HIGH-THROUGHPUT METHODOLOGIES FOR ENZYME INHIBITOR PROFILING WU HAO (B.Sc., SHANDONG UNIVERSITY) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2011 DEDICATION I want to dedicate this thesis to my parents, Wu Xiyuan and Ding Yuan. Without you, I would not be where I am today. You gave me spiritual as well as materials support in my pursuit of knowledge and graduate studies. Your love and care cannot be compared by anything in the world. I also dedicate my work to my girlfriend, Miss Xie Shuyuan, your love, kindness, and encouragements have made this possible.  You are my primary supporter, counselor, friend, and crutch the whole way. i     ACKNOWLDGEMENTS I would like to express my deepest gratitude to my supervisor and mentor A/P Yao Shao Qin. He has brought me into the fascination of Chemical Biology and inspired me throughout my striving for scientific achievements. He has instilled with me unparalleled passion for scientific research, and empowered me to venture into unraveled territories in the Chemical Biology field. His indefatigable spirits, invaluable guidance and professionalism have been deeply engraved upon my mind, and they would be the gleams of light for me to shine through the gloom of scientific unknowns in the years ahead. I am extremely grateful for your mentorship. Beyond science, I admire your professionalism, kindness, and empathy; this truly motivates me to expect the same of myself. My sincere thankfulness and appreciation also extend to my lab-mates in both Chemistry and DBS - Mingyu, Raja, Pengyu, Haibin, Kalesh, Jingyan, Li Lin, Junqi, Chongjing, Xiamin, Zhenkun, Su Ying, Su Ling,   Ching Tian, Jiexun, Xiaohua, Mei Xuan, Mei Ying, Wendy, Li Bing, David, Derek, Joo Leng, Shuyun, Yuhui, Choon Meng, Liang Xian, Kaijia, Souvik, Mahesh, Lay Pheng, Liu Kai, Candy, Liqian, Wei Lin, Wee Liang, Grace, Farhana, Kitty, Cindy, Jigang, Zhengqiu and Xiaoyuan. I would like to take this opportunity to thank each of you for invaluable assistances, fruitful discussions and happy memories over these years. Special thanks goes to my collaborators, Jingyan helped me to carry out the final synthesis wok for 14-3-3 projects as well as part of aldehyde microarray work with cellular events profiling. Pengyu and Lay Pheng advised me in synthetic chemistry and fundamental biological experiments respectively. Pengyu also helped out with the synthesis work with ii     aldehyde microarray project. Jigang had participated in the proteomic work of protein targets identification. Dr. Mahesh revised and commended my journal publications. Their timely assistance, superior team-works, and earnest friendship helped me to tide over the most demanding periods of my research pursuit. Last but certainly not least, I would also like to thank Shandong University for affording the opportunity for studying abroad. I also acknowledge kind support from NUS, through the NUS Research Scholarship. iii     Table of Contents Page Chapter 1. Introduction 1.1 Abstract 1.2 Small Molecule Microarrays: The First Decade and Beyond 1.2.1 Library Design and Synthesis 1.2.1.1 Combinatorial Library Synthesis 1.2.1.2 Microarray Fabrication 1.2.2 Applications of SMMs 1.2.2.1 SMMs to Screen for Enzyme Activities 11 11 1.2.2.2 SMMs to Screen for Binding Profiles and Inhibitors 12 1.2.2.3 Successful Hits Identified Using SMMs 12 1.2.2.4 Recent Applications of SMMs 13 1.3 Summary and Outlook 14 1.4 Project Objectives 17 Chapter 2. A Peptide Aldehyde Microarray for High-Throughput Profiling Cellular Events 18 2.1 Abstract 18 2.2 Introduction 19 2.3 Results and Discussion 22 2.3.1 Synthesis of Peptide Aldehyde and Construction of the Corresponding SMMs 22 iv     2.3.2 Inhibitor Fingerprinting with Pure Enzymes on SMMs 24 2.3.3 Profiling Mammalian Cell Lysttes on SMMs 30 2.3.4 Parasite Lysates and Infected Erythrocytes 2.3.5 2.4 Screened on SMMs 33 Protein Target Identification and Validation 37 Conclusion Chapter 3. 39 Microarray-Assisted High-Throughput Identification of a CellPermeable Small Molecule Binder of 14-3-3 Proteins 41 3.1 Abstract 41 3.2 Introduction 42 3.3 Results and Discussion 45 3.4 3.3.1 Synthesis of Peptide-Small Molecule Hybrid Library 45 3.3.2 Identification of Hybrid Binder with 14-3-3 on SMMs 46 3.3.3 Synthesis Hybride Binders and Competitive Fluorescence Polarization Assay 50 3.3.4 in vitro and in vivo Activities of Compound 2-5 53 3.3.5 Hydrolytic Stability of 2-5 in Cellular Lysates 56 3.3.6 Docked Positions of 2-5 with 14-3-3σ 56 Conclusion Chapter 4. 57 High-Throughput Discovery of Mycobacterium Tuberculosis Protein Tyrosine Phosphatase (MptpB) Inhibitors Using Click Chemistry 59 4.1 Abstract 59 4.2 Introduction 60 4.3 Results and Discussion 61 v     4.3.1 Design and Synthesis of the Bidentate Inhibitors 4.3.2 High-Throughput Screening of Entire Library 4.3.3 4.4 against MptpB 63 Final Hits Selection and Biochemical Evaluations 71 Conclusion Chapter 5. 61 78 Solid-Phase Synthesis of Azidomethylene Inhibitors Targeting Cysteine Proteases 79 5.1 Abstract 79 5.2 Introduction 80 5.3 Results and Discussion 82 5.3.1 Design and Synthesis of Inhibitors 82 5.3.2 High-Throughput Screening of Entire Library against Caspases 5.4 Conclusion Chapter 6. 85 89 Experimental Procedures 90 6.1 General Information 90 6.2 Solution Phase Synthesis 92 6.2.1 General Procedure for Synthesis of Amino Aldehyde 92 6.2.2 Procedure of Synthesis of Immobilization Linker 94 6.2.3 Synthesis of Small Molecule “Hit” Compounds and Corresponding Controls Targeting 14-3-3 Protein 95 6.2.3.1 General Procedure for the Synthesis of Compound I 96 6.2.3.2 General Procedure for the Synthesis of Compound II 96 6.2.3.3 General Procedure for the Synthesis of vi     Compound 1-1 to 2-6 6.3 Solid Phase Synthesis 6.3.1 Procedure for Peptide Aldehyde Library Synthesis 97 103 103 6.3.1.1 Synthesis of Threonyl-Glycyl Resin (TG-resin) 103 6.3.1.2 Loading Fmoc-Amino-CHO onto TG Resin 104 6.3.1.3 Boc-protection of Secondary Amine in Oxazolidine Moiety 6.3.1.4 Peptide Synthesis 104 104 6.3.1.5 Side-chain Deprotection and Cleavage of Peptide Aldehyde from Resin 6.4 6.3.2 Procedure for Peptide Aldehyde Probe Synthesis 106 6.3.3 Synthesis of Peptide-Small Molecule Hybrid Libraries 107 Microarray Work 110 6.4.1 Preparation of Avidin Slides 110 6.4.2 Microarray Preparation for Peptide Aldehyde Library 111 6.4.3 Protein/Proteome Labeling and Screening on Peptide Aldehyde Microarray 6.4.4 6.4.5 112 Microarray Preparation for Peptide-Small Molecule Hybrid Library 6.5 105 114 14-3-3 Protein Labeling and Screening on Peptide-Small Molecule Hybrid Library Microarray 115 6.4.6 Data Extraction and Analysis 115 6.4.7 KD Analysis of Selected High Binders 116 6.4.8 Tyramide Singnal Amplification (TSA) Assay 117 Microplate Screening 117 vii     6.5.1 Microplate Screening of Aldehyde Sub-library against Caspase-3/-7/Cruzain/Rhodesain 117 6.5.2 Competitive Fluorescence Polarization Assay 118 6.5.3 Inhibition Assay against PTPs 118 6.5.4 Ca2+-actived Protease Assay 120 6.5.5 Screening for Inhibition Activity against Caspases 120 6.5.6 IC50 Measurements of Selected Inhibitors against Caspase-1/-3/-7 6.5.7 122 Ki Measurements of Selected Inhibitors against Caspase-1 123 6.6 Cell Culture 123 6.7 Western Blot 124 6.8 Small Molecule Competition Studies 124 6.9 Cell Proliferation Assay 125 6.10 Flow Cytometry Cell Cycle Analysis 126 6.11 Cell Permeability Assay 126 6.12 Live Cell Imaging 128 6.13 Pull-Down and Mass Spectrometry Identification 128 Chapter 7. 7.1 6.13.1 Pull-Down Assay 128 6.13.2 Mass Spectrometric Analysis 129 6.13.3 Mass Data Analysis 130 Concluding Remarks 132 Conclusion 132 Chapter 8. References 136 Chapter 9. Appendix 151 viii     9.1 Supplemental Tables 151 9.2 Supplemental Spectra 186 9.2.1 LC-MS profiles of DEVX-CHO sub-library 186 9.2.2 Selected LC-MS Profiles of 14-3-3 Hybrid Library 188 9.2.3 NMR Spectra of Compound (1-1 to 2-6) 189 9.2.4 ix     D18. Biotin-GG-DEVR-CHO [M/2+H]+   m AU(x1,000) 214nm ,4nm (1.00) [M/2+H]+   3.0 2.0 1.0 0.0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 m in Inten.(x100,000) 421.661 842.317 3.0 422.162 2.0 1.0 343.148 0.0 100 200 300 400 500 600 700 800 900 m /z 9.2.2 Selected LC-MS Profiles of 14-3-3 Hybrid Library C003 mAU 214nm,4nm (1.00) 3000 2000 1000 0.0 2.5 5.0 Inten.(x100,000) 6.0 7.5 10.0 12.5 15.0 534.751 5.0 4.0   3.0 2.0   1.0 391.292 0.0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m /z   C005 mAU 214nm,4nm (1.00) 3000 2000 1000 0.0   2.5 5.0 7.5 10.0 12.5 15.0 Inten.(x100,000) 546.758 2.5 2.0 1.5 1.0 518.245 404.699 395.671 0.5 0.0 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 188     m /z 9.2.3 NMR Spectra of Compound (1-1 to 2-6) O Cl N H H N O O OH N H OH 1-1       189         190         191           192       193         194         195             196         197         198             199         200         201             202           203     [...]... is not restricted to pure proteins and antibodies, but can also be performed with whole cells and crude lysates 1.2.2.1 SMMs to Screen for Enzyme Activities A variety of substrate microarrays have been applied for assessing the activity of enzymes For example, Ellman et al developed coumarin-based peptide microarray to functional profiling protease specificity.82 A similar concept was demonstrated using... Chapter 1 Introduction 1.1 Abstract Microarrays have carved an impressive niche for high- throughput research over the last two decades DNA microarrays, for instance, originally invented and applied in the 1990s, have now become one of the most dominant research tools for genetics and genomics, specifically in the areas of expression profiling, genotyping and copy number variation (CNV) analysis The turn of... using SMMs 15 2.1 Overall strategy of the SMM platform for comparative profiling of biological events 21 2.2 The diverse peptide aldehyde PSIL 23 2.3 Microarray profiles of the peptide aldehyde SMM with 4 different recombinant cysteine proteases 2.4 Microplate inhibitor specificity screening of cysteine proteases determined using the complete diverse inhibitor library 2.5 27 Representative examples... 143-3σ 57 xiv     4.1 Overall strategy of the “click-based” high- throughput discovery of MptpB inhibitors 62 4.2 Inhibition profiles of the click library against MptpB 65 4.3 Colored heatmaps displaying the potency of the ~3500 member PTP inhibitor library against MptpB 4.4 66 Bar graph representing the averaged inhibition potencies from inhibitors assembled using the seven warheads and the Type I... azidomethylene library against three caspases 87 5.4 Inhibition of identified “Hit” 88 5.5 IC50 graphs for other inhibitors against Caspase-1 88 6.1 The spotting format of the aldehyde microarrays used in Chapter 2 112 xv     List of Tables Table Page 1.1 Surfaces and tags developed for SMM fabrication 10 2.1 Inhibitor specificity of cysteine proteases present in this study 29 2.2 Proteins identified by pull-down... 4.2 Comparison between the top 50 inhibitors obtained from the seven sub-libraries 4.3 69 70 IC50 (in µM) and Ki of selected purified inhibitors against MptpB and other PTPs 72 4.4 IC50 plots of selected purified inhibitors against different PTPs 73 4.5 Ki plots of selected purified inhibitors against MptpB 75 4.6 Complete IC50, in µM, (and Ki in µM) of all purified inhibitors against MptpB, other PTPs... O-Benzotriazole-N,N,N’,N’-tetramethyl-uronium-hexafluorophosphate HCl Hydrochloric acid HEPES 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid HPLC High performance liquid chromatography HTS High- throughput screening Hz Hertz hr Hour(s) IC50 Half the maximal inhibitory concentration KD Dissociation constant Ki Inhibition constant LB Luria-bertani LC Liquid chromatography m Multiplet m/z Mass to charge... modes, effectively expanding current capabilities in protein profiling by leveraging on throughput These include small molecule microarray (Chapter 2 and 3) and microplate (Chapters 4 & 5) platforms Chapter 2 describes a novel peptide aldehyde microarray for rapid differentiation of infection stages in cellular level, and characterized the potential enzyme targets of identified compounds Chapter 3 presents... Thiazolidine Ring Formation Glyoxylyl 1,2-Amino Thiol16 Oxime Formation Glyoxylyl Aminoxyl16 Diazirine Various70,71 Aryl Azide Various56 Photolithography Amine Various72 Boronate Formation Boronic Acid Carbohydrate73 Sol-Gel Various74,75 Hydrogel Various76,77 Fluorous Capture C8F17 C8F1778-80 Hydrogen Bond Avidin Biotin50,51,58,81 Covalent Nucleophilic Reaction Schiff-base Formation Amide Formation Diels-Alder... Azidomethylene Inhibitors Targeting Cysteine Proteases”, Org Lett., (2008), 10, 1881 – 1884 2 Srinivasan; R.; Tan, L.P.; Wu, H.; Yao, S.Q.*, “Solid-phase and In Situ Screening of Protein Tyrosine Phosphatase Inhibitors”, Org Lett., (2008), 10, 2295 - 2298 3 Srinivasan; R.; Tan, L.P.; Wu, H.; Yang, P.-Y.; Kalesh, K.A,; Yao, S.Q.*, “HighThroughput Synthesis of Azide Libraries Suitable for Direct “Click” . HIGH- THROUGHPUT METHODOLOGIES FOR ENZYME INHIBITOR PROFILING WU HAO (B.Sc., SHANDONG UNIVERSITY) A THESIS SUBMITTED FOR THE DEGREE. 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid HPLC High performance liquid chromatography HTS High- throughput screening Hz Hertz hr Hour(s) IC 50 Half the maximal inhibitory concentration K D Dissociation. Summary and Outlook 14 1.4 Project Objectives 17 Chapter 2. A Peptide Aldehyde Microarray for High- Throughput Profiling Cellular Events 18 2.1 Abstract 18 2.2 Introduction 19 2.3 Results and Discussion