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Design and Evaluation of Hmp Kinase Analogs As Thiamine

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Design and Evaluation of Hmp Kinase Analogs As Thiamine DESIGN AND EVALUATION OF HMP KINASE ANALOGS AS THIAMINE PATHWAY INHIBITORS by DIEGO A. LOPEZ Presented to the Faculty of the Graduate School of The University of Texas at Arlington in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY THE UNIVERSITY OF TEXAS AT ARLINGTON Summer 2016 Copyright © by Diego A. Lopez 2016 All Rights Reserved ii Acknowledgements This work would not have been possible without the help, support, and motivation of many people. First and foremost, I would like to thank Dr. Frank W. Foss Jr. for his mentorship, guidance, and amazing contribution into my life as a young scientist. I will always look up to him with admiration and follow his academic lessons and life teachings wherever I go. I would like to express my sincere gratitude to my committee members, Dr. Jeon and Dr. Mandal for their continuous support throughout the years and for their insightful suggestions towards the development of the project. In addition, I would like to thank the Organic division professors, Dr. Lovely, Dr. Bugarin, and Dr. Pierce for always being cordial and supportive both inside and outside of the chemistry field. I would also like to thank the numerous people who have contributed into the project, specially Dr. Sumit Bhawal who started the HMP project and developed most of the syntheses present in this study. His hard-work, passion for chemistry, and strive for success will always be remembered by this colleague and friend. I thank all my undergraduate students and members of the Foss’ lab for all the hard work, merit, and joy that have brought into my academic career: Aaron, Andra, Mohammad “Mu”, Pawan, Shakar, Johny, Twi, Nicky, Kevin and Caitlynn. Finally, I would like to thank my family and friends who throughout these years, they have not ceased to push me up to this very moment. My most sincere gratitude to my Calasanz community and high five to Andy, Francisco, Jessica, Cynthia, Hakop, Adam, and Nelli. Lastly, I owe it all to God and my mom for her sacrifice and infinite dedication to me and Alvarito. July 22nd, 2016 viii Abstract DESIGN AND EVALUATION OF HMP KINASE ANALOGS AS THIAMINE PATHWAY INHIBITORS Diego A. Lopez, Ph.D. The University of Texas at Arlington, 2016 Supervising Professor: Frank W. Foss Jr. Current bacterial chemotherapy faces an overwhelming reduction in efficacy due to an increase in bacterial resistance; resulting in a higher clinical demand for novel therapeutics and the investigation for their respective targets. Vitamin pathways exogenous to the human body represent an attractive target for drug development, since the enzymatic machinery is solely and ubiquitously present in many infectious agents. HMP kinase [E.C 2.7.1.49] is a key enzyme in thiamine metabolism. It catalyzes two subsequent phosphorylations of 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP), making it indispensable for microbial survival. Our efforts aimed for a substrate-based approach, where over 50 HMP analogs were synthesized and assayed towards substrate scope and enzyme inhibition. Development of in vitro and in silico analyses were imperative in our search for the needle in a haystack; furthermore, they have aided in the formulation of a structure-activity relationship (SAR) and paved the way for future synthesis. The catalytic activity of HMP was confirmed via a multitude of analytical tools such as UV-Vis, chemiluminescence, liquid chromatography, and mass spectrometry; resulting in the first generation of inhibitors and giving light to a whole new realm of compounds with pro-drug characteristics. ix Table of Contents Acknowledgments ............................................................................................................. vii Abstract ............................................................................................................................. ix List of ilustrations .............................................................................................................. xiii List of tables ..................................................................................................................... xvi Chapter 1 Introduction to antibiotics ................................................................................. 17 1.1 Golden years ......................................................................................................... 17 1.2 Mechanisms of antibiotic action ............................................................................ 19 1.3 Mechanisms of antibiotic resistance ..................................................................... 22 1.3.1 Prevention of access to target ....................................................................... 23 1.3.2 Target modification ........................................................................................ 23 1.3.3 Molecular bypass .......................................................................................... 24 1.3.4 Chemical modification ................................................................................... 25 Chapter 2 Introduction to thiamine biosynthetic pathway ................................................. 27 2.1 Vitamin B1: Biosynthesis and Role in Living Systems ........................................... 27 2.2 The appeal of vitamin pathways as source of novel antibacterials ....................... 32 2.3 HMP kinase structure and function in thiamine biosynthesis ................................ 34 2.4 Thiamine regulation across species ...................................................................... 39 2.5 HMP kinase binding site: substrate & inhibitors .................................................... 40 2.6 HMPK as drug target ............................................................................................. 44 Chapter 3 HMP Kinase Overexpression and Purification ................................................. 47 3.1 Materials and methods .......................................................................................... 49 3.2 Results .................................................................................................................. 51 3.3 Discussion ............................................................................................................. 53 x Chapter 4 Assay Development and Screening of HMP Analog ....................................... 56 4.1 Qualitative TLC assay ........................................................................................... 58 4.1.1 Materials and methods .................................................................................. 58 4.1.2 Results .......................................................................................................... 59 4.1.3 Discussion .................................................................................................... 60 4.2 ADP-Glo chemiluminescence end-point assay ..................................................... 61 4.2.1 Materials and methods .................................................................................. 63 4.2.2 Results .......................................................................................................... 64 4.2.3 Discussion ..................................................................................................... 71 4.3 Enzyme coupled kinetic assay .............................................................................. 82 4.3.1 Materials and methods .................................................................................. 83 4.3.2 Results .......................................................................................................... 85 4.3.3 Discussion ..................................................................................................... 88 4.4 High Performance Liquid Chromatography assay ................................................ 89 4.4.1 Materials and methods .................................................................................. 89 4.4.2 Results .......................................................................................................... 90 4.4.3 Discussion ..................................................................................................... 94 4.5 In vitro whole cell assay and metabolite detection ................................................ 95 4.5.1 Materials and methods .................................................................................. 98 4.5.2 Results ........................................................................................................ 100 4.5.3 Discussion ................................................................................................... 105 4.5.4 Conclusion and future approach ................................................................. 106 Chapter 5 Experimental .................................................................................................. 108 5.1 General procedures ............................................................................................. 106 Appendix A: List of abbreviations ................................................................................... 121 xi Appendix B: NMR spectra .............................................................................................. 124 References ..................................................................................................................... 161 Biographical information ................................................................................................
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