DISCOVERY AND DEVELOPMENT OF SMALL- MOLECULE MODULATORS FOR THE SULFATION OF GLYCOSAMINOGLYCANS and STUDYING THE ROLE OF O-GLCNAC ON CREB THROUGH SEMISYNTHESIS

Thesis by Sheldon Ting Fong Cheung

In Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

CALIFORNIA INSTITUTE OF TECHNOLOGY Pasadena, California

2017 (Defended September 22, 2016) ii

 2017

Sheldon T. Cheung All Rights Reserved iii ACKNOWLEDGEMENTS

First and foremost, I would like to thank my advisor, Prof. Linda Hsieh-Wilson, for

her support and guidance throughout the whole process. Without Linda, I would not have

been able to work on such amazing research and accomplish the work reported here. She

challenged me to be the best scientist possible and I would not be the scientist I am today

without her direction. I would also like to thank my committee members, Prof. Bil Clemons,

Prof. Peter Dervan, and Prof. Dennis Dougherty. Their scientific advice and support

throughout my time here have also been instrumental to my success. Additionally, I would

like to thank all Hsieh-Wilson lab members, past and present. I have been lucky to have such

great colleagues to work with every day that can give day-to-day help and make the graduate

school process that much more enjoyable. I would also like to thank my family: my mom,

dad, brother Sab, sister Liv, mother-in-law Lai-Yoong, father-in-law Chee-Pun, and sister-

in-law Xiao-Xiang. They have all not only been supportive of my studies 3,000 miles away, but have been part of a support network for all parts of my life as well. Last, but certainly not least, I would like to thank my wife Xiao-Lan and son Lewis. They are everything in the world to me and their loving support was what truly allowed me to get through this trying process.

iv ABSTRACT

Glycosaminoglycans (GAGs) are sulfated polysaccharides that play key roles in

many cellular processes, ranging from viral invasion and cancer metastasis to neuronal

development. Their diverse biological activities stem from their complex sulfation patterns, which are tightly regulated in vivo. For instance, the GAG chondroitin (CS) has been shown to undergo regiochemical sulfation during development and after spinal cord injury.

However, few tools exist to modulate specific GAG sulfation patterns and study their importance in different biological contexts. Here, we identified the first cell-permeable small molecule that can selectively inhibit GAG and modify the fine structure of

GAGs. We demonstrate that the inhibitor reduces GAG sulfation in vitro and in cells and reverses CS-E-mediated inhibition of neuronal outgrowth. This small molecule may serve as a useful lead compound or chemical tool for studying the importance of CS and other GAGs in normal biology and disease.

The β-N-acetyl-D-glucosamine (O-GlcNAc) post-translational modification plays a major role in many diseases such as cancer, diabetes, and neurodegenerative disorders, but much is still unknown about its molecular-level influence on protein structure and function.

Although post-translational modifications have been known to induce important structural changes in proteins, notably, no structures of O-GlcNAcylated proteins exist. The challenge of obtaining homogeneous glycoproteins bearing the GlcNAc sugar at defined sites has hindered the structural and biochemical studies of this modification. Here we have utilized a semisynthetic approach to generate a homogeneously O-GlcNAcylated form of cyclic-AMP response element binding protein (CREB) for structural and functional studies.

v TABLE OF CONTENTS

Acknowledgements ...... iii Abstract ...... iv Table of Contents ...... v List of Figures, Tables and/or Schemes ...... vi List of Abbreviations ...... ix Chapter 1: Introduction Glycosaminoglycans and the Role of Sulfation ...... 1 References ...... 14 Chapter 2: Chst15-Antagonist High-Throughput Screen and Validation ...... 18 Introduction ...... 19 Results and Discussion ...... 19 Conclusion ...... 27 Experimental Methods ...... 28 References ...... 32 Chapter 3: Structure-Activity Relationship Analysis of Lead Compounds ...... 34 Introduction ...... 35 Results and Discussion ...... 36 Conclusion ...... 41 Experimental Methods ...... 42 References ...... 69 Chapter 4: Mechanistic and Functional Characterization of Compound 37 ...... 70 Introduction ...... 71 Results and Discussion ...... 72 Conclusion ...... 86 Experimental Methods ...... 87 References ...... 94 Chapter 5: Introduction to O-GlcNAc and Studying its Role in Structure and Function ...... 96 References ...... 105 Chapter 6: Examining the Role of O-GlcNAc on CREB through Semisynthesis ...... 109 Introduction ...... 110 Results and Discussion ...... 110 Conclusion ...... 119 Experimental Methods ...... 120 References ...... 132 Appendix A: Synthesis of Uridine Diphosphate 6-Azido D-Galactose ...... 134 Experimental Methods ...... 135

vi LIST OF FIGURES, TABLES, AND/OR SCHEMES

Chapter 1: Introduction to Glycosaminoglycans and the Role of Sulfation

Figure 1.1 Structures of the major classes of sulfated glycosaminoglycans ...... 3 Figure 1.2 Four major sulfation motifs of chondroitin sulfate ...... 5 Figure 1.3 Illustrative representation of chondroitin sulfate biosynthesis ...... 9

Chapter 2: Chst15-Antagonist High-Throughput Screen and Validation

Figure 2.1 High-throughput assay for the discovery of Chst15 small molecule inhibitors ...... 20 Figure 2.2 Radioactive isotope labeling assay and Chst15 mutational effects on activity ...... 21 Figure 2.3 Optimization of high-throughput assay by fluorimetry ...... 23

Figure 2.4 PAPS and CS-A substrate Km determination by fluorimetry ...... 24 Figure 2.5 Identity of small molecule hits from the fluorescent high-throughput screen ... 25 Figure 2.6 Validation of hit compounds via the fluorescence assay ...... 26 Figure 2.7 Validation of candidate compounds by radioactive isotope labeling assay ...... 27

Chapter 3: Structure-Activity Relationship Analysis of Lead Compounds

Figure 3.1 Comparison of chemical analogs to lead compound 5 (Part I) ...... 37 Scheme 3.1 Synthesis of compound 19 analogs (Route 1) ...... 38 Scheme 3.2 Synthesis of compound 19 analogs (Route 2) ...... 38 Table 3.1 Chst15 Inhibition Relative to 19 of Anilide-Analogs at 25 µM…………… ...... 40 Table 3.2 Chst15 Inhibition Relative to 19 of Vinylbenzene-Analogs at 25 µM ...... 40 Figure 3.2 Comparison of additional chemical analogs to lead compound 19 ...... 41

vii Chapter 4: Mechanistic and Functional Characterization of Compound 37

Figure 4.1 Compound 37 selectively inhibits glycosaminoglycan sulfotransferases...... 73 Figure 4.2 Steady-state kinetic analysis reveals competitive and mixed modes of Chst15 inhibition by 37 ...... 74 Table 4.1 Kinetic parameters of inhibition for Chst15 ...... 75 Figure 4.3 Compound 37 exhibits reversible-covalent inhibitor properties when using βME as a model nucleophile ...... 76 Figure 4.4 Compound 37 inhibitory activity depends on pre-incubation time and shows reversibility ...... 77 Figure 4.5 Representation of astrogliosis after neuronal injury and the potential therapeutic mechanism of small molecule treatment ...... 78 Figure 4.6 Treatment of NIH/3T3 fibroblasts and Neu7 astrocytes with 37 leads to a significant, dose-dependent decrease in CS-E expression ...... 79 Figure 4.7 Treatment of NIH/3T3 fibroblasts with 37 leads to a significant, dose-dependent decrease in HS expression ...... 80 Figure 4.8 CSPGs from Neu7 astrocytes treated with inhibitor 37 show decreased levels of CS-E and other sulfation motifs ...... 81 Table 4.2 Quantification of the percent disaccharide composition of CSPGs secreted from untreated and treated astrocytes ...... 81 Figure 4