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Developing a Bioscavenger Against Organophosphate Nerve Agents

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DEVELOPING A BIOSCAVENGER AGAINST ORGANOPHOSPHATE NERVE AGENTS Reed B. Jacob A Dissertation submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Curriculum in Bioinformatics and Computational Biology Chapel Hill 2017 Approved by: Nikolay V. Dokholyan Brian Kuhlman Alexander Tropsha Timothy C. Elston Matthew R. Redinbo ©2017 Reed B. Jacob ALL RIGHTS RESERVED ii ABSTRACT Reed B. Jacob: Developing a bioscavenger against organophosphate nerve agents (Under the direction of Nikolay V. Dokholyan) Organophosphate poisoning can occur from exposure to agricultural pesticides or chemical weapons. This exposure inhibits acetylcholinesterase resulting in increased acetylcholine levels within the synaptic cleft causing loss of muscle control, seizures, and death. Mitigating the effects of organophosphates in our bodies is critical and yet an unsolved challenge. Weaponized organophosphates are a deadly threat to armed forces and civilians (e.g., exposure to one droplet of VX exceeds the LD 50 of ~1 5 μg/kg) . Here, we present two computational strategies to 1) identify a novel protein acting as a stoichiometric bioscavenger to covalently bind organophosphates, and 2) improve the stability and activity of organophosphate hydrolase as a catalytic bioscavenger through allosteric modulation. We use our first computational strategy, integrating structure mining and modeling approaches, to identify novel candidates capable of interacting with a serine hydrolase probe (either covalently or with equilibrium binding constants ranging from 20 to 120 µM). One candidate Smu. 1393c catalyzes the hydrolysis of the organophosphate omethoate (kcat /K m of -1 -1 -1 3 -1 -1 (2.0±1.3)×10 M s ) and paraoxon (kcat /K m of (4.6±0.8)×10 M s ), V- and G-agent analogs respectively. In addition, Smu. 1393c protects acetylcholinesterase activity from being inhibited by two organophosphate simulants. We demonstrate that the utilized approach is an efficient and iii highly-extendable framework for the development of prophylactic therapeutics against organophosphate poisoning. Organophosphate hydrolase (OPH) degrades many classes of organophosphates and plays an important role in organophosphate remediation. Here, we demonstrate our second computational strategy of protein design to enhance OPH’s enzymatic hydrolysis of organophosphates and test on paraoxon, a G-agent analog, and omethoate, a V-agent analog. We identify five hotspot residues for allosteric regulation and assay these mutants for hydrolysis activity against paraoxon. A number of the predicted mutants exhibit enhanced paraoxon activity -1 -1 over wild type OPH (k cat =907 s ), such as T54I/T199I (1260 s ) while the K m remains relatively unchanged. Computational protein dynamics study reveals four distinct distal regions that display significant changes in conformation dynamics. The enzymatic enhancement of OPH validates a computational design method that is both efficient and readily adapted as a general procedure for enzymatic enhancement. iv This work is dedicated to my father Steve A. Jacob, who passed away before I began this journey. Education was very important to my father and he instilled the same in me. I also want to dedicate this work to my wife Brooke Miel Jacob and my daughter Kara Mae Jacob. It was your encouragement and support that made the many long hours worth it. v ACKNOWLEDGEMENTS Over the last five years I have grown and developed as both a scientist and a person. To start here in Chapel Hill I moved my family from the northwest to an unknown state with no friends or family for support. Luckily, one of the greatest supports for our family has been our church and the friends we made there. We would not have survived these last years without these friends, especially the Conrad’s, who have become family. Of utmost importance is my wife, Brooke Miel Jacob, who has tirelessly been a support. She has been a sounding board listening to all of my ideas and frustrations. She has been a sturdy foundation that has been crucial to our family’s success. No matter what state of mind I presented to her during this time she was there to calm me down, or motivate me as needed. During this five year journey we were blessed with the birth of our first child Kara Mae. It is thinking about the life I desire to create for both my wife and my daughter that has continued to motivate me in times of frustration. Additionally, I would like to thank the Department of Biochemistry and Biophysics, and the rest of the support staff who made it possible for my project to maximize the funding provided for it, especially Marsha Lynn Ray and John Hutton. I would also thank the Program in Molecular and Cellular Biophysics, and the Curriculum in Bioinformatics and Computational Biology, for teaching me many skills, giving feedback for my work and assisting me in becoming a better scientist then when I started. vi The members of my committee who always have expressed their unwavering support and crucial advice to advance my growth and development. My advisor, Nikolay Dokholyan, who encouraged me to think for myself and to think as a scientist. Lastly I want to thank all of those in my life, both educational and personal, who have influenced me in my path. I am excited for what the future will hold as I move forward. vii PREFACE Parts of the work described in this dissertation has been previously published as: Reed B. Jacob, James M. Fay, Cathy J. Anderson, Kenan C. Michaels, and Nikolay V. Dokholyan. “Harnessing Nature’s Diversity: Discovering organophosphate bioscavenger characteristics among low molecular weight proteins.” Sci. Rep. 6, 37175; doi: 10.1038/srep37175 (2016) Reed B. Jacob, Feng Ding, Dongmei Ye, Yazhong Tao, Eric Ackerman, and Nikolay V. Dokholyan. “Computational redesign reveals allosteric mutation hotspots of organophosphate hydrolase that enhance organophosphate hydrolysis.” (In preparation) viii TABLE OF CONTENTS LIST OF TABLES ......................................................................................................................... xi LIST OF FIGURES ...................................................................................................................... xii LIST OF ABBREVIATIONS AND SYMBOLS ........................................................................ xiii CHAPTER 1: CHEMICAL WEAPONS OF MASS DESTRUCTION – A JOURNEY THROUGH HISTORY ....................................................................................................................1 Acetylcholinesterase structure and mechanism .................................................................. 2 Structure/History of organophosphate agents. .................................................................... 5 Effects of OP exposure ....................................................................................................... 8 Current Protective Strategies .............................................................................................. 9 Catalytic Bioscavengers ........................................................................................ 10 Stoichiometric Bioscavengers ............................................................................... 11 CHAPTER 2: DISCOVERY, CHARACTERIZATION, AND INITIAL DESIGN OF LOW MOLECULAR WEIGHT PROTEINS WITH BIOSCAVENGER POTENTIAL .......................14 Results & Methods ............................................................................................................ 16 Computational Workflow overview. .................................................................... 16 Identify candidate scaffolds. ................................................................................. 19 Identification of top-ranked candidates. ............................................................... 22 Protein expression and purification. ..................................................................... 25 OP covalent binding to top candidates.................................................................. 26 ix OP covalent binding to Smu. 1393c active site. ................................................... 29 Smu. 1393c catalytic activity and protection of AChE......................................... 29 Smu. 1393c activity against OPs. ......................................................................... 33 Discussion ......................................................................................................................... 34 CHAPTER 3: COMPUTATIONAL REDESIGN OF ORGANOPHOSPHATE HYDROLASE TO ENHANCE ORGANOPHOSPHATE NERVE AGENT HYROLYSIS .......37 Results ............................................................................................................................... 40 Prediction and experimental validation of single mutations. ................................ 40 Prediction of double and triple mutations with experimental validations. ........... 43 Computational analysis of thermostability and conformational dynamics. .......... 45 Discussion ......................................................................................................................... 49 Methods............................................................................................................................. 51 CHAPTER 4: CONCLUSIONS AND FUTURE DIRECTIONS .................................................55 Stoichiometric bioscavenger ............................................................................................
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