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An Antibiotic Discovery Campaign Targeting Virf, the Main Transcriptional Regulator of Virulence in Shigella Flexneri

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An Antibiotic Discovery Campaign Targeting Virf, the Main Transcriptional Regulator of Virulence in Shigella Flexneri An Antibiotic Discovery Campaign Targeting VirF, the Main Transcriptional Regulator of Virulence in Shigella flexneri By Anthony A. Emanuele A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Medicinal Chemistry) in the University of Michigan 2016 Doctoral Committee: Professor George A. Garcia, Chair Professor Gordon L. Amidon Professor Anna K. Mapp Professor Hollis D. Showalter Professor Ronald W. Woodard A piece of advice that helped me through my last year of graduate school: No amount of regret can change the past, and no amount of anxiety can change the future © Anthony A. Emanuele 2015 For my biggest fan, My Mother (who is more excited than I am about my graduation) ii ACKNOWLEDGEMENTS I would not be where I am today without the support of a lot of people. First and foremost, I would like to thank my advisor, Dr. George Garcia. I cannot thank you enough for all you have done for me over the years. You allowed a young undergraduate student, who had no idea how to do anything, into your lab and gave him a chance to prove himself. Throughout my time in your lab, you have provided me guidance, in science and in life, and have taught what it means to be a good scientist. I could not have asked for a better mentor, thank you for everything. I’d also like to thank the members of my committee for all the advice and support they provided me during the course of my dissertation research: Dr. Hollis Showalter, Dr. Ronald Woodard, Dr. Gordon Amidon, and Dr. Anna Mapp. It would not be right if I did not acknowledge the many former and current members of the Garcia lab that made getting through each day a little easier. Dr. Julie Hurt for dealing with me as an undergraduate; I did not realize how annoying I must have been until after I was in your role, my apologies. Dr. Yi-Chen Chen for being the nicest and kindest person I have ever met in my life; lab morale really dropped off after you left. Dr. Suman Atwal for being someone I could always count on to go to No Thai with. Nathan Scharf for splitting rent and all the awesome baked goods; you should probably switch careers. Maxwell Stefan for making me feel not quite so old and his cheerful personality. Nicholas Ragazzone for being my successor… don’t disappoint me. Lastly, Andrew Pratt (the former adopted lab member) for teaching iii me how to convince people that I am right, even though, I may have no idea what I’m talking about. From the bottom of my heart I’d also like to thank all my friends and family that have supported me throughout my life. Specifically my parents, Leslie and Bernie, I obviously wouldn’t be the man I am today without you; my siblings, Tracy and Michael, for accepting my status as the golden child; and my grandparents. I also have to thank all my friends from back home for always finding a way to keep me grounded and never letting my head get “too” big. Lastly, I’d like to thank my beautiful wife, Katelyn, for all of her support. Over the past year, there were many stressful and discouraging days but, no matter what, you always found a way to make me smile. Words cannot describe how much this meant to me. The research presented in this dissertation was supported by the National Institutes of Health (AI85179), the Pharmacological Sciences Training Program (T32- GM007767), the American Foundation for Pharmaceutical Education, the Rackham Merit Fellowship, and the University Of Michigan College Of Pharmacy. iv TABLE OF CONTENTS DEDICATION…………………………………………………………………………………....ii ACKNOWLEDGEMENTS……………………………………………………………………..iii LIST OF FIGURES……………………………………………………………………………viii LIST OF TABLES…………………………………………………………………...………....xi ABSTRACT……………………………………………………………………………………xiii CHAPTER I. Introduction…………………………………………………………………………..1 The Need for Novel Antibiotics…................................................................1 Shigellosis………………………………………………………………..………2 Shigella Pathogenesis………………………………………………….………4 VirF: Structure, Regulation, and Function…..……………………….……….4 VirF: A Potential Anti-Virulence Target? ....................................................8 Research Objectives…………………………………………………….……...9 References………………….……………………………………………..…...11 II. Small Molecule High-Throughput Screening Campaign Targeting VirF..20 Materials and Methods…………………………………………………..……22 Results…………………………………………………………………….……26 Discussion……………………………………………………………….....…..31 Notes to Chapter II………………………………………………………….…36 References…………………………………………………………………..…36 v Appendix………………………………………………………………………..39 III. Tissue Culture-Based Models of the Shigella flexneri Invasion Process ……………………………………….....………………………………….….…….41 Materials and Methods…………………….....……………………………….42 Results…………………………………………...……………………………..46 Discussion………………………………………...……………………………51 Notes to Chapter III……………………………………………………………57 References…………………………………………..…………………………57 Appendix……………………………………………..…………………………61 IV. The Expression, Purification, and Characterization of VirF………….…..64 Materials and Methods………………………………..………………………66 Results…………………………………………………...……………………..76 Discussion………………………………………………...……………………81 Notes to Chapter IV……………………………………...……………………88 References………………………………………………..……………………88 Appendix…………………………………………………..……………………92 V. Mechanism of Inhibition Determination and Resulting SAR Studies…...94 Materials and Methods………………….…………………………………….96 Results………………………………………………………………………...103 Discussion…………………………………………………………………….116 Notes to Chapter IV………………………………………………………….123 References……………………………………………………………………123 Appendix………………………………………………………………………126 vi VI. HTS of ~20,000 Marine Natural Product Extracts Against Shigella flexneri: Identification of Globomycin and Desferrioxamine E………..131 Materials and Methods……………………………………………………....133 Results.......……………………………………………………………………143 Discussion……………………………………………………....…………….151 Notes to Chapter IV……………………………………………........……….158 References…………………………………………………………………....158 Appendix………………………………………………………………….......162 VII. Concluding Summary...............................................................................188 References...…………………………………………………………………193 vii LIST OF FIGURES Figure I-1. Schematic Representation of Shigella Pathogenesis………………….……5 I-2. Co-crystal Structure of MarA Bound to mar Promoter…………....…………6 II-1. Maps of the Reporter and Positive Control Plasmids Used in the HTS….21 II-2. Flow Chart Depicting the Hit Identification Process for the HTS....………27 II-3. Representative Dose-Response Curves from the Reconfirmation Study …..……………………………………………………………………………….31 II-4. Comparison of Controls for HTS Pilot Screen and 100K Small Molecule Screen ……………………………………………………………….…………32 III-1. S. flexneri 2457T Invasion Assay Results…………………....……………..48 III-2. S. flexneri 2457T Plaque Formation Assay Results…........……………….50 III-3. Size of Plaques from Plaque Formation Assay with S. flexneri 2457T..…51 IV-1. SDS-PAGE Analysis of the MalE-VirF Purification from pMALvirF in E. coli BL21(DE3) Cells…………………………………………………………….…77 IV-2. SDS-PAGE Analysis of the VirF(154-263) Purification from pET19b- VirF(154-263) in E. coli BL21(DE3) Cells…………………………...………78 IV-3. SDS-PAGE Analysis of MalE-VirF Purifications (KS1000 vs. BS103)..…79 IV-4. Electrophoretic Mobility Shift Assay (EMSA) PAGE of MalE-VirF Binding to pvirB DNA Probe……………………………………………….......………82 IV-5. Plot of MalE-VirF Binding to the pvirB DNA Probe…………………………83 V-1. Schematic Depicting Presumptive Steps in the Activation of Transcription by VirF…………………………………………………………………………..95 viii V-2. Electrophoretic Mobility Shift Assay (EMSA) PAGE of MalE-VirF Binding to pvirB DNA Probe in Presence of Small Molecule Hit Compounds…..104 V-3. Characterization of Compound 19615…………………………………..…110 V-4. Maps of Control Plasmids Used in GFP Reassembly Assay……………112 V-5. Maps of Test Plasmids Used in the GFP Reassembly Assay…………..113 V-6. Typical Results Seen in GFP Reassembly Assay………………………..114 V-7. Figure Depicting Deduced SAR…………………………………………….119 VI-1. Flow Chart Depicting the Results of the HTS Campaign………………...137 VI-2. HPLC Trace and Yield of Active Peptidic Molecules……………….........140 VI-3. Crystal Structure (Ellipsoid) of Globomycin A (1)…………………...........146 VI-4. Chemical Structures of Globomycins A-C (1, 2, and 3)………………….147 VI-5. Chemical Structure of Desferrioxamine E…………………………….......147 VI-6. Plots Depicting Fold VirF Activation (solid-line, right y-axis) versus % Growth Inhibition (dotted-line, left y-axis) for Globomycins A-C (1, 2, and 3)…………………………………………………………………………….....148 VI-7. Electrophoretic Mobility Shift Assay (EMSA) of MalE-VirF Binding to pvirB DNA Probe in Presence of Desferrioxamine E (Des)…………………….151 VII-1. Genes Regulated by VirF and their Roles in Pathogenesis....................189 Appendix Figure II-1. Representative Dose-Response Curves From Reconfirmation Stud........40 III-1. Bacterial Toxicity of Compounds….......…………………........……............63 IV-1. Analytical Gel Filtration Results………………………………………………92 IV-2. Negative Controls for FP Assay……………………………………………...93 V-1. EMSA Depicting E. coli RNA Polymerase (RNAP) Binding to the lac Promoter (plac) in the Presence of 19615…………………………………130 VI-1. HRMS Spectrum of Globomycin A (1)……………………………………..178 ix VI-2. HRMS Spectrum of Globomycin B (2)……………………………......……179 VI-3. HRMS Spectrum of Globomycin C (3)……………………………………..180 VI-4. 1H NMR Spectrum of Globomycin A (1) Recorded at 700 MHz (in DMSO- d6)..…………………………………………………………………………….181 VI-5. 13C NMR Spectrum of Globomycin A (1) Recorded at 700 MHz (in DMSO-
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