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Antimicrobial Agents Targeting Early-Stage Isoprenoid

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Antimicrobial Agents Targeting Early-Stage Isoprenoid ANTIMICROBIAL AGENTS TARGETING EARLY-STAGE ISOPRENOID BIOSYNTHESIS: DESIGN, SYNTHESIS AND EVALUATION OF ACETYLPHOSPHONATE INHIBITORS OF DXP SYNTHASE by Ryan J. Vierling A dissertation submitted to The Johns Hopkins University in conformity with the requirements of the degree of Doctor of Philosophy Baltimore, MD October 2014 Abstract The global threat of antibiotic resistant bacteria necessitates renewed efforts at designing antibiotics to tackle this healthcare challenge. Battling or overcoming this hazard will require the united efforts of chemists and microbiologists to discover new targets for antibacterial agents and design new molecular scaffolds to inhibit these unique targets. In addition to finding these new targets, scientists need to better understand the complicated, endogenous defenses of bacteria, including the up-regulation of promiscuous multi-drug resistant (MDR) efflux pumps. One potential new target for antibacterial development is early-stage isoprenoid biosynthesis via the methylerythritol phosphate (MEP) pathway essential to bacteria, parasites and some plants but absent in humans which instead utilize the orthogonal mevalonate pathway. The first catalytic step in the MEP pathway involves the condensation of pyruvate and D-glyceraldehyde 3-phosphate (D-GAP) in a thiamin diphosphate (ThDP)-dependent manner, to generate 1-deoxy-D-xylulose 5-phosphate (DXP) and carbon dioxide. This initial step is catalyzed by the unique biosynthetic enzyme, DXP synthase, and the product represents a metabolic branch point as DXP is the precursor for isoprenoid, thiamin and pyridoxal biosynthesis in bacteria. The importance of these three pathways in bacterial growth and division highlights DXP synthase as an attractive target for antibacterial development. DXP synthase is promiscuous in reference to acceptor substrates and possesses a unique domain arrangement and catalytic mechanism, which involves formation of a ternary complex, suggesting that selective inhibition should be achievable by using unnatural bisubstrate analogs which mimic potential donor and acceptor substrates. ii Towards this goal, we successfully designed, synthesized and evaluated a series of acetylphosphonate inhibitors which interact with the ThDP cofactor to form a phosphonolactylthiamin diphosphate (PLThDP) intermediate, which cannot undergo decarboxylation to form product. Two compounds in this series, butylacetylphosphonate (BAP) and benzylacetylphosphonate (BnAP) were shown to be selective inhibitors of DXP synthase. Further, antimicrobial studies show that these compounds can weakly inhibit bacterial cell growth in complex media. Rescue experiments with downstream metabolites and target overexpression strains confirm that they restrict bacterial growth by a mechanism involving inhibition of DXP synthase. One potential contributing factor to their weak antimicrobial activity is their susceptibility to efflux by the E. coli MDR pump, AcrAB-TolC. We report an observed trend with the length of the alkyl chain of the acetylphosphonate and the potential for uptake and efflux towards defining the optimal properties for a future generation of potent antimicrobial agents targeting DXP synthase. Advisor: Dr. Caren L. Freel Meyers 2nd Reader: Dr. Paul S. Miller iii Acknowledgements This dissertation represents the culmination of a six year, arduous journey through graduate school which was only possible because of the love and support of my family, friends and coworkers. I have deeply appreciated every opportunity to grow and develop as a scientist, and I have been blessed to be surrounded by positive, astoundingly intelligent and insightful individuals throughout my journey. First I would like to thank my own research lab, guided and fostered by our advisor, Professor Caren Freel Meyers. I am grateful for her advice, support and encouragement. She has pushed me for over five years to be a better scientist and communicator. I strive to emulate her scientific passion and organization, and I will always appreciate her advice and sense of humor. I would also like to thank my coworker, Dr. Jessica Mott Smith for being an excellent collaborator and friend. We make an excellent team in the lab and I always admired her work ethic and focus. I would like to thank Dr. Francine Morris for her collaboration and friendship. I am thankful for Sara Sanders agreeing to take on parts of this project and to Alicia DeColli for reading and editing my thesis, and to both of them for their energy and humor. Drs. Kip Bitok, Marie Webster and Leighanne Basta are great friends and I have enjoyed working with and learning from each of them. I would like to thank the members of my thesis committee, Professors Phil Cole, Craig Townsend and Jun Liu for their support, advice and patience. I received so much insight about research and presenting ideas and results from my interactions with them and I am forever grateful. Professor Paul Miller gave freely of his time to read and make recommendations to this thesis and I am grateful to him and to Professor Sean Taverna for attending my thesis defense on short notice. I would also like to thank my undergraduate iv research advisors, Professor Gordon H. Purser and Dr. David J. Vanderah for their support and wisdom in starting my career in chemical research. I have to also thank the institutional support which brought me to Baltimore and started my graduate research at Johns Hopkins. The Chemistry-Biology Interface (CBI) program, which grew under the careful attention of the first Director, Professor Marc Greenberg and continues to flourish under the advisement of the current Director, Professor Steven Rokita. I was welcomed my first day in Baltimore by the academic coordinator Lauren McGhee and I am forever grateful for her advice, organization and friendship. I would also like to thank the NIH for their financial support of the CBI program through our training grant (T32-GM08018901) and for their support of the work in the Freel Meyers lab via an R-O1 grant (GM084998). Baltimore felt much more like home thanks to the friendship of Jessica Popkin, Paul Peters, Dr. Phillip Flanders, Dr. John Sivey, Kristopher Thornsbury and Shawn Lowery, among countless others. I am indebted to them for their humor, company and for always making me feel welcomed. Most of all I want to thank my family for their support and encouragement from the beginning. My sister Kelly and brother Michael are some of my first friends and I could not be more proud of either of them. They are a great support to me and I love them both. My mother and father are truly two of the hardest-working, most loving and supportive parents I could have asked for. Jim Vierling is an excellent role model, father and the epitome of work ethic. Kim Vierling is a tireless mother, ardent supporter of her children and a generally loving and caring individual. I would not be the man I am today without their friendship, love and support. I strive always to live up to their example. v This thesis is dedicated to Ginny Vierling and Art Murphy. vi Table of Contents Chapter 1. Introduction ................................................................................................... 1 1.1 Discovery of antibiotics and development of clinical resistance .............................. 1 1.1.1 A history of antibiotics and their molecular targets ............................................ 3 1.1.2 Efflux pump-mediated resistance ........................................................................ 8 1.2 Isoprenoid biosynthesis is an underutilized target in antibiotic development ......... 11 1.2.1 The discovery of the non-mevalonate pathway for isoprenoid biosynthesis .... 13 1.2.2 Current efforts to target enzymes of the MEP pathway .................................... 15 1.2.3 Regulation in the MEP pathway........................................................................ 18 1.2.4 1-Deoxy-D-xylulose 5-phosphate (DXP) synthase is a favorable target for design of new antibiotics ....................................................................................................... 22 1.3 Reactive substrate mimics as inhibitors of DXP synthase ....................................... 30 References ..................................................................................................................... 32 Chapter 2. Electrophilic phosphonates as selective inhibitors of bacterial DXP synthase ............................................................................................................................ 41 Introduction ................................................................................................................... 41 Results ........................................................................................................................... 48 2.1 Synthesis of unbranched acetylphosphonates (2.1 – 2.2) ........................................ 48 2.2 Synthesis of unbranched acetylphosphonates (2.3 – 2.5) ........................................ 48 2.3 Synthesis of unbranched acetylphosphonates (2.6 – 2.8) ........................................ 49 2.4 Synthesis of phenyl acetylphosphonates.................................................................. 51 2.5 Synthesis of aromatic acetylphosphonates (2.10 – 2.17) ......................................... 52 vii 2.6 Evaluation of acylphosphonates (2.1 – 2.8) as inhibitors of E. coli DXP synthase 54 2.7 Evaluation of acylphosphonates (2.1 – 2.8) as inhibitors of the E1 subunit of porcine
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