Disruption of Ribosome Biogenesis and Induction of Nucleolar Stress by Platinum(II)- based Chemotherapeutics by Matthew Vincent Yglesias A dissertation accepted and approved in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry Dissertation Committee: Michael J. Harms, Chair Victoria J. DeRose, Advisor Michael D. Pluth, Core Member David M. Garcia, Institutional Representative University of Oregon Fall 2024 2 © 2024 Matthew Vincent Yglesias This work is openly licensed via CC BY 4.0. https://creativecommons.org/licenses/by/4.0/ 3 DISSERTATION ABSTRACT Matthew Vincent Yglesias Doctor of Philosophy in Chemistry Title: Disruption of Ribosome Biogenesis and Induction of Nucleolar Stress by Platinum(II)-based Chemotherapeutics Platinum(II) metal complexes—cisplatin, carboplatin, and oxaliplatin—represent a major class of antineoplastics agents used in a majority of cancer treatment regimens throughout the world. Despite their ubiquitous use, the precise mechanisms and targets responsible for cancer cell death are not fully understood. Overcoming these deficiencies will be necessary to address the limitation associated with current Pt-based chemotherapeutics in the clinical setting. Current literature has revealed, unlike cisplatin and carboplatin, oxaliplatin primarily kills cells through disruption of ribosome biogenesis. Ribosome biogenesis is intimately connected to the nucleolus, a phase- separated nuclear condensate, which also functions as a central hub for sensing and coordinating cellular stress response through nucleolar stress response. This work provides insight on the relationship between Pt(II) compounds and disruptions in ribosome biogenesis, and the impact on nucleolar structure. Chapter I summarizes the significance and current understanding of Pt-based chemotherapeutics in the context of ribosome biogenesis and the nucleolus. Chapter II identifies structural and chemical properties of Pt(II) compounds necessary for nucleolar stress induction through a novel immunofluorescence imaging approach for quantifying nucleolar stress. Chapter III applies this framework to a subset of monofunctional Pt(II) compounds which are also shown to induce nucleolar stress. Chapter IV examines spatiotemporal differences in nucleolar stress induced by Pt(II) compounds identified in previous studies—ruling out connections with intracellular accumulation and DNA binding. Chapter V discusses current progress on elucidating the molecular mechanisms for inhibition of rRNA synthesis by oxaliplatin by adapting a ChIP-based sequencing techniques to map the occupancy of RNA Polymerase I machinery along rDNA. Chapter VI provides a comprehensive review on the coordination metal ions with nucleic acids, highlighting recent examples of NMR and x-ray crystallography structures from the literature. This dissertation includes published and unpublished co-authored material. 4 CURRICULUM VITAE NAME OF AUTHOR: Matthew Vincent Yglesias GRADUATE AND UNDERGRADUATE SCHOOLS ATTENDED: University of Oregon, Eugene University of Texas, Austin University of Texas, San Antonio DEGREES AWARDED: Doctor of Philosophy, Chemistry, 2024, University of Oregon Bachelor of Science, Biochemistry, 2014, University of Texas at Austin AREAS OF SPECIAL INTEREST: Platinum Chemotherapeutics Ribosome Biogenesis Immunofluorescence Imaging Chromatin Immunoprecipitation High-throughput Sequencing PROFESSIONAL EXPERIENCE: Graduate Employee, University of Oregon, 2018-2024 Graduate Research Fellow, University of Texas at San Antonio, 2015-2016 Academic Laboratory Technician, Palo Alto College, 2015 GRANTS, AWARDS, AND HONORS: Molecular Biology and Biophysics Training Program NIH Training Grant, University of Oregon, 2020-2022 University of Oregon Promising Scholar Award, University of Oregon, 2018 PUBLICATIONS: Pigg, H. C.; Yglesias, M. V.; Sutton, E. C.; McDevitt, C. E.; Shaw, M.; DeRose, V. J. Time- Dependent Studies of Oxaliplatin and Other Nucleolar Stress-Inducing Pt(II) Derivatives. ACS Chem. Biol. 2022, 17 (8), 2262–2271. DOI: 10.1021/acschembio.2c00399. https://doi.org/10.1021/acschembio.2c00399 5 DeRose, V. J.; Yglesias, M. V. Metal Ion Interactions With DNA, RNA, and Nucleic Acid Enzymes. In Comprehensive Coordination Chemistry III; Elsevier, 2021; pp 968–993. DOI: 10.1016/B978-0-08-102688-5.00112-4. Sutton, E. C.; McDevitt, C. E.; Yglesias, M. V.; Cunningham, R. M.; DeRose, V. J. Tracking the Cellular Targets of Platinum Anticancer Drugs: Current Tools and Emergent Methods. Inorganica Chimica Acta 2019, 498, 118984. DOI: 10.1016/j.ica.2019.118984. Sutton, E. C.; McDevitt, C. E.; Prochnau, J. Y.; Yglesias, M. V.; Mroz, A. M.; Yang, M. C.; Cunningham, R. M.; Hendon, C. H.; DeRose, V. J. Nucleolar Stress Induction by Oxaliplatin and Derivatives. J. Am. Chem. Soc. 2019, 141 (46), 18411–18415. DOI: 10.1021/jacs.9b10319. McDevitt, C. E.; Yglesias, M. V.; Mroz, A. M.; Sutton, E. C.; Yang, M. C.; Hendon, C. H.; DeRose, V. J. Monofunctional Platinum(II) Compounds and Nucleolar Stress: Is Phenanthriplatin Unique? J Biol Inorg Chem 2019, 24 (6), 899–908. DOI: 10.1007/s00775-019-01707-9. Deng, Y.; Yglesias, M. V.; Arman, H.; Doyle, M. P. Catalytic Asymmetric Synthesis of Cyclopentyl β-Amino Esters by [3+2] Cycloaddition of Enecarbamates with Electrophilic Metalloenolcarbene Intermediates. Angew Chem Int Ed 2016, 55 (34), 10108–10112. DOI: 10.1002/anie.201605438. https://doi.org/10.1016/B978-0-08-102688-5.00112-4 https://doi.org/10.1016/j.ica.2019.118984 https://doi.org/10.1021/jacs.9b10319 https://doi.org/10.1007/s00775-019-01707-9 https://doi.org/10.1002/anie.201605438 6 ACKNOWLEDGMENTS I would like to sincerely thank my advisor Dr. Victoria DeRose for her guidance and support over the years. None of this would have been possible without her help and dedication. I would like to thank my committee chair Dr. Michael Harms as a well as my other committee members Dr. Michael Pluth, for and Dr. David Garcia, for their for support and interest in my work and for fostering such a collaborative environment. I would also like all the members of the DeRose lab I’ve had the chance to work with. I would like to thank former members, Dr. Rachael Cunningham, Dr. Emily Sutton, and Dr. Christine McDevitt for being inspiring mentors, collaborators, and friends; and our current lab members—Hannah Pigg, Dillon Willis, Andres Guerrero, Ethan Kimmett, Katelyn Alley, Chris Griffin, Leif Lindberg—for their advice on experiments, random discussions about research, notes on presentation, group meeting snacks, and keeping up a positive lab environment. I would also like to thank all the rotation students and undergraduates I’ve had the opportunity to work with, everyone from my cohort who started this out with me, and all of the friends and colleague I’ve gotten to know over the years for all their support, and camaraderie. I would also like to thank Anna Bartuska for leading the graduate writing circles over the summer and fall, which was incredibly helpful in preparing this document. I would also like to acknowledge my former Dr. Michael Doyle and mentor Dr. Yongming Deng, for helping me gain the skills to get to graduate school. Lastly, I would like to thank my family, especially my mom, dad, aunt and sister for their unrelenting love and support over these many years, who made something like pursuing a PhD even possible for me. This work benefited from access to the University of Oregon high performance computing cluster, Talapas. This investigation was supported in part by a National Institutes of Health Training Grant from Molecular Biology and Biophysics Training Program, T32GM007759 and by grants from the National Science Foundation, CHE 1710721 and CHE 2109255, to Dr. Victoria J. DeRose at the University of Oregon. 7 TABLE OF CONTENTS Chapter Page I. INTRODUCTION .......................................................................................................... 16 Background .................................................................................................................... 16 Cellular uptake and Accumulation of Pt(II) Drugs ................................................ 19 Cellular Effects of Pt Chemotherapeutics ................................................................. 20 Non-canonical Pt Based Chemotherapeutics ........................................................... 21 Ribosome Biogenesis and the Nucleolus .................................................................. 22 The nucleolus as a sensor for cellular stress ................................................... 25 Ribosome biogenesis and Pol I transcription as a chemotherapeutic target ..................................................................................................................... 27 Conclusion ..................................................................................................................... 28 Bridge to Chapter II ...................................................................................................... 29 II. NUCLEOLAR STRESS INDUCTION BY OXALIPLATIN AND DERIVATIVES ............................................................................................................ 30 Nucleolar Stress Induction by Oxaliplatin and Derivatives .................................. 30 Bridge to Chapter III .................................................................................................... 38 III. MONOFUNCTIONAL PLATINUM(II) COMPOUNDS AND NUCLEOLAR STRESS: IS PHENANTHRIPLATIN UNIQUE? ........................ 40 Introduction ................................................................................................................... 40 Results and discussion ................................................................................................. 42 Oxaliplatin and phenanthriplatin cause NPM1 relocalization ................... 42 Picoplatin does not cause NPM1 relocalization ............................................ 44 NPM1 relocalization is not a general property of monofunctional platinum compounds ......................................................................................... 44 Steric bulk is not sufficient to predict NPM1 relocalization ........................ 46 Hydrophobicity is not sufficient for predicting NPM1 relocalization ...... 47 Conclusions .................................................................................................................... 48 Bridge to Chapter IV .................................................................................................... 50 IV. TIME-DEPENDENT STUDIES OF OXALIPLATIN AND OTHER NUCLEOLAR STRESS-INDUCING PT(II) DERIVATIVES ............................... 52 Introduction ................................................................................................................... 52 Results and discussion ................................................................................................. 55 Differences in Kinetics of Nucleolar Stress Induction by Pt(II) Compounds ......................................................................................................... 55 8 Chapter Page Pentaplatin Induces Lower Inhibition of rRNA Transcription ................... 58 Whole Cell and Nuclear Platinum Accumulation Do Not Correlate with Nucleolar Stress Induction by Pt(II) Compounds ............................... 60 Pt(II)-DNA Binding In Vitro and in Cell Does Not Correlate with Nucleolar Stress Induction ................................................................................ 62 Chirality of Pt(II) Compounds Influences the Degree of Nucleolar Stress ..................................................................................................................... 63 Conclusions .................................................................................................................... 66 Bridge to Chapter V ...................................................................................................... 68 V. INFLUENCE OF NUCLEOLAR STRESS INDUCING COMPOUNDS ON THE INTERACTION OF POLYMERASE I TRANSCRIPTION MACHINERY WITH RIBOSOMAL DNA ............................................................ 70 Introduction ................................................................................................................... 70 Results ............................................................................................................................. 73 BMH-21 and oxaliplatin induce reorganization of FC components .......... 73 ChIP-based occupancy assay of Pol I transcription of rDNA ..................... 76 BMH-21 induces Pol I stalling along rDNA ................................................... 79 Pol I and UBF occupancy does not correlate to relative rDNA GC- content .................................................................................................................. 81 Discussion ...................................................................................................................... 82 Bridge to Chapter VI .................................................................................................... 85 VI. METAL ION INTERACTIONS WITH DNA, RNA, AND NUCLEIC ACID ENZYMES ........................................................................................................ 87 Introduction ................................................................................................................... 87 Background: Roles of Metals in DNA and RNA Structure and Function ........................................................................................................ 87 Nucleic Acids as Ligands ............................................................................................ 89 Overview of DNA/RNA Structure ................................................................. 89 Ligand Properties of Bases, Nucleosides, and Nucleotides ........................ 93 Metal Interactions with RNA and DNA Biopolymers ........................................... 95 Properties of Metal Interactions with Oligonucleotides .............................. 95 Scope of Review ................................................................................................. 97 Monovalent Cations: Group I and Thallium(I) ....................................................... 98 Sodium .................................................................................................................. 100 Potassium, Thallium ........................................................................................... 101 9 Chapter Page Rubidium, Cesium .............................................................................................. 102 Group IIA: Magnesium/Calcium/Strontium/Barium ......................................... 102 Transition Metals .......................................................................................................... 104 Chromium ............................................................................................................ 106 Manganese ........................................................................................................... 107 Iron ........................................................................................................................ 108 Cobalt .................................................................................................................... 108 Nickel .................................................................................................................... 110 Copper .................................................................................................................. 110 Zinc, Cadmium ................................................................................................... 110 Transition Metals: Second and Third Row ............................................................... 112 Molybdenum, Ruthenium, Rhodium, Palladium ......................................... 114 Silver ..................................................................................................................... 114 Platinum and Palladium .............................................................................................. 115 Platinum ............................................................................................................... 115 Palladium ............................................................................................................. 123 Tungsten, Rhenium, Iridium, Osmium, Gold, and Mercury ................................ 123 Tungsten, Rhenium, Iridium, Osmium .......................................................... 123 Gold ....................................................................................................................... 124 Mercury ................................................................................................................ 124 Lead ............................................................................................................................. 126 Lanthanides ................................................................................................................... 127 Concluding Remarks .................................................................................................... 128 Bridge to Chapter VII ................................................................................................... 129 VII. CONCLUDING REMARKS ..................................................................................... 130 Summary ........................................................................................................................ 130 Future Directions .......................................................................................................... 131 APPENDICES ..................................................................................................................... 133 A. SUPPLEMENTARY MATERIALS FOR CHAPTER II ...................................... 133 Supplementary Figures and Tables ................................................................. 133 Materials and Methods ...................................................................................... 138 B. SUPPORTING INFORMATION FOR CHAPTER III ........................................ 146 Materials and Methods ...................................................................................... 146 10 Chapter Page Supplemental Figures ........................................................................................ 150 C. SUPPLEMENTARY MATERIALS FOR CHAPTER IV .................................... 151 Methods ................................................................................................................ 151 Supplemental Figures ........................................................................................ 157 Synthesis of Pt(II) Compounds ......................................................................... 159 D. SUPPLEMENTARY MATERIALS FOR CHAPTER V ...................................... 161 Materials and Methods ...................................................................................... 161 Supplemental Figures ........................................................................................ 165 REFERENCES CITED ....................................................................................................... 167 11 LIST OF FIGURES Figure Page 1.1. Chemical structure of Pt (II)-based chemotherapeutics approved worldwide for the treatment of cancer .............................................................. 16 1.2 The mechanism of action for cell death induced by cisplatin .............................. 17 1.3 Chemical structure of monofunctional Pt(II) compounds with demonstrated anticancer properties ............................................................................................ 22 1.4 Ribosome biogenesis in mammalian cells ............................................................... 23 1.5 Nucleolar organization in mammalian cells ........................................................... 24 1.6 Organization of rDNA genes in humans ................................................................ 25 1.7 Disruption of early ribosome biogenesis induces characteristic ‘nucleolar cap’ stress morphology ........................................................................................ 27 2.1. Compounds tested for inducing nucleolar stress via NPM1 relocalization ..... 32 2.2. Nucleolar stress induced by Pt(II) compounds ..................................................... 33 2.3. Quantification of NPM1 relocalization, by Pt(II) compounds ............................ 34 2.4. Size and hydrophobicity correlate with stress induction .................................... 36 2.5. Computed distance measurements and volume representations ...................... 37 3.1. Platinum compounds used in this study ................................................................ 41 3.2. NPM1 relocalization ................................................................................................... 45 3.3. Two ring structural isomers related to phenanthriplatin .................................... 47 3.4. Optimized structures of the platinum(II) compounds ......................................... 48 4.1. Pt(II) compounds used in this study ....................................................................... 53 4.2. Quantification of NPM1 relocalization induced by Pt(II) compounds at various time points ............................................................................................... 57 4.3. Metabolic labeling to measure rRNA synthesis and processing at 3 h treatment ................................................................................................................. 59 4.4. Metabolic labeling to measure rRNA synthesis and processing at 5 h treatment ................................................................................................................. 60 4.5. Platinum content in whole cell, nuclear, and DNA samples measured by ICP-MS .................................................................................................................... 61 4.6. Pt(II) compounds used in isomer-specific NPM1 relocalization studies .......... 64 4.7. Quantification of NPM1 relocalization induced by pentaplatin isomers ......... 64 4.8. Quantification of NPM1 relocalization induced by DACHplatin isomers ....... 65 4.9. Lowest-energy conformations of pentaplatin, 1S,2S- and 1R,2R- configurations ........................................................................................................ 66 12 Figure Page 5.1 Oxaliplatin and BMH-21 induce nucleolar caps containing UBF ....................... 74 5.2. Oxaliplatin and BMH-21 induce condensation of Pol I ....................................... 76 5.3. Effect of platinum and BMH-21 treatment on rDNA occupancy of Pol I ......... 77 5.4. Relative Pol I ChIP-seq signal mapped at defined regions of rDNA ................ 78 5.5. Influence of platinum and BMH-21 treatment on UBF-rDNA occupancy ....... 79 5.6. Relative UBF ChIP-seq signal mapped at defined regions of rDNA ................. 80 5.7. BMH-21 induces Pol I stalling at the 47S promoter .............................................. 80 5.8. BMH-21 induces Pol I stalling downstream of TTF1 binding sites ................... 81 5.9. Pol I and UBF occupancy does not correlate with relative rDNA GC- content ..................................................................................................................... 82 6.1. Metal binding sites of nucleic acids ......................................................................... 90 6.2. Canonical A- and B-form helices adopted by double-stranded RNA and DNA ......................................................................................................................... 91 6.3. Predicted influence and distribution of Mg2+ ions on folding of the SAM-1 riboswitch ............................................................................................................... 92 6.4. Metal nanostructure templated by DNA origami substrate ............................... 93 6.5. Classes of cation interactions and influence on nucleic acid structure ............. 97 6.6. Na+ ion in Hammerhead ribozyme canonical “A9” metal site bound directly to N7 G ..................................................................................................... 100 6.7. Metal sites modeled in crystal structures of a 16-nt RNA duplex ..................... 101 6.8. Mg-F cluster in a 52-nt fluoride-sensing riboswitch from T. petrophila ............. 104 6.9. Crystal structure of L. lactis Mn-sensing riboswitch ............................................. 108 6.10. Crystal structure of NiCo riboswitch from E. bacterium bound to Co(II) ....... 109 6.11. Ultrahigh-resolution structure of Z-DNA in complex with Zn2+ ..................... 111 6.12. Observed binding modes of Cd(II) in ykoY-aptamer domain crystal structures ................................................................................................................ 112 6.13. Crystal structure of RNA duplex containing C-Ag(I)-C base pair ................... 115 6.14. The 2.5 Å crystal structure of DNA Polβcomplexed to monofunctional platinum-carbazole conjugate ............................................................................. 117 6.15. Pt-bound sites observed in 2.8 Å structure of 50S ribosome from T. Thermophilus ........................................................................................................... 122 6.16. Crystal structure of HIV-1 Subtype B DNA duplex bound to Au(III) ............ 124 6.17. DNA duplex containing unexpected C-Hg(II)-T metallobase pairing ............ 125 6.18. High-resolution crystal structure of Pb(II) bound to DNA quadruplex ......... 127 13 Figure Page 6.19. Crystal structure of octameric RNA duplex in complex with Tb(III) .............. 128 A.1. Additional images for compounds tested ............................................................. 134 A.2. Quantification of nucleolar stress induction for Pt(II)-free ligands and solvents used. ......................................................................................................... 135 A.3. Histograms showing NPM1 intensity within a single nucleus in stressed and unstressed cells. ............................................................................................. 136 A.4. DFT-optimized structures of compounds ............................................................. 137 B.1. Concentration dependence of nucleolar stress induced by monofunctional Pt(II) compounds ................................................................................................... 150 C.1. Model oligonucleotide DNA-Pt(II) adduct assays ............................................... 157 C.2. Representative cell images of A549 cells treated with 1S,2S- and 1R,2R- pentaplatin at given treatment times ................................................................. 158 C.3. Representative cell images of A549 cells treated with 1S,2S- and 1R,2R- DACHplatin at given treatment times. ............................................................. 158 D.1. ChIP-seq mapping of Pol I (RPA194) occupancy along rDNA gene in U2OS cells ............................................................................................................... 165 D.2. ChIP-seq mapping of UBF occupancy along rDNA gene in U2OS cells ......... 166 14 LIST OF TABLES Table Page 3.1. Steric bulk measurements for platinum compounds ........................................... 47 3.2. Gibbs free energy of transfer between octanol and water ................................... 49 6.1 Nucleic acid structures with thallium, rubidium and cesium ............................. 99 6.2 Nucleic acid structures with first-row transition metals ...................................... 105 6.3 Nucleic acid structures with second and third row transition metals, lead, and terbium ............................................................................................................ 113 6.4 Nucleic acid structures containing Pt-nucleic acid adducts ................................. 118 A.1. IC50 values in A549 cells for selected compounds at 24 hours ........................... 137 A.2. IC50 values in A549 cells for selected compounds at 48 hours ........................... 137 A.3. Volume and hydrophobicity data .......................................................................... 138 15 LIST OF SCHEMES Scheme Page A.1. NPM1 assay and quantification scheme ................................................................ 133 16 CHAPTER I: INTRODUCTION Background Platinum (Pt) metal complexes represent an important and distinct class of anti- tumor compounds routinely used in chemotherapeutic regimens today. To date three Pt(II)-based chemotherapeutics have been approved worldwide for the treatment of cancers; cisplatin, carboplatin and oxaliplatin (Figure 1.1) (Riddell & Lippard, 2018). Figure 1.1. Chemical structure of Pt (II)-based chemotherapeutics approved worldwide for the treatment of cancer. The first of these Pt(II) complexes, cisplatin [cis-diamminedicholorplatinum(II)], was first described in 1844 by chemist Michele Peyrone (Rottenberg et al., 2021). The cytotoxic and antiproliferative effects of cisplatin were not known until 120 years later when they were unexpectedly discovered by Barnett Rosenberg and Loretta Van Camp, who noticed a distinct inhibition of cell division when studying the effects of electric fields on E. coli utilizing platinum electrodes (Rosenberg et al., 1965; Rottenberg et al., 2021). Subsequent studies identified several Pt compounds generated in situ, including cisplatin, as the causative agent (Rosenberg et al., 1969). Soon after this discovery cisplatin was demonstrated as a potent anti-tumor compound, eventually leading to its approval for medical use in 1978 as the first metal organic coordination complex for the treatment of cancer. Cisplatin is currently used to treat a broad range of cancers including head and neck, lung, breast, cervical, and ovarian, but has been particularly successful in the treatment of testicular cancer where it has improved survival rates from 55% to over 90% (Ghosh, 2019; Wexselblatt et al., 2012). Like many chemotherapeutic agents, cisplatin has a narrow therapeutic range that is primarily limited by patient tolerance to well-known toxic side effects such as nephrotoxicity, ototoxicity, and neurotoxicity (Ghosh, 2019). Despite these limitations, the Pt O H2 N N H2 O O O Oxaliplatin Pt ClH3N H3N Cl Cisplatin Pt OH3N H3N O O O Carboplatin 17 initial success of cisplatin spurred extensive research into characterizing the cellular mechanisms behind Pt-induced cell death. Early investigation into the mechanism of action for cisplatin quickly identified nuclear DNA as the primary cellular target responsible for the cytotoxic effects, characterizing cisplatin as an alkylating agent (Rottenberg et al., 2021). These efforts culminated into a generalized mechanism of action defined by four key steps: (i) cellular uptake, (ii) activation, (iii) DNA binding, and (iv) cellular response leading to cell death (Figure 1.2) (Johnstone et al., 2016; Riddell & Lippard, 2018). Figure 1.2. The mechanism of action for cell death induced by cisplatin. Following uptake into the cell, cisplatin undergoes aquation to form an active cationic species where it may bind to biomolecules present in the cytoplasm or nucleus. Inside the nucleus, a small percentage of cisplatin forms adducts with genomic DNA and trigger the DNA damage response (DDR), which activates several downstream repair and stress pathways. Upon treatment, cisplatin can enter the cell via active transport or simple diffusion. Once inside the cell, the relatively lower [Cl-] concentration inside the cell drives aquation of the two cis-labile chloride ligands to form the active cationic complex cis- [Pt(NH)2(OH2)2] 2+ (Ghosh, 2019). This active aquation species can react with a wide variety of biomolecules, most importantly genomic DNA where it shows affinity towards adjacent guanine nucleobases by forming 1,2-intrastrand crosslinks, 1,2-d(GpG), at the N7 position (Ghosh, 2019; Wexselblatt et al., 2012). The non-labile ligands on Pt-DNA adducts induce distortions in genomic DNA that disrupt DNA function, which eventually trigger the DNA damage response (DDR) in an effort to repair DNA damage (Rottenberg et al., 2021; Wexselblatt et al., 2012). Rapidly dividing cancer cells—which often lack fully functional DNA repair mechanisms—are less able to cope with the extensive DNA damage during replication, eventually leading to DDR activation of cell arrest or apoptosis pathways (Rottenberg et al., 2021). Much of the mechanistic work into 18 cisplatin helped define a structure-activity-relationship (SAR) for Pt chemotherapeutics1 which was followed in the design of second generation compounds: carboplatin and oxaliplatin (Johnstone et al., 2016; Kelland, 2007). Carboplatin, [cis-diammine(cyclobutane-1,1-dicarboxylate)platinum(II)], was initially developed to mitigate the toxic side effects observed in cisplatin treatments. Compared to the dichloro ligands of cisplatin, the less labile bidentate dicarboxylate ligand of carboplatin reduces intracellular aquation rates, effectively limiting off-target binding responsible for the undesirable side effects in cisplatin treatments (Johnstone et al., 2016; Kelland, 2007). The most recently Pt(II) chemotherapeutic to achieve FDA-approval, oxaliplatin, [(1,2-diaminocyclohexane)oxalatoplatinum(II)] was intended to further address toxic side effects in cisplatin treatments and improve efficacy in cisplatin resistant cancers (O’Dowd et al., 2023; Rottenberg et al., 2021). Oxaliplatin maintains a square-planer geometry around the Pt center which is coordinated between a bidentate oxalate ligand and a bidentate 1R,2R-diaminocyclohexane (DACH) ligand (Johnstone et al., 2016; Rottenberg et al., 2021). The labile oxalate ligand improves the water solubility in comparison to the dichloro [Pt(DACH)Cl2] species, while maintaining effective aquation rates (O’Dowd et al., 2023). The 1R,2R-DACH enantiomer was found to be more effective than the 1S,2S- DACH enantiomer in vitro and in vivo. This chirality effect is proposed to be partially due to favorable hydrogen bonding between the NH of 1R,2R-DACH and O6 of 3′-dG in 1,2- d(GpG) crosslinks (Johnstone et al., 2016; O’Dowd et al., 2023). Oxaliplatin has a distinct activity profile compared to cisplatin and carboplatin and is more effective in a distinct subset of cancers, such as gastrointestinal and colorectal cancers. Chemotherapy regimens commonly use oxaliplatin in combination with antimetabolites 5-fluorouracil (5-FU) and folinic acid (FOL), which interfere with DNA synthesis (O’Dowd et al., 2023; Rottenberg et al., 2021). 1 The SAR for Pt chemotherapeutics describes a general structural and chemical component responsible for the cytotoxic and anticancer effects of Pt compound: a neutral square-planer metal complex coordinated to two cis-oriented non-labile ligands (such as amines), and two cis labile ligands (Wexselblatt et al., 2012). 19 Cellular uptake and accumulation of Pt(II) drugs The apparent differences in efficacy, resistance, and side effect profiles, between Pt(II) chemotherapeutics observed in the clinical setting have been widely studied in the context of cellular uptake2 or accumulation of Pt. Decreased cellular accumulation of Pt is shown to correlate with increased resistance in cisplatin, carboplatin, and oxaliplatin, treatments and is considered one of the primary mechanisms by which cells acquire resistances to Pt drugs (Hall et al., 2008; Riddell & Lippard, 2018). Pt(II) compounds move across the cell membrane by a combination of passive diffusion and protein-mediated/active transport. Cisplatin and carboplatin are believed to primarily enter cells through passive diffusion, while oxaliplatin is thought to depend more heavily on active transport mechanisms (O’Dowd et al., 2023; Riddell & Lippard, 2018). However, there is still an incomplete understanding of both modes of transport, (Riddell & Lippard, 2018) and the cellular mechanisms which confer resistance are less well understood (Rottenberg et al., 2021). Several proteins involved in the active transport of organic and/or metal cations, have been reported to interact with Pt and influence intracellular Pt accumulation, many of which belong to the solute carrier (SLC) family of transporters such as, copper transporter 1 (CTR1; SLC31A), organic cation transporters (OCTs; SLC22A) OCT1-3, and multidrug and toxin extrusion antiporters (MATEs; SLC47) MATE1 and MATE2-K. CTR1 expression is shown to correlate with intracellular Pt accumulation and has been implicated in cisplatin resistance mechanisms. CTR1 is downregulated in response to cisplatin treatments and decreased expression is associated with cisplatin resistance in tumor cell lines (Hall et al., 2008; Riddell & Lippard, 2018). However, more recent studies suggest CTR1 may not play a direct role in a Pt uptake (O’Dowd et al., 2023; Riddell & Lippard, 2018). OCT1-3 are bidirectional polyspecific transporters for small organic cations that are differentially expressed among several tissue-types. OCTs promote Pt uptake and are linked to several tissue-specific side effects associated with Pt(II) chemotherapeutics. OCT2 expression in kidney and cochlear hair cells is a key determinant of nephrotoxicity and ototoxicity in cisplatin treatments, which was found to 2 In the literature 'cellular uptake' is commonly used to describe flux in/out of the cell, as well as total intracellular amounts (Hall et al., 2008). To avoid this ambiguity, we focus on cellular accumulation—the net amount of drug or analyte within the cell at a given point in time—and limit 'cellular uptake' to mean an influx, or movement of drug into the cell. 20 be a better substrate for OCT2 compared to carboplatin or oxaliplatin (Hall et al., 2008; Riddell & Lippard, 2018). OCT3 has been observed to transport oxaliplatin, but not cisplatin, and is hypothesized to promote sensitivity of colon cancer to oxaliplatin due to its high expression in the GI tract (Riddell & Lippard, 2018). P-type ATPases, ATP7A and ATP7B, are responsible for sequestering and extruding excess copper in the cell and have been shown to promote Pt efflux. Increased levels of ATP7B in particular are strongly associated with Pt resistance and used as a prognostic marker for oxaliplatin treatment of colorectal cancers (Riddell & Lippard, 2018). MATE1 and MATE2-K are cation efflux transporters associated with Pt efflux mechanisms. MATE2-K, which is expressed in the kidneys, shows strong substrate specificity for oxaliplatin in comparison to cisplatin, and is hypothesized to cause the reduction in nephrotoxicity observed in oxaliplatin treatments (O’Dowd et al., 2023; Riddell & Lippard, 2018). ATP-binding cassette transporters, multidrug resistance proteins (MRP), MRP1, MRP2, and MRP4, have also been shown to transport Pt(II) chemotherapeutics and Pt metabolites. MRP2, which is expressed in the liver and kidneys, is widely implicated in oxaliplatin efflux and its expression is linked to a decrease in accumulation and cytotoxicity (O’Dowd et al., 2023). Cellular uptake and accumulation mechanisms have provided strong insight into Pt sensitivity/resistances mechanisms and demonstrate clear differences in how Pt compounds are handled by the cell. Nevertheless, seemingly contradictory findings highlight how these mechanisms are not fully elucidated, and do not provide a sufficient explanation on their own for the dissimilar efficacies observed in the clinic. Cellular effects of Pt chemotherapeutics From initial research on cisplatin, Pt drugs are understood to be inherently non- specific and have been shown to interact with a broad range of biomolecules including RNAs, proteins, mitochondrial DNA, and sufficiently nucleophilic metabolites (O’Dowd et al., 2023), the biological impacts of which are still poorly understood. The traditional paradigm for Pt chemotherapeutics centers around DDR pathways, and has revealed some differences in the molecular mechanisms of oxaliplatin and cisplatin. The DDR encompasses several mechanisms for the recognition and repair of DNA damage, many of which share overlapping function. Nucleotide excision repair 21 (NER), the primary repair mechanism for processing interstrand crosslinks, is not influenced by the non-labile ligands bound to Pt in DNA adducts. However, mismatch repair (MMR) pathways which can repair cisplatin crosslinks do not detect oxaliplatin crosslinks (O’Dowd et al., 2023; Riddell & Lippard, 2018). Double strand break (DSBs) repair pathways involving homologous recombination (HR) are implicated as a key factor in cisplatin resistance, but deficiencies in HR show little influence on oxaliplatin sensitivity. Similarly, defects in base excision repair (BER) pathways result in sensitivity to cisplatin, but not oxaliplatin (Riddell & Lippard, 2018). Strikingly, RNAi-based genetic analysis revealed that oxaliplatin and phenanthriplatin, but not cisplatin, induce cell death primarily through a disruption of ribosome biogenesis (Bruno et al., 2017). Although both cisplatin and oxaliplatin have been previously demonstrated to induce disruptions in early ribosome biogenesis and inhibit rRNA synthesis, these effects were thought to result downstream of DDR or off- target effects caused by high treatment concentrations (Burger et al., 2010; Hamdane et al., 2015). However, subsequent experiments showed lower Pt accumulation in oxaliplatin treatments compared to cisplatin, which resulted in fewer DSBs when measured by marker γH2AX (Bruno et al., 2017; Schoch et al., 2020). This seminal work led to a paradigm shift for Pt chemotherapeutics (particularly oxaliplatin) towards ribosome biogenesis, and its deeply connected process of nucleolar stress where there has been renewed interest in elucidating the molecular interactions involved in these cell death mechanisms. Non-canonical Pt based chemotherapeutics Pt compounds which fall outside of the traditional SAR are continually investigated in mechanistic studies and drug development. Among these, monofunctional Pt(II) complexes have shown promise as potential anticancer compounds. Unlike bifunctional cis-coordinated Pt(II) chemotherapeutics which can form both monofunctional and bifunctional adducts with DNA, monofunctional Pt(II) compounds contain only one labile ligand which expectedly forms a single bond with DNA (Johnstone et al., 2016). Initial studies found monofunctional Pt(II) compounds such as [Pt(dien)Cl]+ (dien = diethylenetriamine) and [Pt(NH3)3Cl]+ did not display significant anticancer activity, which fell in agreement with traditionally defined SAR. However, 22 monofunctional Pt(II) compounds following the structure cis-[Pt(NH3(Am)Cl]+ (Am = aromatic N-heterocyclic amine), pyriplatin (Am = pyridine) and phenanthriplatin (Am = phenanthridine) (Figure 1.3), were later found to be potent anticancer agents, and shown to form covalent bonds and intercalate with duplex DNA (Johnstone et al., 2016; Wexselblatt et al., 2012). Both pyriplatin and phenanthriplatin are strongly up-taken by the cell, and are thought to work through pathways distinct from cisplatin (Johnstone et al., 2016; Park et al., 2012). Phenanthriplatin was identified to be more similar to oxaliplatin by primarily inducing cell death through a disruption of ribosome biogenesis (Bruno et al., 2017). Figure 1.3. Chemical structure of monofunctional Pt(II) compounds with demonstrated anticancer properties. Phenanthriplatin has been shown to disrupt ribosome biogenesis and induce nucleolar stress. Ribosome biogenesis and the nucleolus Ribosome Biogenesis is the highly conserved and essential process of synthesizing and regulating the production of ribosomes. In eukaryotes, ribosomes are constructed from four ribosomal RNAs (rRNA) in combination with dozens of ribosomal proteins. These are organized into two primary subunits; a large subunit containing the 28S, 5S, and 5.8S rRNA, and a small subunit containing the 18S rRNA (González-Arzola, 2024; Pitts & Laiho, 2022). Ribosome biogenesis primarily takes place in the nucleolus, a non- membrane bound organelle, or biomolecular condensate, within the nucleus (Figure 1.4) (González-Arzola, 2024; Lafontaine et al., 2021). Ribosome biogenesis begins with the rate-limiting transcription of rRNA genes (rDNA) by RNA polymerase I (Pol I) to generate a single pre-rRNA (47S rRNA) containing the 18S, 5.8S, and 28S rRNAs. The 5S rRNA is concomitantly transcribed outside of the nucleolus by RNA polymerase III (González-Arzola, 2024; Pitts & Laiho, 2022). These pre-rRNAs then further cleaved and processed to generate mature rRNA transcripts, which are then assembled with ribosomal proteins to form the two pre-ribosomal subunits (Peña et al., 2017). These pre- Pt ClH3N H3N N Pt ClH3N H3N N PhenanthriplatinPyriplatin + + NO3- NO3- 23 ribosomal subunits translocate into the nucleoplasm where they undergo several maturation steps before being exported to the cytoplasm (Peña et al., 2017). Here, they undergo “functional proof reading” maturation where they combine to form a fully functional ribosome (González-Arzola, 2024; Peña et al., 2017). Figure 1.4. Ribosome biogenesis in mammalian cells. Ribosome biogenesis begins with the initial transcription of 47S pre-rRNA by Pol I, which occurs at the interface of the fibrillar center and dense fibrillar component (FC-DFC) allowing for nascent pre-rRNA to be co-transcriptionally processed. In the DFC, pre- rRNA undergoes further processing and modification. In the granular component (GC) pre-rRNAs are combined with the 5S rRNA and essential ribosomal proteins (RNPs) to assembly the pre-40S and pre-60S ribosomal subunit. Pre-ribosomal subunits undergo further late stage maturation in the nucleoplasm before final export into the cytosol where they combine to form a functional ribosome. The nucleolus is organized in a nested tripartite structure containing three distinct sub-nucleolar compartments which reflect the transcription, processing, and assembly steps of ribosome biogenesis (Figure 1.5) (González-Arzola, 2024; Lafontaine et al., 2021). At the core is the fibrillar center (FC), which is highly enriched in with rRNA 24 transcription machinery and related essential cofactors. Surrounding the FC is the dense fibrillar component (DFC), where pre-rRNA processing occurs (González-Arzola, 2024; Lafontaine et al., 2021). It is generally accepted that pre-rRNA transcription occurs at or near the border of the FC and DFC, which allows for nascent pre-rRNA to be co- transcriptionally processed as it enters the DFC (Hori et al., 2023; Lafontaine et al., 2021). The FC and DFC form a functional module contained within the granular component (GC), where assembly of mature rRNAs with ribosomal proteins takes place, making up the largest component of the nucleolus which demarcates the nucleolus from the nucleus (González-Arzola, 2024; Lafontaine et al., 2021). Figure 1.5. Nucleolar organization in mammalian cells. Multiple complete nucleoli which organize around NORs may be present within the nucleus. The three sub compartments of the nucleolus, the fibrillarin center (FC, red), dense fibrillar component (DFC, blue), and granular component (GC, green) are maintained through liquid-liquid phase separation. Multiple FC-DFC modules exist within a single GC sub- compartment. At the interface of each FC-DFC module are ribosomal rRNA genes (rDNA, orange) under active transcription. A single GC may contain dozens of FC-DFC modules, which form around transcriptionally active rDNA repeats (González-Arzola, 2024; Pitts & Laiho, 2022). These clusters of rDNA repeats, known as nucleolar organizer regions (NORs), are arranged in tandem repeat arrays containing hundreds of copies of rDNA (Figure 1.6) (Pitts & Laiho, 2022). In humans, NORs are located on the short arms of acrocentric chromosomes 13, 14, 15, 21, and 22 (Nurk et al., 2022). The number of repeats can vary substantially between species and individuals, the maintenance and regulation of which is not well understood in mammalian cells (Hori et al., 2023; Pitts & Laiho, 2022). The human genome contains between 200–600 copies of rDNA (Nurk et al., 2022), which can vary between 20–100 copies per NORs (Hori et al., 2023). However, only a fraction of these rDNA repeats are active at any point in time (González-Arzola, 2024; Hori et al., 2023). 25 Figure 1.6. Organization of rDNA genes in humans. rDNA arrays are found in nucleolar organizer regions (NORs) located on the short arm of acrocentric chromosomes. rDNA arrays are organized into tandem repeats of rDNA units, each containing a transcribed region encoding for the 47S pre-rRNA and a non- transcribed intergenic spacer (IGS) region. The IGS contains the spacer promoter (Spacer Pr), enhancer element, 47S promoter (47S Pr) and transcription termination sites (TTF1) for adjacent repeats. External transcribed spacers (5’ETS and 3’ETS) flank each coding region; rDNA genes are separated by internal transcribed spacers (ITS1 and ITS2) The nucleolus as a sensor for cellular stress In more recent decades, the nucleolus has come to be understood as a multifaceted organelle, which not only directly facilitates ribosome biogenesis but is deeply involved in the cellular processes surrounding its regulation. The production of ribosomes is essential and highly coordinated with cell growth and division (González-Arzola, 2024). Perhaps consequently, the nucleolus plays an important role in coordinating cell signaling involved in cell cycle progression, proliferation, and apoptosis.(Yang et al., 2018). More uniquely, the nucleolus functions as sensor of cellular stress through a nucleolar stress response. A broad range of cellular insults can induce nucleolar stress which is defined as a disruption in nucleolar structure or function that leads to activation of downstream cellular stress-response pathways3 (González-Arzola, 2024; Yang et al., 2018). Canonically, nucleolar stress is induced by either rDNA specific DNA damage or 3 Prior to nucleolar stress being understood as a general cell stress sensing phenomenon, the term 'nucleolar stress' was often used synonymously with 'disruption of ribosome biogenesis', since this was only known function of the nucleolus (González-Arzola, 2024). 26 disruption in ribosome biogenesis homeostasis. Other factors which can induce nucleolar stress include nonspecific DNA damage, inhibition in RNA transcription or protein translation, and general stress conditions which can lead to p53 activation (Yang et al., 2018). An important aspect of nucleolar stress is the apparent relocalization of nucleolar proteins which often reflect the inherent relationship between ribosome biogenesis and nucleolar structure. Several nucleolar proteins, such as NPM1, ARF, RPL11, and RPL5, translocate from the nucleolus into the nucleoplasm and cytosol where they have been demonstrated to aid in stabilizing p53 by inhibiting murine double minute 2 (MDM2, HDM2 in humans), causing activation of p53-dependent apoptosis or cell-cycle arrest pathways (Penzo et al., 2019). NPM1 and ARF are also linked to activation of downstream stress response pathways independent of p53. Both NPM1 and ARF can bind the transcription factor c-Myc within the nucleolus, disrupting c-Myc activation of oncogenes involved in cell growth and proliferation (Weeks et al., 2019). ARF has been shown to activate the DDR through ATM (ataxia-telangiectasia mutated)/ATR (ATM- and Rad3-Related) signaling pathways, leading to cell cycle arrest, senescence, or apoptosis (González-Arzola, 2024; Weeks et al., 2019). Translocation of NPM1 from the GC to the nucleoplasm is a well characterized early hallmark of nucleolar stress, although additional distinct nucleolar stress morphologies are observed when nucleolar stress results from disruptions in ribosome biogenesis (Yang et al., 2018). Inhibitors of RNA pol I transcription, such as actinomycin D (ActD) and BMH-21, have been shown to cause segregation of nucleolar subcomponents leading to the formation of 'nucleolar caps', where the FC and DFC phase separate and then condense around the periphery of the GC (Figure 1.7) (Lafontaine et al., 2021; Yang et al., 2018). The function and mechanisms which govern nucleolar restructuring during nucleolar stress are poorly understood, but are thought to depend heavily on the composition and liquid-liquid phase separation properties of the resulting nucleolar condensates (Lafontaine et al., 2021). Another overlooked aspect of nucleolar stress is the kinetics and robustness of nucleolar relocalization, which has been shown to vary significantly among small molecule inhibitors of ribosome biogenesis and in some cases even be reversable (Lu et al., 2018; Pigg et al., 2022). 27 Figure 1.7. Disruption of early ribosome biogenesis induces characteristic 'nucleolar caps' stress morphology. Inhibition of rRNA transcription leads to the formation of 'nucleolar caps'. Ribosome biogenesis and Pol I transcription as a chemotherapeutic target In recent years inhibition of ribosome biogenesis, and more specifically rRNA transcription, has emerged as a potential chemotherapeutic strategy for selectively targeting cancer cells. It is widely acknowledged that cancer cells rely on heightened rates of ribosome synthesis to facilitate the increased demands for cell growth and proliferation. As a result, ribosome biogenesis is often dysregulated in cancer which manifests as elevated rates of rRNA synthesis and greater sensitivity to disruptions in rRNA transcription (Hwang & Denicourt, 2024; Zisi et al., 2022). As the initial and rate- limiting step of ribosome biogenesis, transcription of rRNA by Pol I plays a pivotal role in setting the rate of ribosome biogenesis and has gained interest as a small-molecule target for selective inhibition (Hwang & Denicourt, 2024). Several small molecules and clinically approved drugs have been identified to disrupt Pol I transcription. A majority of these compounds such as ActD, BMH-21, and CX-5461, function as DNA intercalators where they bind to rDNA and disrupt interaction with Pol I machinery (Bruno et al., 2020; Pitts & Laiho, 2022). However, DNA alkylating agents like cisplatin and oxaliplatin, as mentioned above, and antimetabolites such as methotrexate, have also been shown to inhibit rRNA synthesis (Zisi et al., 2022). While, several structural and chemical determinants for Pol I inhibition have been identified among small-molecule inhibitors, the precise mechanisms which dictate Pol I specificity are still poorly understood (Zisi et al., 2022). It is widely suggested that the high affinity of small-molecule inhibitors towards GC-rich regions of the genome, such as rDNA, drive selectivity towards Pol I inhibition (Peltonen et al., 2014; Penzo et al., 2019; Zisi et al., 28 2022). This is not considered to be the only factor contributing to Pol I specificity. rDNA is intrinsically unstable as a consequence of the high repetitiveness and transcriptional rates, and thus is more susceptible to DNA damage, such as DSBs, which are often enriched in the rDNA regions of the genome (González-Arzola, 2024; Hwang & Denicourt, 2024). Recognition and processing of rDNA damage is handled through a distinct nucleolar DDR (n-DDR), which has emerged to describe the unique mechanisms that directly mediate Pol I inhibition in the presence of rDNA DSBs (González-Arzola, 2024). It’s also been hypothesized that chemotherapeutics, such as oxaliplatin and CX- 5461, may preferentially induce rDNA damage which then leads to specific Pol I inhibition via n-DDR (Xuan et al., 2021). Conclusion Within this dissertation, I will address three facets of nucleolar stress induced by Pt(II) compounds. Chapter II will address the chemical and structural properties of oxaliplatin which are necessary for nucleolar stress induction. Here we identify a subset of nucleolar stress inducing Pt(II) compounds which share several chemical and structural properties. Chapter II was originally published in the Journal of the American Chemical Society by Emily C. Sutton, Christine E. McDevitt, Jack Y. Prochnau, Matthew V. Yglesias, Austin M. Mroz, Min Chieh Yang, Rachael M. Cunningham, Christopher H. Hendon, and Victoria J. DeRose (Sutton, McDevitt, Prochnau, et al., 2019). Chapter III expands this inquiry to monofunctional Pt(II) compounds and describes how spatial orientation and steric bulk specifically factor into nucleolar stress induced by monofunctional Pt(II) compounds. Chapter III was published the Journal of Biological Inorganic Chemistry by Christine E. McDevitt, Matthew V. Yglesias, Austin M. Mroz, Emily C. Sutton, Min Chieh Yang, Christopher H. Hendon, and Victoria J. DeRose (McDevitt et al., 2019). In chapter IV we address potential variability in the robustness and kinetics of nucleolar stress induced by Pt (II) analogs of oxaliplatin. This chapter demonstrates spatiotemporal differences in nucleolar stress between Pt(II) compounds, which were not explained by trends in cytotoxicity, DNA platination or intracellular Pt accumulation. Chapter IV was previously published in ACS Chemical Biology and co-authored by Hannah C. Pigg, Matthew V. Yglesias Emily C. Sutton, Christine E. McDevitt, Michael 29 Shaw, and Victoria J. DeRose (Pigg et al., 2022). Chapter V explores the biological and molecular interactions involved in Pol I inhibition by oxaliplatin and their connection to nucleolar stress. In this chapter we describe the recent progress in applying chromatin immunoprecipitation and DNA sequencing technique to map the rDNA occupancy of Pol I machinery. Chapter V contained unpublished work by Matthew V. Yglesias. Lastly, Chapter VI will review the interactions of coordinate metal ions with DNA, RNA, and nucleic acid enzymes, with a focus on insights gained from more recently published NMR and x-ray crystallography structures. Chapter VI was co-written with Victoria J. DeRose and first published in Comprehensive Coordination Chemistry III (Victoria J. DeRose & Yglesias, 2021). Bridge to Chapter II This introductory chapter establishes the current and historical significance of Pt- based chemotherapeutics in the medical fields, and their influence in cancer biology and inorganic chemistry. A current knowledge of the mechanisms of action for Pt chemotherapeutics, and potential mechanisms underlying side-effect and resistance/sensitivity profiles, was discussed, emphasizing how novel insights into these interactions are translatable to the clinical setting and will be needed to overcome the limitations of current Pt chemotherapeutics. This chapter also provides a summary on ribosome biogenesis and the nucleolus, in the context of nucleolar stress and its connection to Pt compounds, including the finding that some Pt chemotherapeutics induce cell death through a disruption in ribosome biogenesis. Ribosome biogenesis and Pol I are importantly highlighted as potential targets for novel chemotherapeutic agents, necessitating a greater understanding of these mechanisms. Chapter II will describe an immunofluorescence assay for determining nucleolar stress which was developed and used to identify chemical and structural properties necessary for Pt(II) analogs of oxaliplatin to induce nucleolar stress. 30 CHAPTER II: NUCLEOLAR STRESS INDUCTION BY OXALIPLATIN AND DERIVATIVES This chapter has been previously published as: Sutton, E. C.; McDevitt, C. E.; Prochnau, J. Y.; Yglesias, M. V.; Mroz, A. M.; Yang, M. C.; Cunningham, R. M.; Hendon, C. H.; DeRose, V. J. Nucleolar Stress Induction by Oxaliplatin and Derivatives. J. Am. Chem. Soc. 2019, 141 (46), 18411–18415. https://doi.org/10.1021/jacs.9b10319. E.C.S. and C.E.M. share co-first authorship. E.C.S., C.E.M. and V.J.D. wrote the original manuscript with input from all authors. Nucleolar Stress Induction by Oxaliplatin and Derivatives The chemotherapeutic agent cisplatin has inspired the synthesis and investigation of thousands of Pt(II) analogs (Kelland, 2007). Of these, only two other compounds— carboplatin and oxaliplatin—have met FDA standards for medical use. Until recently, it was believed that the cytotoxicity of these compounds could be attributed solely to their DNA crosslinking abilities and subsequent induction of the DNA damage response (DDR), a known trigger of apoptotic pathways (Wexselblatt et al., 2012). As the body of research on Pt(II) reagents has grown, a more complex picture has emerged of the mechanisms of action behind these ubiquitous drugs (Wexselblatt et al., 2012). A striking recent discovery is that oxaliplatin, but not cisplatin or carboplatin, cause cytotoxicity via disruptions in ribosome biogenesis rather than DDR (Bruno et al., 2017). Ribosome biogenesis occurs in the nucleolus, a conserved and highly structured membrane-less organelle in eukaryotes. Disruptions of the nucleolus or ribosome biogenesis trigger the nucleolar stress response, which leads to cell death or senescence via activation of the tumor suppressor p53. Because its molecular mechanisms are not fully understood, and due to its potential role as a chemotherapeutic target, this fascinating stress process is an area of intense interest in the fields of molecular biology and medicine (Boulon et al., 2010; Farley-Barnes et al., 2018; Tsai & Pederson, 2014; Woods et al., 2015). The specificity of oxaliplatin as a nucleolar stress inducer is intriguing when considered alongside other data indicating a relationship between Pt(II) compounds and the nucleolus (Pickard & Bierbach, 2013). Post-treatment fluorescent labeling of clickable https://doi.org/10.1021/jacs.9b10319 31 Pt(II) drug analogs has shown localization of these compounds to the nucleolus, (Pickard & Bierbach, 2013; Wirth et al., 2015) and there is significant evidence that Pt(II) compounds associate with ribosomes and ribosomal RNA (Hostetter et al., 2012; Melnikov et al., 2016; Osborn et al., 2014; Plakos & DeRose, 2017; Rijal & Chow, 2008; Saunders & DeRose, 2016; Sutton, McDevitt, Yglesias, et al., 2019). The structural determinants and molecular mechanisms by which only specific Pt(II) compounds may cause a nucleolar stress response are not understood. Here, we explore properties of oxaliplatin and other Pt(II) compounds and find that a narrow window of derivatives are able to induce nucleolar stress. The results define a set of constraints for Pt(II) compounds to induce this unique cell death pathway. We selected Pt(II) compounds to test a variety of properties including steric bulk, hydrophobicity, cross-linking ability, and ligand orientation (Figure 2.1). The extent of nucleolar stress was measured by nucleophosmin (NPM1) imaging (Figure 2.2 and A.1). Translocation of NPM1 from the granular component (GC) of the nucleolus to the nucleoplasm is a hallmark of the nucleolar stress response (Rubbi & Milner, 2003; Yang et al., 2016). NPM1 translocation has been shown to be a necessary, but not sufficient, feature of p53-mediated cell death upon nucleolar stress (Yang et al., 2016) and thus is a robust and appropriate marker. A549 cells were selected for this study as they are well- established to have a characteristic nucleolar stress response resulting in p53-mediated apoptosis (Bursac et al., 2014; Nicolas et al., 2016). 32 Figure 2.1. Compounds tested for inducing nucleolar stress via NPM1 relocalization in mammalian cells. Cells were treated for 24 h with a given compound prior to fixation and secondary immunofluorescence to detect NPM1 (Figure 2.2 and A.1). The extent of NPM1 redistribution was quantified using an image analysis pipeline (Figure A.1) to calculate the coefficient of variation (CV) of NPM1 intensity in each cell (Figure 2.3). The uniform distribution of NPM1 in cells undergoing nucleolar stress yields a low CV, as seen in positive control samples treated with known stress inducer, actinomycin D (ActD) (Figure 2.3). In addition to the observation of NPM1 redistribution, we noted a change in the shape of nucleoli from eccentrically shaped aggregates to round, sphere-like structures upon stress induction (Figure 2.2). As predicted, (Bruno et al., 2017; McDevitt et al., 2019) oxaliplatin (2) induces robust redistribution of NPM1, similar to the positive control (ActD), while NPM1 distribution in cisplatin (1) and carboplatin (3) treated cells more closely resembles that of the no-treatment control (Figure 2.2 and 2.3). 33 Figure 2.2. Nucleolar stress induced by Pt(II) compounds. NPM1 (green) relocalization following 24 h treatment in A549 cells. Treatment concentrations are 10 µM except for ActD (5 nM). Scale bar = 10 µm. We note that for cisplatin-treated cells, a small amount of NPM1 redistribution was observed at this treatment concentration. This is likely because the 24 h IC50 value for cisplatin (12.8 µM, Table A.1), is close to the treatment concentration, which may result in a subset of cisplatin-treated cells experiencing abnormal NPM1 distribution downstream of other cell death pathways, such as those mediated by the DDR.4 Oxaliplatin, by contrast, shows robust NPM1 relocalization at treatment concentrations well below the 24 h IC50 value (81.5 µM, Table A.1), suggesting that nucleolar stress significantly precedes cell death pathways (Bruno et al., 2017). NPM1 relocalization at concentrations below IC50 4 This model is supported by previously published data demonstrating that cisplatin causes significantly more DNA damage than oxaliplatin(Chaney et al., 2005; Faivre et al., 2003; Woynarowski et al., 2000) and that DDR-mediated cell death occurs upon cisplatin treatment, but not oxaliplatin treatment.(Bruno et al., 2017) Additionally, data from our lab shows that DNA damage occurs at early time points in cisplatin- treated cells, prior to observed NPM1 distribution in oxaliplatin-treated cells (unpublished). 34 value was observed with other stress-inducing compounds, some of which did not exhibit significant cell death until 48 h of treatment (Tables A.1 and A.2). Thus, observation of nucleolar stress at 24 h does not necessarily predict measured toxicity. Figure 2.3. Quantification of NPM1 relocalization induced by Pt(II) compounds. Treatment conditions as in Figure 2.2; replicates, CV calculations, and boxplot presentation as described in Appendix A. For each treatment data set, boxes represent median, first, and third quartiles, and vertical lines are the range of data with outliers defined in the Appendix A. Oxaliplatin is distinct from cisplatin and carboplatin in both labile and nonlabile Pt(II) ligands. The labile, chelating oxalate ligand of oxaliplatin delays aquation and therefore biomolecule cross-linking (Jerremalm et al., 2002) in comparison with cisplatin. We exchanged the labile and nonlabile ligands of oxaliplatin and cisplatin with DACHPt (4), and DOAP (5). We found that 4, which has the nonlabile DACH ligand of oxaliplatin and labile chloride groups of cisplatin, induces robust nucleolar stress. By comparison, 5, which possesses the nonlabile ammine ligands of cisplatin and the labile oxalic acid ligand of oxaliplatin, does not induce stress (Figure 2.2 and 2.3). The oxalic acid ligand alone also had no influence on NPM1 redistribution, nor did the DACH ligand by itself (Figure A.2 and A.3). From this, we concluded that the nonlabile DACH ligand of oxaliplatin is responsible for the nucleolar stress response. 35 We next considered whether cross-linking of biomolecules by the Pt(II) compound is necessary for the induction of nucleolar stress. An alternate hypothesis is that the charged Pt(II) acts as a targeting agent to facilitate transport of the DACH moiety to the nucleolus where it disrupts nucleolar processes without forming a Pt(II)-DACH lesion on a biomolecule. DACH-En (6) retains the DACH ligand but is unable to form cross-links with biomolecules due to replacement of the oxalic acid with an ethylene diamine ligand (Figure 2.1 6). This positively charged compound did not induce stress (Figure 2.2 and 2.3), suggesting that cross-linking of Pt(II) to cellular targets is necessary to induce a nucleolar stress response. To refine requirements of the Pt(II) ligands that cause nucleolar stress, we examined the effects of steric bulk by testing PtEn (7), PtMeEn (8), and pentaplatin (9). Compound 7 possesses a nonlabile ethylenediamine ligand. This small molecule did not induce stress in A549 cells (Figure 2.2 and 2.3), indicating that a chelating diamine ligand, a common feature between 7, 4, and oxaliplatin, is not sufficient to induce stress. The addition of a methyl group to generate the bulkier 8 was also not sufficient to induce stress (Figure 2.3, A.2, and A.3). Compound 9 possesses a five-membered ring that places its volume between the non-stress-inducing 8 and the stress-inducing six-membered 4 (Figure 2.4.A). Compound 9 was found to induce nucleolar stress (Figure 2.2), although with a slightly higher resultant CV than positive controls or oxaliplatin (Figure 3). These results suggest that bulk may be an important metric lending towards the ability of Pt(II) compounds to induce nucleolar stress. Using computed values for volume, we conclude that as a general trend Pt(II) compounds with more steric bulk are more likely to induce nucleolar stress (Figure 2.4.A, y-axis). Compound length, or steric reach, also generally appears to correlate with stress induction (Figure 2.4.A, x-axis). Some exceptions to this trend are discussed below. 36 Figure 2.4. Size and hydrophobicity correlate with stress induction, with some exceptions. A| Ligand reach, or farthest distance from the Pt center, compared to computed volume measurements. B| Hydrophobicity of Pt compounds determined by the water/octanol partition coefficient. Compounds with a higher partition coefficient (log P) are more hydrophobic than those with a lower log P. Log P measurements and calculations of compound volume and Pt-edge distance are described in the Appendix A. The chair confirmation of the DACH ligand is not essential for stress induction. BenzaPt (10), in which the DACH cyclohexane is replaced with a planar aromatic ring (Figure 2.1, 10), also induces robust NPM1 redistribution (Figure 2.2 and 2.3). Like compound 4, 10 is more hydrophobic than the simpler diam(m)ine compounds. To estimate the relative hydrophobicity of our compounds of interest, we measured their water/octanol partition coefficients (Table A.3). All of the stress-inducing compounds were found to be relatively hydrophobic (Figure 2.4.B), leading to the conclusion that hydrophobicity, like steric bulk, positively correlates with stress induction. Similarly to steric bulk, however, exceptions to this trend were observed. Picoplatin (11), APP (12), and azidoplatin (13) do not cause NPM1 relocalization despite being similar or higher in terms of size and hydrophobicity to compounds that do not cause nucleolar stress (Figure 2.4). These exceptions may provide insight into the elements responsible for causing stress. 37 One particularly interesting comparison is the ligand orientation between 12 and 10 (Figure 2.5.A). These two Pt(II) compounds both have an aromatic ring but differ in the orientation of the ring relative to the Pt(II) and, by extension, ring orientation relative to a biomolecule to which the compound is bound. While benzaPt causes nucleolar stress, AP does not. Similarly, 11 does not cause nucleolar stress despite having volume and reach similar to other compounds (Figure 2.4.A and 2.5). These results demonstrate a critical role for ring orientation in the ability of Pt(II) compounds to induce nucleolar stress. Figure 2.5. A| Computed distance measurements and volume representations for nonstress-inducing compounds 11 and 12 alongside stress-inducing compounds 4 and 10. B| Ball and stick drawings of nonstress-inducing compound 8 alongside stress-inducing 4, 9, and 10. The observation that 13 does not cause stress is of interest as this compound has extended volume and has previously been shown to localize to the nucleolus (Wirth et al., 2015). Thus, nucleolar localization, even when combined with relatively high hydrophobicity and larger bulk and length, is not sufficient to induce nucleolar stress. 38 Taken together, the results described provide significant insight into the structural determinants of nucleolar stress induction among Pt(II) compounds. We conclude that there is an important role for ligand orientation and a general correlation between steric bulk and stress induction (Figure 2.5). The differential responses induced by these compounds have clinical implications as the three currently FDA-approved Pt(II) chemotherapeutics are known to have different treatment and side effect profiles. Other important differences between these compounds have been observed in the literature. For example, oxaliplatin is noted to cause immunogenic cell death (ICD), while cisplatin does not (Englinger et al., 2019; Siew et al., 2015; Terenzi et al., 2016). Although this contrast is also observed in nucleolar stress, connections between ICD and nucleolar stress are not well-studied. Oxaliplatin has also been shown to cause changes in the size of neuronal nucleoli correlating with peripheral neuropathy,(McKeage et al., 2001) a common side effect associated with oxaliplatin chemotherapy regimens. The relationship between nucleolar stress and platinum- induced neurotoxicity has not been explored. Additionally, there is some evidence that p53 mutations in colon cancer cell lines result in resistance to oxaliplatin-mediated cell death (Toscano et al., 2007). This may be of interest given oxaliplatin’s use in colon cancer treatments and p53’s role in nucleolar stress-induced cell death. Further study is warranted to provide clarification on the molecular mechanisms by which these compounds induce such different responses in the cell. For example, the stress-inducers may be interfering with progression of ribosome biogenesis,(Bursac et al., 2014; Rubbi & Milner, 2003) disrupting an intermolecular interaction of NPM1 that sequesters it in the nucleolus,(Yang et al., 2016) altering biophysical properties of nucleic acids,(Keck & Lippard, 1992; Malina et al., 2007) or globally perturbing the biomolecular interactions that maintain nucleolar integrity. More work is needed to understand this fascinating biological stress process and to define the specific properties of Pt(II) compounds that cause it. Bridge to chapter III In this chapter we sought to identify structural and chemical components of Pt(II) compounds necessary for the induction of nucleolar stress, hypothesizing that inherent 39 differences in the chemical nature of oxaliplatin and cisplatin are responsible for disparate cell death pathways induced by these two chemotherapeutics. By designing a quantitative immunofluorescence-based assay to measure relocalization of NPM1—an analog for nucleolar stress and disruption of ribosome biogenesis—we performed a structure function study utilizing a strategic library of Pt(II) compounds. Chapter III expands this framework to a set of non-canonical monofunctional Pt(II) compounds based on phenanthriplatin, which has also been shown to induce cell death through disruption of ribosome biogenesis. 40 CHAPTER III: MONOFUNCTIONAL PLATINUM(II) COMPOUNDS AND NUCLEOLAR STRESS: IS PHENANTHRIPLATIN UNIQUE? This chapter was originally published as: McDevitt, C. E.; Yglesias, M. V.; Mroz, A. M.; Sutton, E. C.; Yang, M. C.; Hendon, C. H.; DeRose, V. J. Monofunctional Platinum(II) Compounds and Nucleolar Stress: Is Phenanthriplatin Unique? J Biol Inorg Chem 2019, 24 (6), 899–908. https://doi.org/10.1007/s00775-019-01707-9. C.E.M. and V.J.D. wrote the original manuscript with input from all authors. Introduction Platinum-based drugs are an important class of chemotherapeutics. After the initial discovery of the anti-proliferative capabilities of cisplatin, the drug was FDA approved in 1978 and continues to be in significant use over 40 years later (Kelland, 2007; Rosenberg et al., 1965). Two additional Pt(II) compounds were subsequently approved by the FDA, carboplatin in 1989 and oxaliplatin in 1996. Improvements upon these three drugs have been attempted and some new compounds even entered into clinical trials, but none have been approved by the FDA (Kelland, 2007). The three FDA-approved drugs are all considered classical platinum compounds. The characteristics of classical compounds are a result of early structure-activity relationship (SAR) studies that determined the necessary properties for platinum compounds to exhibit anti-proliferation activity (Johnstone et al., 2014). These required components are that the platinum compound be square planar, have a neutral overall charge, and contain two non-labile cis-am(m)ines and two labile cis anionic ligands (Johnstone et al., 2014). Although these rules led to the drugs that are used today, research into compounds that would not be within a traditional SAR study have produced non-classical platinum drugs with anti-proliferative activity. These non- classical compounds include Pt(IV) prodrugs, monofunctional platinum compounds, trans-platinum, polyplatinum, and tethered platinum, complexes (Johnstone et al., 2014; Sutton, McDevitt, Yglesias, et al., 2019). One of the most effective and well-studied non- classical compound is the monofunctional Pt(II) compound, phenanthriplatin (Figure 3.1) (Johnstone et al., 2014; Park et al., 2012). In addition to having only a single exchangeable https://doi.org/10.1007/s00775-019-01707-9 41 anionic ligand, the N-heterocyclic ligand of phenanthriplatin and others of this class, such as picoplatin (Figure 3.1), is perpendicular to the square-planar Pt ligand plane. Figure 3.1. Platinum compounds used in this study Phenanthriplatin has exhibited 7–40× higher activity in the NCI-60 human tumor cell line screen when compared to other platinum chemotherapeutics (Johnstone et al., 2014; Park et al., 2012). Phenanthriplatin has higher cellular uptake than cisplatin or pyriplatin (Park et al., 2012). In addition, the phenanthridine ligand of phenanthriplatin may facilitate rapid DNA binding though reversible intercalation between nucleobases before Pt-DNA binding occurs (Almaqwashi et al., 2019). Studies have also revealed some of the biological targets of phenanthriplatin. It has been shown to act as a topoisomerase II poison (Riddell et al., 2016). Phenanthriplatin lesions were also demonstrated to inhibit RNA Polymerase II activity,(Kellinger et al., 2013) but allows DNA polymerase η bypass (Gregory et al., 2014). Overall, these studies have shown that phenanthriplatin can affect biological processes in a variety of ways, and this has led researchers to suggest that the effectiveness of the compounds is through multiple cellular pathways (Facchetti & Rimoldi, 2019). In a recent study, the classical platinum compound oxaliplatin and non-classical platinum compound, phenanthriplatin were both shown to induce ribosome biogenesis 42 stress as the primary pathway to induce cell death (Bruno et al., 2017). This surprising observation is in contrast with cisplatin and carboplatin, which were shown to cause cell death through DNA damage as expected for classical compounds. The ability to induce nucleolar stress shared between oxaliplatin and phenanthriplatin is perplexing considering the major structural differences between the two compounds. We endeavored to determine whether there were structural similarities between these two molecules which would explain this similar activity and determine whether the ability to induce nucleolar stress was inherent to the family of non-classical monofunctional platinum(II) compounds5. To do this, we synthesized a suite of monofunctional and related platinum compounds (Figure 3.1) and analyzed their ability to cause nucleolar stress by measuring nucleophosmin (NPM1) relocalization. We further compared structural and electronic properties of these compounds based on DFT calculations. We find that phenanthriplatin, but not related quinoplatin or isoquinoplatin, induce nucleolar stress as measured by NPM1 relocalization in human lung carcinoma A549 cells. Although phenanthriplatin has the largest total volume and hydrophobicity of the compounds tested, quinoplatin and isoquinoplatin may have similar potential to disrupt intermolecular interactions based on Pt-ligand distances. We conclude that the unique ability of phenanthriplatin to induce nucleolar stress is conferred by the acridine ring. The ligand disposition of these monofunctional N-heterocyclic Pt(II) compounds is sufficiently different from oxaliplatin to suggest that separate properties of oxaliplatin and phenanthriplatin lead to their abilities to both cause nucleolar stress. Results and discussion Oxaliplatin and phenanthriplatin cause NPM1 relocalization A previous study examining cell death mechanisms of phenanthriplatin (1) and oxaliplatin (2) has shown that both compounds cause cell death through ribosome biogenesis stress (Bruno et al., 2017). In this study, we monitored NPM1 relocalization from the nucleolus to the nucleoplasm, which is a hallmark of nucleolar stress (Yang et al., 2016). Under non-stress conditions NPM1 is localized to the nucleolus; however, NPM1 is distributed throughout the nucleoplasm during nucleolar stress. We set out to 5 Monofunctional Pt(II) compounds following the structure cis-[Pt(NH3(Am)Cl]+ (Am = aromatic N- heterocyclic amine). 43 measure the extent of NPM1 relocalization when cells were treated with platinum compounds with cyclic ligands and either monofunctional or bifunctional substitution properties. We first examined NPM1 relocalization following treatment with oxaliplatin and phenanthriplatin. Actinomycin D (ActD), a known ribosome biogenesis stress inducer, caused NPM1 relocalization to the nucleoplasm while the negative no-treatment control showed NPM1 localization in the nucleoli (Figure 3.2.A). Both 1 and 2 caused relocalization of NPM1 throughout the nucleus, confirming their ability to cause nucleolar stress as previously reported (Bruno et al., 2017). To determine the extent of nucleolar stress, we quantified the heterogeneity of nuclear NPM1 intensity distribution by its coefficient of variation (CV). The CV is calculated as the standard deviation in pixel intensity corresponding to NPM1-based immunofluorescence within the nucleus, normalized by the mean intensity. In cells that are undergoing nucleolar stress, NPM1 is relatively evenly diffused throughout the nucleus, leading to homogeneous intensities and a small CV value. Histograms of representative NPM1 immunofluorescences in cell images show an even population of pixel intensities within the nucleus for compounds that cause NPM1 relocalization (Figure 3.2.B). For cells that are not undergoing stress, NPM1 is concentrated in the nucleolus while being absent in the nucleoplasm, resulting in a heterogeneous population of pixel intensities and a high CV value. Histograms of NPM1 immunofluorescences from compounds that do not cause NPM1 relocalization show a bimodal distribution in pixel intensities which would result in a large CV (Figure 3.2.B). The CV was calculated for individual cells and averaged for each treatment condition. Corresponding to our representative NPM1 images (Figure 3.2.A), compounds that caused no NPM1 redistribution had a median CV around 1 (when normalized to the no-treatment control) while compounds that caused NPM1 relocalization had a median CV at or lower than, 0.6 (normalized to the no-treatment control).6 NPM1 relocalization was observed upon treatment with 1, 2, and ActD (Figure 3.2.C). Additionally, treatment with phenanthridine ligand alone is not sufficient to induce nucleolar stress (Figure 3.2.C). 6 In this study we observed that cells undergoing nucleolar stress—when determined by relocalization of NPM1—had an average CV ≤ 0.6 when normalized to the untreated cells. 44 Picoplatin does not cause NPM1 relocalization There are large structural differences between oxaliplatin and phenanthriplatin; however, these disparate compounds are both able to activate nucleolar stress pathways whereas cisplatin does not. Both the DACH ligand of oxaliplatin and the phenanthridine ligand of phenanthriplatin add significant steric bulk in comparison to cisplatin. However, phenanthriplatin is a monofunctional compound. In addition, unlike the case of oxaliplatin, in phenanthriplatin, the phenanthridine ring is oriented perpendicular to the square-planar Pt ligand plane (Park et al., 2012). Picoplatin (3) is one compound that bridges these differences in that the 2-picoline ligand is oriented perpendicular to the square-planar]Pt ligand plane (Chen et al., 1998). 3 is also a classical bifunctional platinum compound and enabled us to determine whether the added ligand bulk regardless of orientation was sufficient to induce NPM1 relocalization. In A549 cells threated with 3, NPM1 did not relocalize to the nucleoplasm (Figure 3.2.A) as quantified by a median normalized CV of approx. 1 (Figure 3.2.C), indicating that 3 does not cause nucleolar stress. NPM1 relocalization is not a general property of monofunctional platinum compounds. After determining that the bifunctional compound, 3, did not cause NPM1 relocalization despite having some similarities to oxaliplatin in terms of added ring and steric bulk, we next examined the properties of non-classical monofunctional platinum compounds. We synthesized three additional monofunctional compounds that have a pyridine or quinoline ligand to test whether nucleolar stress was inherent to ring- containing monofunctional platinum(II) compounds as a whole or whether it was a phenomenon only exhibited by phenanthriplatin. 45 Figure 3.2. NPM1 relocalization. A| Representative images for each platinum treatment. DAPI (grey) shows the nucleus of A549 cells. NPM1 (green) is evenly distributed in positive control actinomycin D (ActD), and also in cells treated with oxaliplatin, and phenanthriplatin, indicating nucleolar stress. NPM1 is localized to the nucleolus in untreated cells, and cells treated with isoquinoplatin, quinoplatin, pyriplatin, and picoplatin. Scale bar = 10 µm. Cells were treated with 10 µM platinum at 24 h with the exception of phenanthriplatin and phenanthridine which were used at 0.5 µM. B| Representative histograms for individual cells. In untreated negative control and pyriplatin-treated cells, large populations of pixels are found at low and high intensity. NPM1 localization throughout the nucleoplasm is seen following oxaliplatin and phenanthriplatin treatment with pixel intensity centered around 0.4. C| Coefficient of variation for platinum treatments. CV values for individual nuclei are plotted for each treatment group. Box plot center line represents the median, and the bottom and top limits represent the first and third quartile, respectively. The CV from each cell is normalized to the mean CV from the no-treatment control sample. Populations that have NPM1 relocalization have a median CV of around 0.6 while populations without NPM1 relocalization are around 1. 46 We had tested 3 and determined that the perpendicular orientation of the picoline ligand is not sufficient to cause NPM1 relocalization. To further explore the influence of ligand orientation and the binding mode of monofunctional Pt compounds, we next tested pyriplatin (4). Similar to 3, 4, contains a single aromatic ring. However, unlike 3, 4, has more possible orientations of the aromatic ring due to the lack of steric interference involving the methyl group of the 2-picoline ligand (Johnstone & Lippard, 2014). In addition, 4 is more similar to 1 in being a monofunctional compound with an overall positive charge. Following a 24 h treatment at 10 µM, 4 did not cause NPM1 relocalization and samples had a median normalized CV of approx. 1 (Figure 3.2). From this we concluded that the ability to cause NPM1 relocalization was not inherent to the class of monofunctional Platinum(II) compounds containing N-heterocyclic ligands. We next considered whether steric bulk was a factor in NPM1 relocalization by examining the influence of the addition of a second ring. We synthesized the structural isomers quinoplatin (5) and isoquinoplatin (6) (Figure 3.3), to test whether a second aromatic ring would be sufficient to cause NPM1 relocalization. We tested these compounds and determined that neither 5 nor 6 caused increased NPM1 relocalization7, with NPM1 intensities from cells treated with both compounds having a median normalized CV of approx. 1 (Figure 3.2). From this we concluded that for monofunctional Pt(II) compounds, the steric bulk from a second ring alone does not induce NPM1 relocalization regardless of ring orientation. This added further evidence that NPM1 relocalization was not an inherent property of this non-classical class of platinum compounds and was unique to 1 under these conditions. Steric bulk is not sufficient to predict NPM1 relocalization From our data, we have determined that phenanthriplatin and oxaliplatin are unique to our suite of compounds. We next examined whether there are any trends present in steric bulk that could explain whether compounds caused NPM1 relocalization. The molecular structure for all platinum(II) compounds investigated in 7 Our conclusions here were ultimately based on the visual lack of well-defined NPM1 relocalization in cell images when compared to 1, 2, and ActD (Figure 3.2.A). We did however notice, CV measurements in cells treatments with 5 were slightly lower on average compared to untreated cells (norm. CV = 0.85, Figure 3.2.B). This prompted a subsequent concentration dependence experiment (Figure B.1) where we found that 5 did not induce significant levels of NPM1 redistribution even at high concentrations (20 µM). 47 this study were optimized using DFT (Figure 3.4) and two variables were calculated to assess steric bulk. First, the volume of the optimized, non-hydrolyzed structure is obtained by sampling the respective electrostatic potential (Table 3.1). 2 and 1 were the compounds with the largest volume; however, this included the aquation-labile ligands which accounts for a large portion of oxaliplatin’s volume. Figure 3.3. Two-ring structural isomers related to phenanthriplatin Second, the magnitude of the maximum vector between the platinum center and the surface of the compound, where the surface of the compound is defined as the extent to which electrostatic potential permeates in space was calculated (Table 3.1). No trend was found with these distance measurements. Oxaliplatin, which causes NPM1 relocalization had similar distance to that of isoquinoplatin which did not cause NPM1 relocalization (Table 3.1). Thus, while phenanthriplatin exhibits the largest steric bulk, it does not have the maximum steric reach from platinum to the surface of the compound. Table 3.1. Steric bulk measurements for platinum compounds in order of increasing volume. Compound Volume (Å3) Maximum Pt-to-surface distance (Å) Pyriplatin (4) 24.91 6.27 Picoplatin (3) 27.69 6.21 Isoquinoplatin (6) 31.21 8.37 Quinoplatin (5) 33.89 7.15 Oxaliplatin (2) 34.13 6.78 Phenanthriplatin (1) 37.25 7.72 48 Figure 3.4. Optimized structures of the platinum(II) compounds are displayed at an isosurface level of 0.25 e/Å−3 for each compound, as implemented in VESTA. This illustrates the volume of the molecule that is reported. The distances between the platinum atom and the surface of each compound are shown with the corresponding vector. All measurements are reported in angstrom (Å). Hydrophobicity is not sufficient for predicting NPM1 relocalization Hydrophobicity of the non-labile ligand may be an important factor for interrupting biomolecular interactions, or in partitioning into cellular compartments or subregions of the nucleolus. We examined if there was a trend in hydrophobicity that would explain why 2 and 1 cause NPM1 relocalization while all other compounds in our library did not. We used our optimized structures to calculate ∆Gwater-octanol (Table 3.2). As expected, compounds with more aromatic rings were more hydrophobic and had a more positive ∆Gwater-octanol while compounds with less rings showed the opposite trend. 1 is more hydrophobic than all other tested compounds except 3, which does not cause NPM1 relocalization and is the most hydrophobic compound tested with a Gibbs solvation energy of 2.54 kcal/mol. Overall, this measure of hydrophobicity was not able to produce a trend that provides a satisfactory explanation for why oxaliplatin and 49 phenanthriplatin cause NPM1 relocalization while other platinum(II) compounds did not. Therefore, we can conclude that hydrophobicity alone is not sufficient for causing NPM1 relocalization. Table 3.2. Gibbs free energy of transfer between octanol and water Compound ∆Gwater-octanol (Kcal/mol) Oxaliplatin (2) -4.92 Pyriplatin (4) -1.92 Isoquinoplatin (6) -0.607 Quinoplatin (5) -0.34 Phenanthriplatin (1) 0.94 Picoplatin (3) 2.54 Conclusions This work aimed to find a structural relationship between oxaliplatin and phenanthriplatin which would provide information on necessary and sufficient structural components required for these platinum compounds to induce cell death via nucleolar stress. In comparison with cisplatin, which does not cause nucleolar stress, oxaliplatin and phenanthriplatin both have significantly larger ring-containing ligands. Phenanthriplatin is also a monofunctional Pt(II) compound. To explore this question, we synthesized a library of ring-containing platinum compounds, most being monofunctional Pt(II) compounds. This library was tested for the ability to induce nucleolar stress by monitoring NPM1 relocalization and quantifying the resulting images. First, we tested oxaliplatin and phenanthriplatin to confirm that they caused NPM1 relocalization in agreement with previous literature proposing that they cause nucleolar stress (Bruno et al., 2017). We then tested whether a heterocyclic ligand plane would be sufficient by testing picoplatin and found that picoplatin did not cause nucleolar stress as measured by NPM1 relocalization. Thus, for bifunctional platinum compounds, a ligand ring is insufficient to cause nucleolar stress. We investigated the importance of ligand ring number and distribution in other compounds of the monofunctional platinum(II) class by testing pyriplatin, quinoplatin, and isoquinoplatin. None of these compounds caused NPM1 relocalization, indicating that phenanthriplatin was unique in this class of monofunctional Pt(II) compounds. We note that this limited study has been performed at a single concentration and treatment 50 time for all compounds. It is possible that longer treatment time or higher concentrations might lead to different effects, and this is being explored in further studies. None of the non-phenanthriplatin compounds cause significant levels of nucleolar stress at relatively high (10 µM) treatment concentrations compared to phenanthriplatin (0.5 µM), indicating that they are in a different class than phenanthriplatin in terms of activities. We performed DFT calculations to optimize structures and calculate the solvent- dependent difference in Gibbs free energy between water and n-octanol, a measure of hydrophobicity. To further investigate structural characteristics, we calculated the maximum distance from the platinum atom to the surface of each structure and volume from DFT-optimized structures. We found no correlation between this distance and the ability to cause NPM1 relocalization. Further, there was no strong correlation between the solvent-dependent difference in Gibbs free energy between water and octanol for compounds that were able to induce NPM1 relocalization. In view of these results, we suggest that phenanthriplatin is a unique compound in the monofunctional platinum(II) compound class in its ability to cause NPM1 relocalization. We suggest that the addition of a third aromatic ring in phenanthriplatin may play a large role in differentiating phenanthriplatin from other monofunctional platinum(II) compounds we tested for inducing nucleolar stress. The presence of a third aromatic ring increases steric bulk both above and below the square-planar platinum ligand plane. Additionally, a third ring increases hydrophobicity and provides intercalation potential to phenanthriplatin (Almaqwashi et al., 2019) in comparison to quinoplatin and isoquinoplatin. Phenanthriplatin exhibited the largest volume and was the most hydrophobic compound of the monofunctional platinum(II) compounds but did not exhibit the longest distance from the platinum atom to the