acid-arabinogalactan-peptidoglycan complex, a significant part of the mycobacterial cell wall. The mounting level of biochemical understanding of this pathway and its importance in bacterial cell wall biosynthesis indicates that it is not only a relevant target but also an accessible one. Of the four enzymes crucial to this biosynthetic pathway, one was chosen as the primary focus: dTDP-D-glucose-4,6- dehydratase (RmlB). There are 3 steps in the reaction mechanism of RmlB: oxidation of the C4 position of dTDP-D-glucose to form a 4-keto structure, dehydration of the C6 position via the elimination of water and a subsequent reduction to result in a 6-deoxy product. Crystal structures of this particular enzyme, dTDP-D-glucose 4,6-dehydratase (RmlB), complexed with single substrates or substrate analogs have provided a foundation for these studies, enabling the rational design of a small library of potential inhibitors. Twelve mechanism-based inhibitors of RmlB are proposed. These compounds reflect the current understanding of the mechanism and mimic the sugar portion of the sugar-nucleotide substrate at various steps throughout the reaction mechanism. Each of the proposed inhibitors is designed to inhibit one of the specific steps of the mechanism. While the intention of this project is to synthesize each compound in this library from commercially available starting materials in 15 steps or less, the primary goal of this particular dissertation is to synthesize 3 of the 12 proposed inhibitors from the commercially available starting material 1,5-anhydro-D-glucitol. The long term goal of this work is to produce these compounds in significant amounts in order to test their efficacy in an animal model of mycobacterial infection. Thesis Supervisor: John M. Essigmann Title: Professor of Chemistry and Toxicology Acknowledgements Above all, I would like to take the time to sincerely thank my advisor, John Essigmann, for everything he has done for me over the past 4 years. I had the good fortune of having John as my undergraduate advisor, and from that time, he has been a consistent source of information and support. In the past year and a half in particular, he has given me a number of opportunities that I greatly appreciate-it is thanks to him that I had a wonderful time helping to teach the Biotechnology and Engineering course in Bangkok, and the amazing experience of spending a few months in Thailand. It was also John who had the great idea to attempt this research project this year, and I have only become even more excited about the project as the year progressed; all of these experiences have been a big part of my professional and personal growth over the past year, and I am grateful for his influences. I am eternally indebted to A. Nicole Dinaut, whose constant support and encouragement over the past months has been invaluable. She has not only provided guidance with every one of my chemistry dilemmas, but has also been a close friend, always celebrating our successes and dismissing our setbacks with smiling optimism. I am grateful to Kaushik Mitra for sharing his practical expertise, Denise Rodrigues for her help with my syntheses and 2D NMRs, Uday Sharma for synthetic words of wisdom and his assistance with hydrogenations and Will Neeley for sharing suggestions on technical matters. Many other lab members were a pleasure to work with, and I thank them for their moral support and friendship. I would like to thank the students of BEH.214 during the spring of 2002 for their assistance in putting the project together, and, in particular, Hector Hernandez for working with me on the final project for the class. I would like to thank Drs. Mathuros Ruchirawat and Somsak Ruchirawat for providing me the opportunity to stay and work at the Chulabhorn Research Institute in Bangkok, Thailand and Kim Bond Schaefer for continual administrative support while I was away. I appreciate the financial support from the NIH Training Grant in Environmental Toxicology along with help from Professor Douglas Lauffenburger of the Biological Engineering Division; both helped make my trip to Thailand a reality. Finally, I thank my family for their unconditional love and support. My mother and grandparents have encouraged every one of my endeavors, often making personal sacrifices for my benefit. They have instilled in me a love of learning, the confidence to pursue my goals relentlessly, and the desire to do something meaningful. Table of Contents Design and Synthesis of Inhibitors of dTDP-D-glucose 4,6-dehydratase (RmlB), an Enzyme Required for dTDP-L-Rhamnose Production in M .Tuberculosis.......................... 1 A b stract .................................................................................................................................... 2 Acknowledgements ........................................................................................................... 3 Table of Contents..................................................................................................................... 5 List of Fig u res........................................................................................................................... 7 List of Abbreviations ......................................................................................................... 8 Chapter 1. Introduction................................................................................................... 10 1.1 Overview ..................................................................................................................... 10 1.2 History and Epidemiology of TB Infection W orldwide ....................................... 11 1.3 Infection with Mycobacterium Tuberculosis....................................................... 15 1.3.1 Characteristics of Mycobacterium Tuberculosis.................... 15 1.3.2 The M ycobacterial Cell Wall ......................................................................... 16 1.3.3 Transmission, Infection and Disease Progression ......................................... 17 1.4 Host-Pathogen Interactions and the Immune Response ...................................... 18 1.4.1 M acrophage Activation and Response ........................................................... 19 1.4.2 Factors increasing susceptibility.....................................................................24 1.5 Current Treatments and the BCG Vaccine........................................................... 25 1.5 .1 Ison iazid ............................................................................................................... 2 5 1.5.2 Rifampin ......................................................................................................... 26 1.5.3 Ethambutol ....................................................................................................... 26 1.5.4 Pyrazinamide................................................................................................... 27 1.5.5 Streptomycin .................................................................................................. 27 1.5.6 Second line drugs............................................................................................ 27 1.5.7 Bacillus Calmette-Gudrin Vaccine ..............................................................