Preclinical Evaluation of Protein Disulfide Isomerase Inhibitors for the Treatment of Glioblastoma By Andrea Shergalis A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Medicinal Chemistry) in the University of Michigan 2020 Doctoral Committee: Professor Nouri Neamati, Chair Professor George A. Garcia Professor Peter J. H. Scott Professor Shaomeng Wang Andrea G. Shergalis [email protected] ORCID 0000-0002-1155-1583 © Andrea Shergalis 2020 All Rights Reserved ACKNOWLEDGEMENTS So many people have been involved in bringing this project to life and making this dissertation possible. First, I want to thank my advisor, Prof. Nouri Neamati, for his guidance, encouragement, and patience. Prof. Neamati instilled an enthusiasm in me for science and drug discovery, while allowing me the space to independently explore complex biochemical problems, and I am grateful for his kind and patient mentorship. I also thank my committee members, Profs. George Garcia, Peter Scott, and Shaomeng Wang, for their patience, guidance, and support throughout my graduate career. I am thankful to them for taking time to meet with me and have thoughtful conversations about medicinal chemistry and science in general. From the Neamati lab, I would like to thank so many. First and foremost, I have to thank Shuzo Tamara for being an incredible, kind, and patient teacher and mentor. Shuzo is one of the hardest workers I know. In addition to a strong work ethic, he taught me pretty much everything I know and laid the foundation for the article published as Chapter 3 of this dissertation. The work published in this dissertation really began with the initial identification of PDI as a target by Shili Xu, and I am grateful for his advice and guidance (from afar!). His preliminary experiments to validate PDI as a target made this work possible. I must thank all of the co-authors of the work in this dissertation, including Ana Kyani, Bikash Debnath, and Armand Bankhead III for their assistance with computational chemistry and bioinformatic analysis, Soma Samanta for her guidance, Yuting Kuang for her graduate school advice, and Urarika Luesakul for teaching me how to make really cool figures in Adobe Illustrator. Ding Xue and Suhui Yang were the lead chemists responsible for synthesizing and characterizing most of the compounds for my ii dissertation, and I am forever grateful for their triumphs and struggles. Dan Lu, Ziwei Liu, Shuai Mao, and Yuning Shen also prepared compounds and were very helpful. Additionally, Christine Cuthbertson, Shuai Hu, and Maha Hanafi are incredible lab mates and survivors. All of the postdoctoral researchers in the Neamati lab over the years, including Tiangong Lu and Yibin Xu, have been incredibly helpful and have brought a bright and cheerful atmosphere into the lab. Of course, much of this would not be possible without the help of all the talented undergraduate students in the Neamati lab, including Kirin Cromer, Hannah Driks, and Amina Tanweer, who sacrificed time they could be studying to come in and perform screening campaigns. I also want to thank our collaborators, specifically Bonnie Liu, an incredibly talented biochemist, for preparing many of the cell lines and biochemical tools that we used to study PDI, Jenn Schmidt, for helping me with protein mass spectrometry experiments, and the Ljungman lab for all their Bru-seq efforts. I want to extend a special thank you to the members of the Stuckey lab, including Dr. Stuckey, Jennifer Meagher, Elyse Petrunak, and Pryia Chinnaswamy for teaching me the basics of crystallography and being an amazing support system as I troubleshooted my crystallography project (even though I never succeeded). Finally, I want to thank my fiancé for his patience and support. I could not have done this without him. My parents have also been very supportive, even though I am pretty sure they still think I am just getting a master’s degree. Furthermore, volleyball and soccer games were a welcome break from the lab, and, in addition to lab mates, Nick Ragazzone, Lindsey Drake, Amy Fraley, Sumit Bandekar, Sarah Burris, and everyone in our cohort made graduate school more fun. iii Table of Contents ACKNOWLEDGEMENTS ............................................................................................................ ii LIST OF FIGURES ........................................................................................................................ v LIST OF TABLES .......................................................................................................................... x LIST OF ABBREVIATIONS ....................................................................................................... xii ABSTRACT .................................................................................................................................. xv CHAPTER I Current Challenges and Opportunities in Treating Glioblastoma ............................. 1 CHAPTER II Protein Disulfide Isomerase ................................................................................... 75 CHAPTER III Discovery and Mechanistic Elucidation of a Class of PDI Inhibitors for the Treatment of Glioblastoma ........................................................................................................... 96 CHAPTER IV Design, Synthesis, and Biological Evaluation of Novel Allosteric Protein Disulfide Isomerase Inhibitors .................................................................................................................... 131 CHAPTER V Characterization of Alpha-Aminobenzylphenol Analogues in Glioblastoma Cell Lines ............................................................................................................................................ 188 CHAPTER VI Concluding Summary ......................................................................................... 264 iv LIST OF FIGURES Figure Figure I-1 Classification of brain tumors as reported from the Central Brain Tumor Registry of the United States. .................................................................................................................................. 6 Figure I-2 Common characteristics of glioblastoma. ...................................................................... 8 Figure I-3 Canonical gliomagenesis mediators EGFR, P53, and RB1 are important for cancer signaling. ......................................................................................................................................... 9 Figure I-4 Signaling pathways involving membrane proteins upregulated in GBM as determined by LC-MS/MS and iTRAQ. .......................................................................................................... 11 Figure I-5 Twenty genes associated with reduced survivability in the TCGA GBM patient cohort profiled with RNASeq expression data......................................................................................... 15 Figure I-6 Hierarchical clustering of 20 genes ............................................................................. 17 Figure I-7 Expression of 20 genes significantly associated with reduced survivability in GBM across 33 TCGA diseases.............................................................................................................. 22 Figure I-8 The blood-brain barrier protects the brain from foreign material with a layer of endothelial cells bound by adherens junctions (i.e. vascular endothelial (VE)-cadherin) and tight junctions (i.e. junction adhesion molecules (JAMs), endothelial cell adhesion molecule (ESAM), claudins, and occludins). ............................................................................................................... 42 Figure I-9 CNS MPO Version 2 scores were calculated for 73 GBM drug candidates. .............. 46 v Figure II-1 Domain structure of PDI family members. ................................................................ 80 Figure II-2 PDI structure ............................................................................................................... 81 Figure II-3 Role of PDI in the endoplasmic reticulum. ................................................................ 83 Figure II-4 Multifunctional roles of the PDI family. .................................................................... 84 Figure II-5 PDI plays an important role in various disease states. ............................................... 87 Figure III-1 Discovery of 35G8. ................................................................................................... 99 Figure III-2 35G8 destabilizes PDI............................................................................................. 100 Figure III-3 DFO decreases the potency of 35G8. ..................................................................... 105 Figure III-4 Effects of 35G8 treatment on cellular pathways. .................................................... 108 Figure III-5 Effect of 35G8 treatment on RNA synthesis in U87MG cells. ............................... 110 Figure III-6 ROS induction of 35G8 analogues at (A) 4 hours, (B) 6 hours, and (C) 24 hours. 111 Figure III-7 PAINS characteristics of 35G8 do not interfere with PDI activity in the insulin turbidity assay. ............................................................................................................................ 113 Figure III-8 Venn diagram for the genes with greater than two-fold change