University of Kentucky UKnowledge Theses and Dissertations--Pharmacy College of Pharmacy 2018 DISCOVERY OF NEW ANTIMICROBIAL OPTIONS AND EVALUATION OF AMINOGLYCOSIDE RESISTANCE ENZYME- ASSOCIATED RESISTANCE EPIDEMIC Selina Y. L. Holbrook University of Kentucky, [email protected] Digital Object Identifier: https://doi.org/10.13023/etd.2018.310 Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Holbrook, Selina Y. L., "DISCOVERY OF NEW ANTIMICROBIAL OPTIONS AND EVALUATION OF AMINOGLYCOSIDE RESISTANCE ENZYME-ASSOCIATED RESISTANCE EPIDEMIC" (2018). Theses and Dissertations--Pharmacy. 89. https://uknowledge.uky.edu/pharmacy_etds/89 This Doctoral Dissertation is brought to you for free and open access by the College of Pharmacy at UKnowledge. It has been accepted for inclusion in Theses and Dissertations--Pharmacy by an authorized administrator of UKnowledge. For more information, please contact [email protected]. STUDENT AGREEMENT: I represent that my thesis or dissertation and abstract are my original work. Proper attribution has been given to all outside sources. I understand that I am solely responsible for obtaining any needed copyright permissions. I have obtained needed written permission statement(s) from the owner(s) of each third-party copyrighted matter to be included in my work, allowing electronic distribution (if such use is not permitted by the fair use doctrine) which will be submitted to UKnowledge as Additional File. I hereby grant to The University of Kentucky and its agents the irrevocable, non-exclusive, and royalty-free license to archive and make accessible my work in whole or in part in all forms of media, now or hereafter known. I agree that the document mentioned above may be made available immediately for worldwide access unless an embargo applies. I retain all other ownership rights to the copyright of my work. I also retain the right to use in future works (such as articles or books) all or part of my work. I understand that I am free to register the copyright to my work. REVIEW, APPROVAL AND ACCEPTANCE The document mentioned above has been reviewed and accepted by the student’s advisor, on behalf of the advisory committee, and by the Director of Graduate Studies (DGS), on behalf of the program; we verify that this is the final, approved version of the student’s thesis including all changes required by the advisory committee. The undersigned agree to abide by the statements above. Selina Y. L. Holbrook, Student Dr. Sylvie Garneau-Tsodikova, Major Professor Dr. David J. Feola, Director of Graduate Studies DISCOVERY OF NEW ANTIMICROBIAL OPTIONS AND EVALUATION OF AMINOGLYCOSIDE RESISTANCE ENZYME- ASSOCIATED RESISTANCE EPIDEMIC DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Pharmacy at the University of Kentucky By Selina Yijia Li Holbrook Lexington, Kentucky Director: Dr. Sylvie Garneau-Tsodikova, Associate Professor of Pharmaceutical Sciences Lexington, Kentucky 2018 Copyright © Selina Yijia Li Holbrook 2018 ABSTRACT OF DISSERTATION DISCOVERY OF NEW ANTIMICROBIAL OPTIONS AND EVALUATION OF AMINOGLYCOSIDE RESISTANCE ENZYME-ASSOCIATED RESISTANCE EPIDEMIC The extensive and sometimes incorrect and noncompliant use of various types of antimicrobial agents has accelerated the development of antimicrobial resistance (AMR). In fact, AMR has become one of the greatest global threat to human health in this era. The broad-spectrum antibiotics aminoglycosides (AGs) display excellent potency against most Gram-negative bacteria, mycobacteria, and some Gram-positive bacteria, such as Staphylococcus aureus. The AG antibiotics amikacin, gentamicin, kanamycin, and tobramycin are still commonly prescribed in the U.S.A. for the treatment of serious infections. Unfortunately, bacteria evolve to acquire resistance to AGs via four different mechanisms: i) changing in membrane permeability to resist drugs from entering, ii) upregulating efflux pumps for active removal of intracellular AGs, iii) modifying the antimicrobial target(s) to prevent drugs binding to their targets, and iv) acquiring resistance enzymes to chemically inactivate the compounds. Amongst all, the acquisition of resistance enzymes, AG-modifying enzymes (AMEs), is the most common resistance mechanism identified. Depending on the chemistry each enzyme catalyzes, AMEs can be further divided into AG N-acetyltransferases (AACs), AG O-phosphotransferases (APHs), and AG O-nucleotidyltransferases. To overcome AME-related resistance, we need to better understand these resistance enzymes and further seek ways to either escape or inhibit their actions. In this dissertation, I summarized my efforts to characterize the AAC(6') domain and its mutant enzymes from a bifunctional AME, AAC(6')-Ie/APH(2")-Ia as well as another common AME, APH(3')- IIa. I also explained my attempt to inhibit the action of various AAC enzymes using metal salts. In an effort to explore the current resistance epidemic, I evaluated the resistance against carbapenem and AG antibiotics and the correlation between the resistance profiles and the AME genes in a collection of 122 Pseudomonas aeruginosa clinical isolates obtained from the University of Kentucky Hospital System. Besides tackling the resistance mechanisms in bacteria, I have also attempted to explore a new antifungal option by repurposing an existing antipsychotic drug, bromperidol, and a panel of its derivatives into a combination therapy with the azole antifungals against a variety of pathogenic yeasts and filamentous fungi. KEYWORDS: aminoglycoside-modifying enzyme (AME), enzyme engineering, substrate promiscuity, enzyme kinetics, minimum inhibitory concentrations (MICs), drug combination. Selina Yijia Li Holbrook June 27, 2018 Date DISCOVERY OF NEW ANTIMICROBIAL OPTIONS AND EVALUATION OF AMINOGLYCOSIDE RESISTANCE ENZYME- ASSOCIATED RESISTANCE EPIDEMIC By Selina Yijia Li Holbrook Dr. Sylvie Garneau-Tsodikova Director of Dissertation Dr. David J. Feola Director of Graduate Studies June 27, 2018 DEDICATION In memory of my mother who brought me into this world, for throughout your entire life you demonstrated sacrificial and unconditional love for me. For my husband and best friend who shares this life with me, for you love me, serve me, lead me, help me, and support me. ACKNOWLEDGEMENTS Throughout my journey as a graduate student I have been blessed with numerous people who helped and guided me to become a better scientist. Without the time, patience, and expertise they invested in me, I would not be able to accomplish my degree. First of all, I would like to thank my Ph.D. advisor, Dr. Sylvie Garneau-Tsodikova, who has devoted enormous amount of time, funding, ideas, patience, and enthusiasm throughout my Ph.D. study and research. She teaches me to think with creativity, to execute with scientific accuracy, to observe with unbiased eyes, and to overcome difficulties with perseverance. She inspires me to challenge myself on a daily basis to achieve a better self and also sets an exceptional role model for an outstanding scientist and educator who serves both science and the people around her with a humble heart. With her guidance and funding, my Ph.D. study was made possible. I would also like to give genuine gratitude to Dr. Matthew S. Gentry, Dr. Steve G. Van Lanen, Dr. Jürgan Rohr, and Dr. Chang-Guo Zhan for their selfless service on my Ph.D. committee. Their encouragement, inspiring comments, and thoughtful questions have helped guide me to be a better researcher. I would like to thank Dr. Gentry specially for his input into training me in cell culturing and immunohistochemistry techniques. Furthermore, I would like to thank Dr. Oleg V. Tsodikov who, as a teacher and collaborator, provided his expertise in structural biology and enzyme dynamics in many of my projects. iii His contribution broadened the scope and impact of my studies that without whom I would not be able to accomplish. My gratitude also goes to all past and present S.G.-T. and O.V.T. lab members for all their support and encouragement throughout my study. I would like to thank Dr. Keith D. Green especially, for the time and energy he spent on training me lab techniques, teaching me scientific knowledge, and helping me in times of trouble shooting. I thank Taylor A. Lundy for her hugs. The support of all lab mates goes beyond friendship. Last but not least, I would like to thank all parties that provided financial support throughout my graduate study, including the University of Kentucky College of Pharmacy Department of Pharmaceutical Sciences, the National Institutes of Health, the University of Kentucky Presidential fellowship, which provided funding from 2016 to 2017. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS.………………………………………..………………….....iii LIST OF TABLES.………………………………………………………………….…..xiii LIST OF FIGURES.……………………………………………………………………..xvi LIST OF ABBREVIATIONS.………………………………………………………....xxiii Chapter 1. New trends in the use of aminoglycosides.…………………………………….1 1.1. INTRODUCTION.………………………………………………………………..1 1.2. AMINOGLYCOSIDES MODE OF ACTION: BINDING TO THE RIBOSOME.7 1.2.1. Aminoglycosides binding to the A-site.………………………….…………7 1.2.2. Aminoglycosides binding to h69.…………………………………….……10 1.3. AMPHIPHILIC AMINOGLYCOSIDES.………………………………….……12 1.4. AMINOGLYCOSIDES AS ANTIFUNGAL AGENTS……………………...…14 1.5. READING THROUGH THE FAULTY “STOPS” AT THE PREMATURE TERMINATION CODONS.……………………………………………...……16 1.5.1. Cystic