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University of Windsor Scholarship at UWindsor Electronic Theses and Dissertations Theses, Dissertations, and Major Papers 2016 Exploiting Cancer Cell Mitochondria as a Therapeutic Strategy: Structure Activity Relationship Analysis of Synthetic Analogues of Pancratistatin Dennis Ma University of Windsor Follow this and additional works at: https://scholar.ca/etd Recommended Citation Ma, Dennis, "Exploiting Cancer Cell Mitochondria as a Therapeutic Strategy: Structure Activity Relationship Analysis of Synthetic Analogues of Pancratistatin" (2016). Electronic Theses and Dissertations.ca/etd/5744 This online database contains the full-text of PhD dissertations and Masters’ theses of University of Windsor students from 1954 forward. These documents are made available for personal study and research purposes only, in accordance with the Canadian Copyright Act and the Creative Commons license—CC BY-NC-ND (Attribution, Non-Commercial, No Derivative Works). Under this license, works must always be attributed to the copyright holder (original author), cannot be used for any commercial purposes, and may not be altered.

Any other use would require the permission of the copyright holder. Students may inquire about withdrawing their dissertation and/or thesis from this database. For additional inquiries, please contact the repository administrator via email (scholarship@uwindsor.ca) or by telephone at 519-253-3000ext. Exploiting Cancer Cell Mitochondria as a Therapeutic Strategy: Structure Activity Relationship Analysis of Synthetic Analogues of Pancratistatin Dennis Ma A Dissertation Submitted to the Faculty of Graduate Studies through the Department of Chemistry and Biochemistry in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Windsor Windsor, Ontario, Canada © 2016 Dennis Ma Exploiting Cancer Cell Mitochondria as a Therapeutic Strategy: Structure Activity Relationship Analysis of Synthetic Analogues of Pancratistatin By Dennis Ma APPROVED BY: ______________________________________________ S.

Ray, External Examiner Manchester University ___________________________________________ P. Vacratsis Department of Chemistry and Biochemistry ___________________________________________ M. Boffa Department of Chemistry and Biochemistry ___________________________________________ M. Crawford Department of Biological Sciences ___________________________________________ S.

Pandey, Advisor Department of Chemistry and Biochemistry May 25, 2016 Declaration of Co-authorship & Previous Publication I. Declaration of Co-Authorship I hereby declare that this dissertation incorporates material that is result of joint research as follows: This dissertation incorporates the outcome of joint research efforts undertaken in collaboration with Daniel Tarade, Tyler Gilbert, Christopher Pignanelli, Fadi Mansour, Scott Adams, Colin Curran, Alexander Dowhayko, Megan Noel, Melissa Cowell, Manika Gupta, Ian Tuffley, Sabrina Ma, Phillip Tremblay, Kevinjeet Mahngar, Pardis Akbari-Asl, Sergey Vshyvenko, Jonathan Collins, Tomas Hudlicky, and James McNulty under the supervision of Dr. In all cases, experimental design, execution, data analysis, interpretation, and manuscript preparation were performed by the author. All authors have read and approved the final manuscript prior to submission.

Collaboration with James McNulty is covered in Chapters 2 and 4 of this dissertation; this contribution was through provision of chemical compounds and write-up of synthesis details. Collaboration with Sergey Vshyvenko, Jonathan Collins, and Tomas Hudlicky is covered in Chapters 2-5 (and Appendices A and B) of this dissertation; this contribution was through synthesis and provision of chemical compounds and write-up of synthesis details. Collaboration with Phillip Tremblay, Kevinjeet Mahngar, and Pardis Akbari-Asl is covered in Chapters 2 and 3 (and Appendix A) of this dissertation; there was partial contribution to experimental execution and data interpretation. Collaboration with Daniel Tarade, Tyler Gilbert, Christopher Pignanelli, Fadi Mansour, Scott Adams, Colin Curran, Alexander Dowhayko, Megan Noel, Melissa Cowell, Manika Gupta, Ian Tuffley, and Sabrina Ma is covered in Chapters 4 and 5 of this dissertation; there was partial contribution to experimental execution and data interpretation.

I am aware of the University of Windsor Senate Policy on Authorship and I certify that I have properly acknowledged the contribution of other researchers to my dissertation, and have obtained written permission from each of the co-authors to include the above materials in my dissertation. I certify that, with the above qualification, this dissertation, and the research to which it refers, is the product of my own work. Declaration of Previous Publication This dissertation includes 4 original papers that have been previously published: CHAPTER 2 Ma D, Tremblay P, Mahngar K, Akbari-Asl P, Collins J, Hudlicky T, McNulty J, Pandey S. A Novel Synthetic C1 Analog of 7-deoxypancratistatin Induces Apoptosis in p53 Positive and Negative Human Colon Cancer Cells by Targeting the Mitochondria: Enhancement of Activity by Tamoxifen.

Investigational New Drugs 2012;30(3):1012-27. CHAPTER 3 Ma D, Tremblay P, Mahngar K, Collins J, Hudlicky T, Pandey S. Selective Cytotoxicity Against Human Osteosarcoma Cells by a Novel Synthetic C-1 Analogue of 7-deoxypancratistatin is Potentiated by Curcumin. APPENDIX A Ma D, Tremblay P, Mahngar K, Akbari-Asl P, Collins J, Hudlicky T, Pandey S.

Induction of Apoptosis and Autophagy in Human Pancreatic Cancer Cells by a Novel Synthetic C-1 Analogue of 7-deoxypancratistatin. APPENDIX B Ma D, Collins J, Hudlicky T, Pandey S. Enhancement of apoptotic and autophagic induction by a novel synthetic C-1 analogue of 7-deoxypancratistatin in human breast adenocarcinoma and neuroblastoma cells with tamoxifen. CHAPTERS 4 & 5 These chapters are manuscripts in preparation for submission.

Declaration of Previous Publication (Continued) I certify that I have obtained written permission from the copyright owners to include the above published materials in my dissertation. I certify that the above material describes work completed during my registration as graduate student at the University of Windsor. I declare that, to the best of my knowledge, my dissertation does not infringe upon anyone’s copyright nor violate any proprietary rights and that any ideas, techniques, quotations, or any other material from the work of other people included in my dissertation, published or otherwise, are fully acknowledged in accordance with the standard referencing practices. Furthermore, to the extent that I have included copyrighted material that surpasses the bounds of fair dealing within the meaning of the Canada Copyright Act, I certify that I have obtained a written permission from the copyright owners to include such materials in my dissertation.

I declare that this is a true copy of my dissertation, including any final revisions, as approved by my doctoral committee and the Graduate Studies office, and that this dissertation has not been submitted for a higher degree to any other University or Institution. v Abstract Distinct characteristics, including decreased dependence on mitochondrial respiration and high levels of oxygen radicals, provide opportunities for cancer targeting. We have shown the compound pancratistatin (PST) to selectively induce apoptosis in cancers by mitochondrial targeting. However, its low availability in nature was limiting its preclinical development.

Various PST analogues were synthesized to circumvent this limitation. In this dissertation, these analogues were screened and several had comparable or greater anti- cancer activity compared to PST. The analogue, SVTH-7, demonstrated the most potent anti-cancer activity, followed by SVTH-6 and -5 in vitro and in vivo. These compounds had greater efficacy than PST, 7-deoxyPST analogues, and multiple standard chemotherapeutics, and were found to induce apoptosis in cancer cells by acting on cancer cell mitochondria.

Furthermore, the anti-cancer effects of PST analogues were enhanced when used with agents known to target cancer cell mitochondria and oxidative vulnerabilities, including tamoxifen, curcumin, and piperlongumine. Interestingly, functional complex II and III of the electron transport chain were required for SVTH-7 to inflict its pro-apoptotic effects on cancer cells, suggesting exploitation of a mitochondrial vulnerability by SVTH-7. Therefore, these findings demonstrate a novel approach to treat cancer by exploiting cancer cell mitochondria with PST analogues alone or in combination with other compounds. These PST analogues have high therapeutic potential and this work will lay the groundwork for the identification and characterization of distinct mitochondrial features of cancer cells.

vi Acknowledgements I would like to thank my mentor and supervisor, Dr. Siyaram Pandey, for taking me into his lab in the summer of 2007 as an undergraduate student and giving me the opportunity to work in his lab for all these years to the completion of my undergraduate research project and this dissertation. He was the first to spark my interest in research and his guidance, expertise, patience and encouragement have been invaluable to my growth as a scientist. He is an example of having all the right intensions as a researcher to benefit others, and genuinely cares and advocates for his students.

Thank you to my doctoral committee, including Dr. Siyaram Pandey, Dr. Panayiotis Vacratsis, to Dr. Michael Boffa, Dr.

Michael Crawford, and Dr. Sidhartha Ray for their valuable input, critical evaluation of my work, and encouragement during my graduate studies. Thank you for the heartfelt local support to this work and our lab by the Knights of Columbus Chapter 9671 (Windsor, ON), the Pajama Angels, and the Couvillon Family, in memory of Kevin Couvillon. Your kindness, generosity, and encouragement have been extremely motivational to us and is greatly appreciated.

Thank you for believing in us. Thank you to the Lotte & John Hecht Memorial Foundation for funding the pancratistatin project and the Jesse & Julie Rasch Foundation for supporting the natural health products research in our laboratory. vii My graduate studies have been funded by a Vanier Canada Graduate Scholarship, a CIHR Frederick Banting and Charles Best Canada Graduate Scholarship, and an Ontario Graduate Scholarship. Thank you to our collaborators, Dr.

Tomas Hudlicky, Sergey Vshyvenko and Jonathan Collins of Brock University, for synthesizing and providing the synthetic derivatives of pancratistatin for this project. Thank you to Dr. James McNulty, for providing natural pancratistatin to our laboratory, as well as combretatstatin A4 (CA4) analogues for the synthetic CA4 project. Thank you to Dr.

Yi Wang at Wenzhou Medical University in China for providing synthetic analogues of curcumin for the synthetic curcumin project. Thank you to Dr. James Gauld in the capacity as both a mentor and collaborator. Having you next door was like having a second supervisor.

You have gone out of your way and have done so much for me and it is greatly appreciated; please also give my regards to Oscar and Frank. Thanks to Dan Simard and Mohamed Aboelnga for performing computation studies for my projects. Also, thanks to the rest of the Gauld Group, that I have gotten to know, for their support and friendship from across the hall: Phil De Luna, Bogdan Ion, Grant Fortowsky, Hisham Dokainish, Sameer Jafar, Wanlei Wei, Travis Dewolfe, Danielle, Rami Gherib, Eric Bushnel. Thank you to Dr.

Michael Boffa, Dr. Sirinart Ananvoranich, Dr. Bulent Mutus, Dr. Barbara Zielinski, and Dr.

Porter for the use of their lab equipment. Thank you to our secretaries in the Department of Chemistry and Biochemistry, Marlene Bezaire, Elizabeth Kickham, Cathy Wilson, and Michelle Miglietta for keeping our department running and for your support and encouragement. All of viii your work is much appreciated. Special thanks to Marlene for going out of her way to let me know about deadlines and scholarship opportunities; you are the reason why I am graduating as planned.

You have gone above and beyond, moving the whole world around for me, and your help throughout these years has been extremely valuable and greatly appreciated. Also thank you to Beth for addressing all my financial matters and concerns with general lab maintenance. Thank you to the Chemical Control Center crew, including Jerry Vriesacker, Alina Jaworska-Sobiesiak, Tina Lepine, Francis Arnaldo, and Sherri Menard, for all your assistance in obtaining lab supplies and materials. Special thanks to Jerry for dealing with all my extra ordering concerns in a very timely and efficient manner.

Special thanks to Janice Tubman, Elizabeth Fidalgo da Silva, Dorota Lubanska, Joe Lichaa, Bob Hodge, Elaine and Linda Sterling for their technical support.