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The Discovery and Evaluation of Inhibitors of the Keap1-Nrf2 Protein-Protein Interaction

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The Discovery and Evaluation of Inhibitors of the Keap1-Nrf2 Protein-Protein Interaction THE DISCOVERY AND EVALUATION OF INHIBITORS OF THE KEAP1-NRF2 PROTEIN-PROTEIN INTERACTION Rowena Melanie Hancock July 2012 A thesis submitted for the degree of Doctor of Philosophy of University College London Department of Pharmaceutical and Biological Chemistry UCL School of Pharmacy 1 Plagiarism Statement This thesis describes research conducted at University College London, School of Pharmacy between October 2008 and October 2011 under the supervision of Dr Geoff Wells. I certify that the research described is original and that any parts of the work that have been conducted by collaboration are clearly indicated. I also certify that I have written all the text herein and have clearly indicated by suitable citation any part of this dissertation that has already appeared in publication. Signature Date 2 Table of Contents Acknowledgements ............................................................................................................... 7 Abbreviations ......................................................................................................................... 8 Abstract ................................................................................................................................. 11 1. Introduction: Keap1-Nrf2 protein-protein interaction ................................................. 12 1.1 Cancer ........................................................................................................................ 12 1.2 The Hallmarks of cancer .......................................................................................... 12 1.3 Carcinogenesis ......................................................................................................... 14 1.4 Chemoprevention ..................................................................................................... 15 1.5 Cancer chemopreventive agents ........................................................................... 16 1.6 Clinical trials of chemopreventive agents ............................................................. 17 1.7 The need for new chemopreventive agents ......................................................... 19 1.8 Xenobiotic metabolism and DNA protection ......................................................... 20 1.9 Natural products with cytoprotective properties ................................................... 21 1.9.1 Isothiocyanates .................................................................................................. 23 1.9.2 Diallyl sulphides ................................................................................................. 24 1.9.3 Phenolic compounds ........................................................................................ 25 1.9.4 Dithiolethiones ................................................................................................... 26 1.9.5 Flavonoids .......................................................................................................... 27 1.9.6 Carotenoids ........................................................................................................ 28 1.10 Nrf2 and Keap1 ....................................................................................................... 28 1.11 Structure of Keap1 and Nrf2 and their interaction ............................................. 30 1.12 Characterising the protein-protein interaction .................................................... 33 1.13 Sequestosome-1 (p62) .......................................................................................... 35 1.14 Prothymosin α ......................................................................................................... 38 2. Introduction: Protein-protein interactions .................................................................... 41 2.1 Protein-protein interaction misconceptions ........................................................... 41 2.2 Targeting protein-protein interactions .................................................................... 43 2.3 Approaches to design and generate PPI inhibitors ............................................. 44 2.4 Protein-protein interaction case studies ................................................................ 46 2.4.1 The discovery of a small-molecule inhibitor of the p53-MDM2 interaction – a PPI success story .................................................................................................... 46 2.4.2 Inhibition of the β-catenin/Tcf complex – an example of failure of small molecule design in targeting a PPI ........................................................................... 50 2.5 Conclusions and future perspectives..................................................................... 51 3 3. Introduction: Protein and Peptide drugs ...................................................................... 52 3.1 Insulin ......................................................................................................................... 52 3.2 Adaptation of peptides for oral administration ...................................................... 53 3.3 Drug development using a peptide as a lead compound ................................... 55 3.3.1 Case study: ACE inhibitors .............................................................................. 55 4. Project aims ..................................................................................................................... 58 5. Linear peptides to inhibit the Keap1-Nrf2 protein-protein interaction ..................... 63 5.1 Introduction to solid phase peptide synthesis ...................................................... 63 5.2 Results: Fluorescein-labelled peptides ................................................................. 66 5.2.1 Peptide synthesis .............................................................................................. 66 5.2.2 Characterisation of the final compounds ....................................................... 72 5.2.3 Biological evaluation ......................................................................................... 73 5.3 Competitive binding assays .................................................................................... 81 5.3.1 Synthesis of unlabelled peptide inhibitiors .................................................... 81 5.4 Modifications to the native ETGE sequence ........................................................ 86 5.4.1 Synthesis of inhibitors ....................................................................................... 86 5.4.2 Characterisation of the final compounds ....................................................... 87 5.4.3 Biological Evaluation ......................................................................................... 88 5.5 Further modifications to the native ETGE sequence .......................................... 91 5.5.1 Synthesis of inhibitors ....................................................................................... 92 5.5.2 Characterisation of the final compounds ....................................................... 92 5.5.3 Analysis of the final compounds ..................................................................... 92 5.6 Modifications to the phenylalanine residue ........................................................... 96 5.6.1 Synthesis of inhibitors ....................................................................................... 96 5.6.3 Analysis of the final compounds ..................................................................... 97 5.7 DLG motif peptides ................................................................................................... 98 5.7.1 Synthesis of inhibitors ....................................................................................... 99 5.7.3 Analysis of the final compounds ..................................................................... 99 5.8 Modifications to the native DLG sequence ......................................................... 100 5.8.1 Synthesis of inhibitors ..................................................................................... 100 5.8.2 Characterisation of the final compounds ..................................................... 100 5.8.3 Analysis of the final compounds ................................................................... 101 5.9 Inhibitors based on the binding sequences of Prothymosin α and Sequestosome-1 ........................................................................................................... 102 5.9.1 Analysis of the final compounds ................................................................... 102 4 5.10 Hybrid peptides of the ETGE and sequestosome-1 binding motifs .............. 106 5.11 Summary................................................................................................................ 108 6. Cell penetrating peptides ............................................................................................. 113 6.1 Arginine rich peptides ............................................................................................ 113 6.2 Amphipathic helical peptides ................................................................................ 113 6.3 Selecting a cell penetrating strategy .................................................................... 113 6.3.1 Synthesis of cell penetrating peptides ......................................................... 114 6.3.2 Characterisation and analysis of the final compounds
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