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Kinetic Analysis and Inhibition Studies of Iron-Dependent Histone Demethylases" (2014)

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Kinetic Analysis and Inhibition Studies of Iron-Dependent Histone Demethylases Washington University in St. Louis Washington University Open Scholarship Arts & Sciences Electronic Theses and Dissertations Arts & Sciences Winter 12-15-2014 Kinetic Analysis and Inhibition Studies of Iron- Dependent Histone Demethylases Barbara Gordon Cascella Washington University in St. Louis Follow this and additional works at: https://openscholarship.wustl.edu/art_sci_etds Part of the Chemistry Commons Recommended Citation Cascella, Barbara Gordon, "Kinetic Analysis and Inhibition Studies of Iron-Dependent Histone Demethylases" (2014). Arts & Sciences Electronic Theses and Dissertations. 349. https://openscholarship.wustl.edu/art_sci_etds/349 This Dissertation is brought to you for free and open access by the Arts & Sciences at Washington University Open Scholarship. It has been accepted for inclusion in Arts & Sciences Electronic Theses and Dissertations by an authorized administrator of Washington University Open Scholarship. For more information, please contact [email protected]. WASHINGTON UNIVERSITY IN ST. LOUIS Department of Chemistry Dissertation Examination Committee: Liviu M. Mirica, Chair Michael Gross Joseph Jez John-Stephen Taylor Timothy Wencewicz Kinetic Analysis and Inhibition Studies of Iron-Dependent Histone Demethylases by Barbara Gordon Cascella A dissertation presented to the Graduate School of Arts and Sciences of Washington University in partial fulfillment of the requirements for the degree of Doctor of Philosophy December 2014 St. Louis, Missouri © 2014, Barbara Gordon Cascella Table of Contents List of Figures ..................................................................................................................................................... v List of Schemes ............................................................................................................................................... viii List of Tables ..................................................................................................................................................... ix List of Abbreviations ......................................................................................................................................... x Acknowledgments............................................................................................................................................ xii Abstract ............................................................................................................................................................ xiv Chapter 1: Introduction .................................................................................................................................... 1 1.1 Covalent Modification of Chromatin ............................................................................................. 1 1.2 Dynamic Methylation and Demethylation of Histone Lysine Residues ................................... 6 1.3 Catalytic Mechanisms of Histone Lysine Demethylation ........................................................... 7 1.3.1 Flavin-Dependent Histone Demethylases ............................................................................ 7 1.3.2 Iron-Dependent Histone Demethylases .............................................................................. 10 1.4 HDMs in DNA Repair ................................................................................................................... 15 1.5 HDMs in Cancer ............................................................................................................................. 16 1.5.1 Flavin-Dependent Histone Demethylases .......................................................................... 16 1.5.2 Iron-Dependent Histone Demethylases .............................................................................. 17 1.6 Project Goals.................................................................................................................................... 19 1.7 References ........................................................................................................................................ 20 Chapter 2: Kinetic Analysis of Iron-Dependent Histone Demethylases: α-Ketoglutarate Substrate Inhibition and Potential Relevance to the Regulation of Histone Demethylation in Cancer Cells ...................................................................................................................................................... 28 2.1 Introduction ..................................................................................................................................... 28 2.2 Methods ............................................................................................................................................ 29 2.2.1 Protein Expression and Purification .................................................................................... 29 2.2.2 Formaldehyde Dehydrogenase Coupled Fluorescence Assay .......................................... 31 2.2.3 Dioxygen Consumption Assay.............................................................................................. 33 2.2.4 MALDI-TOF Mass Spectrometry Assay ............................................................................ 34 2.2.5 Docking Studies of KDM4A and KDM4C with αKG ..................................................... 35 2.2.6 Crystallography ........................................................................................................................ 35 ii 2.3 Results and Discussion ................................................................................................................... 36 2.4 Conclusions ...................................................................................................................................... 54 2.5 References ........................................................................................................................................ 55 Chapter 3: Survey of Simple Primary- and Secondary-Substrate Analog Inhibitors of Iron-Dependent Histone Demethylases ......................................................................................... 61 3.1 Introduction ..................................................................................................................................... 61 3.2 Methods ............................................................................................................................................ 66 3.2.1 Synthesis of Primary Substrate Analogs .............................................................................. 66 3.2.2 Synthesis of Secondary Substrate Analogs .......................................................................... 70 3.2.3 MALDI-TOF Mass Spectrometry Inhibition Assay ......................................................... 74 3.2.4 Enzyme-Templated Cycloaddition Reaction ...................................................................... 75 3.3 Results and Discussion ................................................................................................................... 75 3.4 Conclusions ...................................................................................................................................... 84 3.5 References ........................................................................................................................................ 85 Chapter 4: Inhibition of JmjC-Histone Demethylases with a Modified Histone H3 Peptide .............. 89 4.1 Introduction ..................................................................................................................................... 89 4.2 Methods ............................................................................................................................................ 92 4.2.1 Synthesis of a Modified Histone H3 Peptide...................................................................... 92 4.2.2 MALDI-TOF Mass Spectrometry Assays ........................................................................... 93 4.2.3 Docking Studies of ARK(Nε- bis(propargyl))ST with KDM4A ...................................... 95 4.3 Results and Discussion ................................................................................................................... 96 4.4 Conclusions ................................................................................................................................... 107 4.5 References ..................................................................................................................................... 108 Chapter 5: Inhibition and Crystallography Studies of KDM4A with the Small Molecule JIB-04 .... 112 5.1 Introduction .................................................................................................................................. 112 5.2 Methods ......................................................................................................................................... 114 5.2.1 Synthesis of JIB-04 .............................................................................................................. 114 5.2.2 Inhibition of KDM4A by JIB-04 ....................................................................................... 115 5.2.3 Docking Studies of KDM4A with JIB-04 Isomers ........................................................ 117 5.2.4 Crystallography ....................................................................................................................
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