Western University Scholarship@Western Electronic Thesis and Dissertation Repository 5-17-2019 10:30 AM Non-Traditional Metallation of Metallothioneins with Xenobiotic Therapeutic Metals Daisy L. Wong The University of Western Ontario Supervisor Stillman, Martin J. The University of Western Ontario Graduate Program in Chemistry A thesis submitted in partial fulfillment of the equirr ements for the degree in Doctor of Philosophy © Daisy L. Wong 2019 Follow this and additional works at: https://ir.lib.uwo.ca/etd Part of the Analytical Chemistry Commons, Inorganic Chemistry Commons, Medicinal-Pharmaceutical Chemistry Commons, and the Other Chemistry Commons Recommended Citation Wong, Daisy L., "Non-Traditional Metallation of Metallothioneins with Xenobiotic Therapeutic Metals" (2019). Electronic Thesis and Dissertation Repository. 6338. https://ir.lib.uwo.ca/etd/6338 This Dissertation/Thesis is brought to you for free and open access by Scholarship@Western. It has been accepted for inclusion in Electronic Thesis and Dissertation Repository by an authorized administrator of Scholarship@Western. For more information, please contact [email protected]. Abstract The rise of the Anthropocene has seen more global pollution than before in history. With the explosion of consumer electronics in the last half century, the rise of metal pollution from their extraction and disposal results in the unnatural introduction of heavy and rare metals into the ecosystem. Organisms have a metal defense protein, metallothionein, which has multiple roles in essential metal regulation and protection against toxic metal exposure. However, these modern heavy metals prominent in electronics are not found biologically and their interactions in the body are generally unknown. Some of these metals are employed as therapeutic agents in the treatment of cancers, and as such this Thesis describes an investigation of therapeutic agents as models for heavy metal pollution to provide insight into the mechanisms of metal metabolism. Using electrospray ionization mass spectrometry and spectroscopic techniques, the binding of human metallothionein with the exotic metals platinum and rhodium is explored. Platinum and rhodium bind readily to human metallothionein, raising concerns for toxicity. Keywords Metallothionein, metal regulation, metalloprotein, ietalation, metallochaperone, glutathione, binding constants, kinetic rate constants, pH-dependence, metal-thiolate clusters, cisplatin, metallodrugs, Platinum Group Metals, rhodium, oxidation, drug resistance, heavy metal pollution, environmental toxicity, anthropogenic waste, xenobiotic metals, electronic waste, Molecular Mechanics, Molecular Dynamics, Time- Dependent Density Functional Theory, Molecular Orbital Theory, UV-visible Absorption Spectroscopy, Circular Dichroism Spectroscopy, Native Electrospray Ionization Mass Spectrometry, Denaturant, Non-platinum cancer therapeutics, Non-traditional ietalation. i Summary for Lay Audience Metals are everywhere. Some metals are required by humans and organisms as nutrients, while some can be extremely toxic. These toxic metals can be deadly depending on amount of exposure, often causing heavy damage to cells and tissues. Life has adapted to light levels of toxic metal exposure with proteins that are involved in metal binding - a group of proteins known as Metallothioneins. When exposed to toxic metals, cells will increase the production of this protein, metallothionein, to counteract the increased exposure. These metallothionein proteins capture the toxic metal and isolate it from performing its toxic activity. This response also can occur in some cancer cells, in response to metal-based therapy (cisplatin, a platinum-based drug), where the metal in the drug triggers the cancer cell to have an aggressively defensive response. At the chemical level, metallothionein acts to break apart the drug molecule and isolate the toxic metal for safe excretion from the body. Recent research in constructing robust molecular frameworks for these drugs to improve cancer treatment efficacy may be able to bypass Metallothionein’s defensive nature in these aggressive cancers. Using an extremely precise analytical methods known as mass spectrometry that show the changes in the exact mass as the protein breaks down the metal from the drug. While metal-based drugs are a specific type of toxic metal exposure, there is a fear of the increased impact of human activity on ecosystems, climate, and the environment will result in toxic metal exposure to all life in ways that has not been done before. These involve toxic metals that have never had a biological role and are found in major electronics and consumer goods that are now a standard of living. The implications of these cancer drug analyses with metallothioneins are extended to address this issue of toxic metal pollution and its effects. ii Co-Authorship Statement This thesis contains material from previously published manuscripts. Dr. Martin Stillman is a coauthor of all the published papers and supervised Daisy Wong. For all chapters that were published, Ms. Daisy Wong wrote the initial draft of the paper. Dr. Martin Stillman was involved in all levels of publication and had major roles in both editing and revising the published manuscripts. For all Chapters except Chapter 2 and 6, Ms. Daisy Wong was solely responsible for acquiring all of the data, preparing the figures, and drafting the manuscripts with guidance and assistance from Dr. Martin Stillman. The conceptual experimental design for the experiments in Chapter 2 were carried out Ms. Natalie Korkola, Ms. Daisy Wong, and Dr. Martin Stillman. Ms. Natalie Korkola carried out the sample preparation, experimental procedures, data collection, and creating the figures with assistance and guidance from Dr. Martin Stillman and Ms. Daisy Wong. Ms. Daisy Wong wrote the paper for publication. Editing of the draft was carried out by Ms. Daisy Wong, Ms. Natalie Korkola, and Dr. Martin Stillman. Ms. Natalie Korkola is a coauthor of the associated manuscript. The computational calculations reported, and related discussion in Chapter 6 were carried out by Ms. Angel Zhang. Ms. Daisy Wong carried out the sample preparation, experimental procedures, data collection, and creating the figures with assistance and guidance from Dr. Martin Stillman. Ms. Angel Zhang created figures for the calculated results. Ms. Daisy Wong wrote the paper for publication. Mr. Abayomi Faponle and Dr. Sam P. de Visser carried out initial calculations that led to some of the experiments performed in Chapter 6. Editing of the draft was carried out by Ms. Daisy Wong, Ms. Angel Zhang and Dr. Martin Stillman. Ms. Angel Zhang is listed as a coauthor on the associated manuscript. For their contributions, Mr. Abayomi Faponle and Dr. Sam P. de Visser (University of Manchester, UK) are also listed as coauthors on the associated manuscript. iii Acknowledgments First and foremost, I thank my supervisor and friend Professor Martin Stillman, for introducing me to bioinorganic chemistry and providing me the opportunity to pursue my own original research. I am forever grateful for the mentorship and camaraderie he has given over the years, as well as the many amazing opportunities to network and present my research to the international community. I have been a member of the Stillman Bioinorganic Group for almost a decade and have watched many members in transit. I would like to thank former group members Dr. Tyler Pinter, Dr. Gordon Irvine, Dr. Duncan Sutherland, and Dr. Mike Tiedemann for my initial training. I am grateful to have worked closely with Ms. Angel Zhang, Ms. Judith Scheller, Ms. Lina Heinlein, and Dr. Dorothee Ott, and will treasure the many wonderful memories we all shared. A special thank you goes to Ms. Angel Zhang for teaching me the wonders of computational chemistry. I am thankful to have worked with the upcoming members of the group Ms. Natalie Korkola, Ms. Amelia Yuan, Ms. Adyn Melenbacher, and Mr. Riley Hooper, I believe you are all talented individuals and I wish you the best in whichever paths you carve for yourselves. This Thesis could not have been completed without the support of the staff at the Electronic Shop at UWO, as the miracles they work keep our equipment and computers running smoothly. Special thanks to Mr. Doug Hairsine for the maintenance, training, and advice on the operation of the ESI-MS. Thank you to Dr. Chris Levy (UWO), Matt Werm (The Grad Club, UWO), Andrew Wall (former Electronic Shop), Matthew Pelletier, Alfred Nataprawira, and Andrew Day (and his cats, Hobbes and Caprica) for your encouragement and unwavering support. And lastly, thank you to my mum and dad, for without them, I would not be here today. iv Table of Contents Abstract ................................................................................................................................ i Co-Authorship Statement ................................................................................................... iii Acknowledgments .............................................................................................................. iv Table of Contents ................................................................................................................ v List of Tables ..................................................................................................................... xi List of Figures ..................................................................................................................