METHODS OF BLOCKING EFFLUX PUMP ACTIVITY IN ESCHERICHIA COLI Thesis Submitted to The College of Arts and Sciences of the UNIVERSITY OF DAYTON In Partial Fulfillment of the Requirements for The Degree of Master of Science in Chemistry By Erich Nicholaus Auer Dayton, Ohio May 2018 METHODS OF BLOCKING EFFLUX PUMP ACTIVITY IN ESCHERICHIA COLI Name: Auer, Erich Nicholaus APPROVED BY: Matthew E. Lopper, Ph. D. Faculty Advisor Associate Professor Justin C. Biffinger, Ph. D. Committee Member Assistant Professor Doug Daniels, Ph. D. Committee Member Executive Director & Research Professor ii ABSTRACT METHODS OF BLOCKING EFFLUX PUMP ACTIVITY IN ESCHERICHIA COLI Name: Auer, Erich Nicholaus University of Dayton Advisor: Dr. Matthew E. Lopper Overuse of antibiotics throughout the developed world has contributed significantly to the emergence of antibiotic-resistance in bacteria. Although this helps bacteria it is extremely harmful to humans. As bacteria adapt to the antibiotics, current treatments become ineffective and bacterial infections can become life threatening. One of the mechanisms that bacteria, such as E. coli, use are efflux pumps. Efflux pumps are capable of taking potentially harmful substances, like antibiotics, from inside the bacterial cell to outside of the cell. This allows the bacterium to escape from the damaging effects of the antibiotics. My hypothesis was that natural products, small molecule compounds, or DNA aptamers would be able to bind to some part of the tripartite structure of the AcrA-AcrB –TolC efflux pump and block its activity. The use of natural products and small molecule compounds as potential inhibitors allowed me to determine what substances and structural targets are not viable options. The use of DNA aptamers did show significant potential in blocking efflux pump activity. iii ACKNOWLEDGMENTS I would like to start by thanking Dr. Matthew Lopper, my advisor, for allowing me to be a part of his research lab for the past three years. He has been there for every step of this journey and I would not have finished this thesis without his guidance. I would also like to thank the Office for Graduate Academic Affairs for the financial support. In addition, I would like to thank the University of Dayton Chemistry Department for support throughout the past five years. Finally, I would like to thank all of the people I have worked with under the direction of Dr. Lopper including: Donald Mckenna, Robert Leszcynski, Sarah Baxter, CJ Moellering, and Venicia Hawach. iv TABLE OF CONTENTS ABSTRACT……………………………………………………………...………………iii ACKNOWLEDGMENTS………………………………………………………………..iv LIST OF FIGURES………………………………………………………………………vi LIST OF TABLES…………………………………………………………………….....vii LIST OF ABBREVIATIONS…………………………………………………………..viii CHAPTER 1: INTRODUCTION………….……………………………………………...1 CHAPTER 2: IN SILICO DOCKING OF SMALL MOLECULE COMPOUNDS………7 2.1 Introduction…………………………………………………………………..7 2.2 Experimental Methods………………………………………….…………….8 2.3 Results and Discussion…………………………………………….………..10 2.4 Conclusions……………………………………………………….…………18 CHAPTER 3: NATURAL PRODUCTS…………………………….…………………..19 3.1 Introduction……………………………………………………………….…19 3.2 Experimental Methods………………………………………………………20 3.3 Results and Discussion……………………………………………………...21 3.4 Conclusions………………………………………………………………….23 CHAPTER 4: APTAMERS………………..……………………………..……………...24 4.1 Introduction………………………………………………………………….24 4.2 Experimental Methods……………………………………………………....25 4.3 Results and Discussion……………………………………………………...29 4.4 Conclusions………………………………………………………………….31 CHAPTER 5: GENERAL CONCLUSION…………………………………………......33 REFERENCES…………………………………………………………………………..36 v LIST OF FIGURES Figure 1: Efflux pump overexpression leads to antibiotic resistance…..………………...3 Figure 2: Model of Gram-positive and Gram-negative bacteria efflux pumps…...………4 Figure 3: Binding Sites chosen for in silico efflux pump screening….………………….12 Figure 4: Efflux assay showing inhibition using ZINC06716957…………………….…18 Figure 5: Results of efflux assays with plant extracts……...…………………………….23 Figure 6: Efflux assay showing inhibition using ssDNA aptamers.…………..…………31 vi LIST OF TABLES Table 1: Small molecule compounds……….…………………………………………..15 Table 2: Mass of plant extracts…………………………………………………………21 vii LIST OF ABBREVIATIONS DNA Deoxyribonucleic Acid E. coli Escherichia coli O.D. Optical density PBS Phosphate-buffered saline CPZ Chlorpromazine DMSO Dimethylsulfoxide EtBr Ethidium bromide ABI-PP AcrAB/ MexAB-specific inhibitor of pyridopyrimidine derivative a.u. Arbitrary fluorescence intensity units SELEX Systematic Evolution of Ligands by Exponential enrichment ssDNA Single-stranded deoxyribonucleic acid viii CHAPTER 1 INTRODUCTION Antibiotic resistance is a developing problem throughout the world. As society progresses and people use more antibiotics, pathogens are able to mutate in order to avoid detection and destruction. Part of the persistence and survival of bacteria, including Escherichia coli, is the ability to confer resistance over time. Without this ability, they would struggle to survive for long periods of time.1 As host organisms have evolved and developed natural antimicrobial mechanisms, bacteria have responded with their own protective processes.3 The speed of adaptation within bacterial populations has been so rapid that within two years of the development of penicillin several bacterial strains had already developed resistance.4 This rate of adaptation has not slowed down since it first observed 70 years ago. Currently there are strains of Staphylococcus aureus, Pseudomonas aeruginosa, and other types of bacteria that are resistant to many antibiotics that are on the market.2 The development of resistance to current antibiotics is a major concern for the health care industry due to the higher costs and the higher risk of permanent damage caused by these drug-resistant pathogens.4 Bacteria can develop resistance to antibiotics in three different ways: acquired, adaptive, and intrinsic resistance.4,5 Acquired resistance refers to any advantage that an organism has gained over a parent, whether it is through the incorporation of DNA or from mutations that occur.6 Adaptive resistance occurs when the microbe changes the 1 way it expresses genes in response to stimuli. The last way in which microbes can develop resistance is through intrinsic resistance. Intrinsic resistance includes the mechanisms that exist with in an organism from the start. Efflux pumps are part of the intrinsic resistance since they are present in bacterial cells as a functional tool used for many purposes. They also become part of the acquired resistance through mutations that effect the levels to which the efflux pumps are expressed. This mutation is when they truly become problematic in our public healthcare system.4,5,7 Efflux pumps are thought to be used in the normal metabolism of bacteria, through functions such as the transport of amino acids, glucose, and cellular signaling.2 These efflux pumps also have the ability to pump out potentially harmful substances such as antibiotics, allowing them to be readily repurposed in resistance mechanisms. In drug resistant bacteria, the expression of efflux pumps increases in order to pump out as much of the antibiotic as possible (Figure 1).2,3 E. coli, along with many types of bacteria, has the ability to remove an extremely wide range of compounds from within the cell. This allows bacteria to be highly resistant to antibiotic agents due to the ability to easily pump out these compounds using the efflux pump apparatus.7 Development of new potential therapies that use chemical efflux pump inhibitors, in combination with available antibiotics, as a viable option to antimicrobial resistant bacteria.2,3 2 Figure 1. Efflux pump overexpression leads to antibiotic resistance: As a potentially harmful substance (red) builds up inside of a bacterial cell, the cell is able to use efflux pumps (blue/ purple) to prevent accumulation of the substance in the cell and protect itself from damage. The end result is a cell that has pumped out the majority of harmful compounds as shown on the right. Efflux pumps differ in structure depending on the type of bacteria in which they occur. Gram positive bacteria usually have efflux pumps that are a single polypeptide within the cytoplasmic membrane (Figure 2A).2,3,6 Meanwhile gram negative bacterial efflux pumps consist of three parts: an inner membrane protein, an outer membrane protein and a connector that bridges the two (Figure 2B).2 This focused on a gram- negative RND-type pump which consists of AcrA, AcrB, and TolC. AcrA acts as the adaptor protein connecting AcrB, the inner membrane transporter, and TolC, the outer membrane transporter.9 TolC is an outer membrane protein that is connected directly to AcrA and AcrB. AcrA bridges the intermembrane space, this is known as a membrane- fusion protein and acts as a barrier for where TolC and AcrB fit together within the membrane. 3 A B TolC AcrA AcrB Figure 2: Model of Gram-positive and Gram-negative bacteria efflux pumps: (A) An efflux pump in a gram-positive bacteria (B) The E. coli efflux pump in a Gram-negative bacteria that bridges the inner and outer membrane of the cell, consisting of AcrA (dark blue), AcrB (purple) and TolC (light blue). These three protein structures, working in conjunction with one another, form a channel that uses the energy of the proton-motive force to pump substrates out using conformational changes that push them through the protein channel.11 The complex that forms is capable of