UUttaahh SSttaattee UUnniivveerrssiittyy DDiiggiittaallCCoommmmoonnss@@UUSSUU All Graduate Theses and Dissertations Graduate Studies 5-2013 NNoovveell AAmmiinnooggllyyccoossiiddeess:: BBiiooaaccttiivvee PPrrooppeerrttiieess aanndd MMeecchhaanniissmm ooff AAccttiioonn Sanjib K. Shrestha Utah State University Follow this and additional works at: https://digitalcommons.usu.edu/etd Part of the Biology Commons RReeccoommmmeennddeedd CCiittaattiioonn Shrestha, Sanjib K., "Novel Aminoglycosides: Bioactive Properties and Mechanism of Action" (2013). All Graduate Theses and Dissertations. 2073. https://digitalcommons.usu.edu/etd/2073 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@USU. It has been accepted for inclusion in All Graduate Theses and Dissertations by an authorized administrator of DigitalCommons@USU. For more information, please contact [email protected]. NOVEL AMINOGLYCOSIDES: BIOACTIVE PROPERTIES AND MECHANISM OF ACTION by Sanjib K. Shrestha A dissertation submitted in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Biology Approved: __________________ _____________________ Jon Y.Takemoto, PhD Michelle M. Grilley, PhD Major Professor Committee Member ___________________ ____________________ Charles D. Miller, PhD Dennis L.Welker, PhD Committee Member Committee Member ___________________ ____________________ Cheng-Wei Tom Chang, PhD Mark R. McLellan, PhD Committee Member Vice President for Research and Dean of Graduate School UTAH STATE UNIVERSITY Logan, Utah 2013 ii Copyright © Sanjib K. Shrestha 2013 All Right Reserves iii ABSTRACT Novel Aminoglycosides: Bioactive Properties and Mechanism of Action by Sanjib K. Shrestha, Doctor of Philosophy Utah State University, 2013 Major Professor: Dr. Jon Y. Takemoto Department: Biology Fungicide discovery is relatively neglected when compared to the investment in the development of antibacterial, antiviral, and anti-cancer therapeutics. Due to extensive use of currently available fungicides in agriculture and medicine, resistance is emerging among plant and animal pathogenic fungi. This necessitates the search for novel antifungal agents that are effective and less toxic and that do not promote resistance. FG08 and K20 are novel aminoglycoside analogs synthesized from kanamycin B and A, respectively. The antimicrobial properties of these analogs were tested in vitro against a wide range of agriculturally and clinically important fungal pathogens. Both compounds showed broad-spectrum antifungal properties, but they did not inhibit bacteria such as Escherichia coli and Staphylococcus aureus. The hemolytic activities and cytotoxicities of FG08 and K20 were also evaluated. They showed no toxicity or lowered toxicity against animal cells at their antifungal minimum inhibitory concentrations (MICs). iv The fungicidal mechanisms of action of FG08 and K20 were examined using intact cells of Saccharomyces cerevisiae, Cryptococcus neoformans, hyphae of Fusarium graminearum. FG08 and K20 caused SYTOX Green dye uptake and potassium efflux by intact cells, indicating that they increase plasma membrane permeability. FG08 and K20 also caused leakage of pre-loaded calcein from small unilamellar vesicles (SUVs) composed of lipids that mimic the lipid composition of fungal membranes, further suggesting increased membrane permeability as their mechanism of action. The synergistic interactions of K20 with six azoles (such as itraconazole, and fluconazole) were investigated against a wide array of fungal pathogens. The in vitro results revealed strong synergy between K20 and azoles against plant and human pathogenic fungi. Their synergies were furthered confirmed by time kill curves and disk diffusion methods. In conclusion, FG08 and K20 are broad-spectrum antifungal agents that do not inhibit bacteria. At their antifungal MICs, they are not toxic to animal cells, but they inhibit fungi by interacting with the fungal plasma membrane, leading to pore formation. These novel aminoglycoside analogs appear attractive for applications as fungicides in agriculture and medicine. (151 pages) v PUBLIC ABSTRACT Novel Aminoglycosides: Bioactive Properties and Mechanism of Action by Sanjib K. Shrestha, Doctor of Philosophy Utah State University, 2013 Major Professor: Dr. Jon Y. Takemoto Department: Biology More than 90% of crop diseases are due to fungal infections, which globally cause enormous economic losses. Fungal infections in humans and animals have also become a serious concern, especially in immunocompromised individuals. However, only a few new fungicides have been introduced since the mid-1980s, and concerns with inconsistent and declining effectiveness due to resistance, environmental impacts, animal and human toxicity, and costs continue to challenge the use of available fungicides. There is a serious and persistent need for new antifungal agents that are more effective, eco-friendly, less toxic, and available at reasonable cost. This study reveals two novel aminoglycosides, FG08 and K20, that are synthesized from kanamycin B and A, respectively. Kanamycin B and A are aminoglycoside antibiotics that kill bacteria. In contrast, FG08 and K20 showed broad- spectrum antifungal activities against a wide range of agriculturally and clinically important fungal pathogens, but they did not inhibit bacteria. They showed no or lowered vi toxicities against animal cells at their antifungal minimum inhibitory concentrations (MICs). They inhibit fungi by interacting with the fungal plasma membrane, leading to pore formation. K20 can be produced on a large scale, which makes it feasible to develop as a fungicide for commercial application. Additionally, K20 showed synergistic antifungal interaction with azoles against various fungi. Azoles are today’s most widely used antifungal agents to treat fungal infections in agriculture and medicine. In conclusion, this research has helped discover a new class of broad spectrum fungicides, which appear attractive for application as crop disease protectants and therapeutics against serious fungal infections in immunocompromised humans. vii DEDICATION This dissertation is dedicated to my loving parents, Sanat K. Shrestha and Niranjan K. Shrestha, and my uncle Prof. Dr. Krishna K. Shrestha. viii ACKNOWLEDGMENTS Foremost, I would like to express my sincere gratitude to my advisor, Dr. Jon Y. Takemoto, for the continuous support of my PhD study and research, for his motivation, enthusiasm and immense knowledge. I appreciate his willingness to permit me the freedom to explore different avenues of research while I was working in his laboratory. I deeply appreciate his persistence in pushing me to re-examine, rethink, and rewrite so as to produce the best work possible. I have been amazingly honored to have him as an advisor; without his support this dissertation could not have been completed. I would specially like to thank Dr. Michelle Grilley for her endless guidance, insightful comments, and constant encouragement throughout my doctoral program. I would like to convey my deepest appreciation and respect to all members of my committee, Dr. Cheng-Wei Tom Chang, Dr. Bradley R. Kropp (former committee member), Dr. Charles Miller, and Dr. Dennis Welker (new committee member) for their support, time, understanding, and encouragement through all these years. I owe special thanks to Lynnette Takemoto for her technical support and personal advice. I am also grateful for the support from Dean Scott Hinton, Dr. Ronald Sims, and Dr. Dong Chen of the Synthetic Biomanufacturing Institute, USU. I also thank and acknowledge all of my lab mates in Dr. Takemoto’s laboratory who made my work here greatly enjoyable. Special thanks to Christine Dhiman, Thomas Anderson, Dr. Mekki Bensaci, and Yukie Kawasaki. I would also like to thank my colleagues in the Dewald and Chang laboratories, Dr. Sitaram Harihar, Dr. Almut Volmer, Dr. Marina Fosso, and Jaya Shrestha for their friendship and help. ix I would like to thank my parents for their endless love, support, and sacrifices they have done for me. I am extremely grateful to my elder sisters, Menuka Shrestha and Medina Shrestha, for their constant support and encouragement they have provided me during these years. In addition, I would like to deeply appreciate my sweet brother, Rajeev K. Shrestha, for taking good care of our parents, business, and everything back home. I am very fortunate to have him as a brother. Special thank go to my dear son, Sanil Shrestha, for being my strength and origin of happiness. Last but not least, I wish to thank my beloved wife, Nika Shrestha, for her unconditional love, support, and patience. She was always there consoling me and stood by me through the good times and bad. Without you, my dear, I could not have achieved this. Sanjib K. Shrestha