ACTIVITY-BASED CHEMICAL PROTEOMICS PROFILING OF NATURAL PRODUCTS AND DRUG-LIKE SMALL MOLECULES YANG PENGYU (M Sc., Chinese Academy of Sciences) A THESIS SUBMITTED FOR THE DEGREE OF DOCTORATE OF PHILOSOPY DEPARTMENT OF CHEMISTRY NATIONAL UNIVERSITY OF SINGAPORE 2011 ii This thesis is dedicated to my parents, my wife and my daughter Acknowledgements It is my great pleasure to take this opportunity to express my acknowledgements to all the people who have helped or encouraged me during my PhD project First, I would like to express my deepest appreciation to my supervisor Prof Yao Shao Q., for his support and guidance throughout the research His continued support led me to the right way His intensity, creativity, passion and dedication to science is admirable Prof Yao has allowed me great freedom in developing projects to work on in his lab and has always been supportive of the collaborations that led to much of my work The diverse, interdisciplinary nature of Prof Yao’s research program is one of the things that drew me to his group in the first place, and I have learned much by being in such an environment Special thanks to my collaborators for their excellent work including: Wang Min and Prof He Cynthia Y for their patience and support from my first days learning T brucei to my final days working out these manuscripts; Mun Hong and Prof James M Lear for their contribution on library contruction Many researchers provided me their reagents or expertise I would like to thank them here: Prof Christopher J Chang (University of California, Berkeley) for the bacterial His-AGT plasmid, mammalian plasmids FLAG-Cox8A-SNAP, FLAG-H2B-SNAP, FLAG-KDEL-SNAP, FLAG-NK1R-SNAP, mCherry-Cox8A, and mCherry-KDEL; Dr Conor R Caffrey and Prof James H McKerrow (University of California, San Francisco) for cruzain and rhodesain as well as anti-rhodesain and iv anti-TbCatB; Prof Siu Kwan Sze (Nanyang Technological University, Singapore) for his support for LC-MS/MS experiments I am truly grateful to all the labmates past and present Especially I would like to thank Liu Kai, Raja, Wu Hao, Mingyu, Grace, Li Lin, Shen Yuan, Su Ling, and Mei Yin for their help and contribution I would like to thank Hongyan, Haibin, Lay Pheng, Liqian, Candy, Jinyan, Su Ying, Xiamin, Chongjing, Zhenkun, and Xiaoyuan for their friendship It is my great pleasure to study in such a friendly lab atmosphere I would like to thank the members of my thesis committee: Prof Lu Yixin, Prof Tan Choon Hong and Prof James M Lear for their input and helpful suggestions of my research projects I also acknowledge kind support from NUS for providing me research scholarship Thanks also go to Department of Chemistry administrative staffs for their supports, especially Suriawati Bte Sa'Ad for her help on all kinds of issues Finally, I am grateful to my family and friends for their constant support and encouragement I own my parents and parents-in-law for their endless love and support for my study I am in debt to my brother and my sister, who takes care of my parents when I study abroad I am deeply indebted to my wife Baiyun, for her endless love, support, and encouragement at all times Without her support, this thesis work is impossible My love to my daughter Guyu, cheers me up with her cute smile, her endless curiosity, her naughty requests, and sometimes even her cries I would like to thank my friends – Zhibin, Lifa, Yunben, Yujun and their families – for their friendship and help v Table of Contents Page Dedication iii Acknowledgements iv Table of Contents vi Summary x List of Publications xii List of Abbreviations xv List of Figures xxi List of Schemes xxv List of Tables xxvi Chapter 1: Introduction 1.1 Drug Target Identification – An Overview 1.2 Activity-Based Protein Profiling (ABPP) 1.2.1 Introduction 1.2.2 Activity-Based Probe Design 1.2.3 Bioorthogonal Chemistry in ABPP 10 1.2.4 Application of ABPP for Target ID of Natural Products 17 1.2.5 Application of ABPP for Imaging of Protein Activities 20 Chapter 2: Activity-Based Proteome Profiling of Potential Cellular Targets 24 of OrlistatTM Abstract 25 vi 2.1 Introduction 26 2.2 Results and Discussion 28 2.2.1 Design of Orlistat-like Probes 28 2.2.2 Retrosynthetic Analysis of THL-R 31 2.2.3 Synthesis of three Orlistat-like Probes 32 2.2.4 Effects on Cell Proliferation, Phosphorylation of eIf2 36 and Activation of Caspase-8 2.2.5 In Situ and In Vitro Proteome Profiling 39 2.2.6 Cellular Target Identification and Validation 43 2.2.7 Cellular Imaging 48 2.2 Conclusion Chapter 3: Chemical Modification and Organelle-Specific Localization of 50 51 Orlistat-Like Natural-Product-Based Probes Abstract 52 3.1 Introduction 53 3.2 Results and Discussion 56 3.2.1 Design of a Library of Orlistat-Like Probes 56 3.2.1 Synthesis of Sixteen Orlistat-Like Probes 59 3.2.2 Biological Screening 62 3.2.3 In Situ Proteome Profiling and Target Identification 63 3.2.4 Design and Synthesis of an AGT/SNAP-Orlistat 74 Bioconjugate as the Organelle-Targetable Probe vii 3.3 Conclusion Chapter 4: Parasite-Based Screening and Proteomic Profiling Reveal 80 82 OrlistatTM, an FDA-Approved Drug, as a Potential Anti-Trypanosoma brucei Agents Abstract 83 4.1 Introduction 84 4.2 Results and Discussion 88 4.2.1 Trypanocidal Activities of Orlistat-Like Probes in T 88 4.2.2 Comparative in Situ Proteomic Profiling of T brucei 96 4.2.3 Putative Target Identification and Validation of Both 101 brucei PCF and BSF Trypanosomes 4.2.4 Cellular Uptake and Morphological Changes of T brucei 109 upon Probe Treatment 4.3 Conclusion Chapter 5: Activity-Based Chemical Proteomics Cellular Target Profiling 115 117 of K11777, a Clinical Cysteine Protease Inhibitor Abstract 118 5.1 Introduction 118 5.2 Results and Discussion 120 5.2.1 Design of K11777-like Probes 120 5.2.1 Synthesis of K11777-like Probes 123 viii 5.2.2 Effects on Trypanocidal Activities of Probes 126 5.2.3 In Situ Proteome Profiling and Target Identification 127 5.2.4 Cellular Imaging 132 5.3 Conclusion Chapter 6: Design, Synthesis and Biological Evaluation of Potent 135 136 Azadipeptide Nitrile Inhibitors and Activity-Based Probes as Promising Anti-Trypanosoma brucei Agents Abstract 137 6.1 Introduction 138 6.2 Results and Discussion 142 6.2.1 Design and Synthesis of Aza-nitriles 142 6.2.2 Biological screening 147 6.2.3 Design and Synthsis of Activity-based Probes 154 6.2.4 In Situ Proteome Profiling 156 6.2.5 Cellular Imaging 162 6.3 Conclusion 166 Chapter 7: Concluding Remarks 168 Chapter 8: Materials and Methods 169 Chapter 9: References 288 Appendix 312 Appendix 323 Appendix CD ix Summary Assigning the cellular target(s) of bioactive small molecules, whether the compounds are discovered by cell-based phenotypic or target-based screens of chemical libraries, remains an ongoing challenge The ability of accurately and thoroughly determining of SMtarget interaction profiles as well as mapping of metabolic and signaling pathways on the proteomic scale would therefore be more illuminating, as it could provide invaluable biological insights for a drug candidate by both understanding the primary mechanism-of-action, and at the same time, side effects due to unexpected “off-target” interactions at a very early stage of drug development, which should help to reduce the attrition rate in development In many cases such a capability could find new potential therapeutic value for an established drug as well as it could also offer strong clues for compound optimization in order to maximize the therapeutic potential and minimize potential cellular toxicity of a drug The data may also serve to define previously unknown protein functions, based on the phenotypes induced by compounds Recent advances in chemical proteomics (or activity-based proteomics), a multidisciplinary research area integrating biochemistry and cell biology with organic synthesis and mass spectrometry, have enabled a more direct and unbiased analysis of a drug’s mechanism of action in the context of the proteomes as expressed in the target cell or the tissue of interest In this thesis, I describe the design and synthesis of OrlistatTM-like natural product-based probes (Chapter 2, & 4), K11777-like drug candidate-based probes (Chapter 5) and azanitrile-containing small molecules (Chapter 6), determination of x 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 ... describe the design and synthesis of OrlistatTM -like natural product -based probes (Chapter 2, & 4), K11777 -like drug candidate -based probes (Chapter 5) and azanitrile-containing small molecules (Chapter... 1.2 Activity- Based Protein Profiling (ABPP) 1.2.1 Introduction 1.2.2 Activity- Based Probe Design 1.2.3 Bioorthogonal Chemistry in ABPP 10 1.2.4 Application of ABPP for Target ID of Natural Products. .. Application of ABPP for Target ID of Natural Products Natural products, and their derivatives and mimics, have rendered an enormous contribution to the treatment of diseases such as infentions and cancers