Ph.D. Thesis Netrajoshi
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UC Santa Cruz UC Santa Cruz Electronic Theses and Dissertations Title Allosteric and inhibitory investigations of human 15- Lipoxygenases Permalink https://escholarship.org/uc/item/38j7h7xz Author Joshi, Netra Publication Date 2013 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA SANTA CRUZ ALLOSTERIC AND INHIBITORY INVESTIGATIONS OF HUMAN 15- LIPOXYGENASES A dissertation submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY in CHEMISTRY by NETRA SUBHASH JOSHI June 2013 The Dissertation of Netra Subhash Joshi is approved: -------------------------------------------------- Professor Theodore R. Holman -------------------------------------------------- Professor Glenn Millhauser (Chair) --------------------------------------------------- Professor Pradip Mascharak ---------------------------------------------- Tyrus Miller Vice Provost and Dean of Graduate Studies Copyright © by Netra S. Joshi 2013 ii Table of Contents List of Figures.………………………………………………………………………..iv List of Tables………..………………………………………………………………...v Abstract……………………………………………………………………………….vi Acknowledgements………………………………………………………………...viii Chapter 1 Introduction……………………………………………………………………1 References……………………………………………………………………29 Chapter 2 Inhibition of 12/15-lipoxygenase as therapeutic strategy to treat stroke………………………………………………………………………....47 References……………………………….…………………………………...74 Chapter 3 Potent and selective inhibitors of human reticulocyte 15-Lipoxygenase- 1 as anti-stroke therapies.....................................…………………………..……..79 References…………………………………………………………………..118 Chapter 4 Investigations into the allosteric and pH effect on substrate specificity of human epithelial 15-Lipoxygenase-2……………………………………….126 References…………………………………………………………..............164 iii List of Figures Chapter 1 Figure 1.1…………………………………………………………….………………24 Figure 1.2……………………………………………………………….……………25 Figure 1.3………………………………………………….…………………………26 Figure 1.4…………………….………………………………………………………27 Figure 1.5…………………….………………………………………………………28 Chapter 2 Figure 2.1…………………………………………………………………………….66 Figure 2.2…………………………………………………………………………….68 Figure 2.3…………………………………………………………………………….70 Figure 2.4…………………………………………………………………………….72 Figure 2.5…………………………………………………………………………….74 Chapter 3 Figure 3.1…………..……………………………………………………………….105 Figure 3.2…………………………………………………………………………...106 Figure 3.3………………………………………………………………………...…107 Figure 3.4………………………………………………………………………...…108 Figure 3.5………………………………………………………………………...…109 Figure 3.6………………………………………………………………………...…110 Figure 3.7………………………………………………………………………...…111 Chapter 4 Figure 4.1…………..……………………………………………………………….148 Figure 4.2…………………………………………………………………………...149 Figure 4.3………………………………………………………………………...…150 Figure 4.4………………………………………………………………………...…151 Figure 4.5………………………………………………………………………...…152 Figure 4.6………………………………………………………………………...…153 Figure 4.7………………………………………………………………………...…154 Figure 4.8………………………………………………………………………...…155 Figure 4.9………………………………………………………………………...…156 Figure 4.10……………………………………………………………………...…..157 iv List of Tables Chapter 3 Table 3.1…………..………………………………………………………………..112 Table 3.2…………………………………………………………………………....113 Table 3.3………………………………………………………………………...….114 Table 3.4………………………………………………………………………...….115 Table 3.5………………………………………………………………………...….116 Table 3.6………………………………………………………………………....…117 Chapter 4 Table 4.1…………..………………………………………………………………..158 Table 4.2…………………………………………………………………………....159 Table 4.3………………………………………………………………………...….160 Table 4.4………………………………………………………………………...….161 Table 4.5………………………………………………………………………...….162 v Abstract ALLOSTERIC AND INHIBITORY INVESTIGATIONS OF 15- HUMAN LIPOXYGENASES by Netra S. Joshi This dissertation focuses on two isoforms of 15-human lipoxygenase, reticulocyte 15-LOX-1 and epithelial 15-LOX-2, that differ considerably in their tissue expression, reaction specificity, auto-inactivation and response to the allosteric effectors. These 15-LOX isoforms exert their function in the cells by reacting with a variety of endogenous fatty acid substrates and producing hydroperoxy products, which regulate the inflammatory process in the cells. These 15-LOX products can participate in opposing roles in human diseases, and their ratios are known to regulate cell-cell adhesion processes in cells, playing a vital role in inflammation, cancer and thrombosis. Therefore, understanding the factors that affect the substrate specificity of these two LOX isozymes is critical to comprehend their exact role in human diseases. Furthermore, 15-LOX-1 has been recently implicated in stroke related damage via hydroperoxidation of fatty acid containing membranes and its ability to degrade mitochondrial membrane when triggered by reactive oxygen species. Studies indicate the potential of 15-LOX-1 inhibitors as neuroprotectors against stroke related damage. However drug discovery for stroke therapeutics is a long, tedious process with high chance of failure and presents a constant search for potent, selective inhibitors vi This dissertation investigates the discovery of a novel chemotype for 15-LOX- 1 inhibitors. It describes in detail the initial screening, characterization and optimization of potent and selective inhibitors of 15-LOX-1. The results of the in-vivo and in-vitro studies demonstrate the potential of this chemotype as a stroke therapeutic. Finally, the factors that regulate the substrate specificity of human epithelial 15-LOX-2 are also investigated here. The findings demonstrate that both pH and LOX products alter the kinetic parameters of C20 and C18 fatty acid substrates differentially, with both pH and LOX products activating the C20 kinetics but both inhibit C18 kinetics, resulting into a significant increase in the C20/C18 substrate specificity ratio. These alterations in the product ratio may present important consequences in human disease progression due to the distinct biological effects of these products. vii Acknowledgments I would like to take this opportunity to thank with gratitude my research advisor, Prof. Ted Holman for giving me a valuable opportunity to work in his lab. You have always been a very patient, understanding and helpful mentor. I remember the second quarter when I had a severe neck injury and I was not even sure if I could continue my studies here at UC Santa Cruz. I can’t thank you enough for taking it easy on me that time and helping me pursue my dream of doing a Ph.D. Your guidance, approachable and friendly nature, made it easier for me to continue my studies and I will always be indebted to you. I would also like to thank my thesis reading committee, Prof. Glenn Millhauser (Chair) and Prof. Pradip Mascharak, for their patience, and timely review of my Ph.D. progress. I learnt a lot from both of your classes during the first year and that enhanced my interest further in this field. I now want to extend my thanks to all the past and current Holman Lab members I worked with and whose support made my lab work experience enjoyable. Thank you for your efforts to accommodate my restricted schedule in the lab; it made my life easier. I am especially thankful to Dr. Aaron Wecksler, Dr. Eric Hoobler, Brian Jameson and a dear colleague of 5 years, Late Ken Ikei for their support. I also want to convey my thanks to my two undergraduates- Quang Tran and Max Stefan. Working with you both helped me to develop my mentoring skills. Qiangli Zhang aka Li, from Mass spectrometry facility is another person in the department whom I am thankful to for patiently helping me schedule, run and analyze my samples on LTQ. viii Finally, I need to thank the most important people in my life for their continued support and blessings without which this journey towards my goal would have been impossible. Big thanks with gratefulness to my parents, Seema and Subhash Joshi, for their sacrifices, encouragements and the efforts that made me the person I am today. I will be indebted to you forever. Special thanks to my wonderful husband Vishwas Hardikar for his love, support, patience, and encouragement during my endeavor here. It means more than I could describe. Thanks to my brother, Vaibhav Joshi for helping me with the initial admission process by promptly arranging and sending transcripts, recommendation letters from India. Your support and best wishes are greatly appreciated. Thanks to my sister, Priyanka Joshi for letting me decompress from time to time. I also would like to thank my late Father-in- law Vinayak Hardikar for the encouragement and prompt help for arranging study material for my preparatory exams. And the most thanks, but not the least by any means, to my son Jay Hardikar for being the most wonderful and loving person in my life despite my crazy schedule and impatience. Thank you for accepting me as a working mom and I hope that one day I will make up for the time we lost due to my studies when you were young. Your presence always makes me forget my worries and I love you more than you could ever imagine. ix The text of this document includes permitted reprints of the following previously published material: Yigitkanli K, Pekcec A, Karatas H, Pallast S, Mandeville E, Joshi N, Smirnova N, Gazaryan I, Ratan RR, Witztum JL, Montaner J, Holman TR, Lo EH, van Leyen K., Inhibition of 12/15-lipoxygenase as therapeutic strategy to treat stroke, Ann Neurol. 2013 Jan;73(1):129-35. The co-author, Theodore R. Holman, listed in this publication directed and supervised the research which forms the basis for