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Epigenetic Therapeutics in Malaria

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Epigenetic Therapeutics in Malaria Epigenetic therapeutics in malaria: A chemical biological approach towards the validation of histone lysine methyltransferase inhibition in Plasmodium falciparum Alexandra Lubin PhD Thesis Supervisor: Dr Matthew Fuchter Department of Chemistry, Imperial College London March 2018 Declaration of Originality I confirm that the work presented within this thesis is entirely my own, conducted under the supervision of Dr Matthew J. Fuchter, at the Department of Chemistry, Imperial College London, unless otherwise stated. All work performed by others has been acknowledged within the text and referenced where appropriate. Copyright Declaration The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. 2 Abstract A growing threat from drug resistance means there is an urgent need for new therapeutics and novel mechanisms of action for antimalarial drug discovery. Epigenetic mechanisms, including histone methylation, are vital throughout the Plasmodium lifecycle, and could provide exciting new targets. BIX-01294 and a series of diaminoquinazolines are putative Plasmodium histone lysine methyltransferase inhibitors, with exciting antimalarial properties, although robust evidence for their molecular targets is lacking. To this end, a well-developed SAR for this series allowed for the development of small-molecule photo-crosslinkable probes to investigate the targets. These probes effectively label Plasmodium falciparum lysates and show similarities with the target profiles of BIX-01294 and the diaminoquinazoline series. Initial pull-down proteomics experiments with two different probes identified 45 common proteins from different classes, many of which are essential, highlighting the suitability of the developed probes as valuable tools for target identification in Plasmodium falciparum. 3 Acknowledgements First and foremost, I would like to thank my supervisor Dr Matt Fuchter, for giving me the opportunity to work on this project and his continued support throughout. I have thoroughly enjoyed my PhD and have been lucky to carry out such interesting and varied research. I would like to thank Sandeep for helping so much when I first started, and Ainoa for helping develop the project and for her support whilst I wrote this thesis. Her help and support has been invaluable. I would also like to thank the whole Fuchter group, who have been helpful, supportive, and a lot of fun to be around, and especially Hannah, Luiza, Katie and Melis, who have kept me sane and made the last three years so enjoyable. A special thanks to the laboratory of Artur Scherf at the Pasteur Institute in Paris, who have collaborated heavily on this project throughout. Particular thanks to Patty, who provided invaluable time, materials, data and advice. I would also like to thank Professor Jake Baum and the members of his group, who allowed me space in their life sciences lab and helped me learn the biological techniques needed for this project, and Professor Ed Tate and his group for their help and support with the proteomics, and for gifting the valuable capture reagents used in this work. Finally, I would like to thank my mum for her continuous support, and David, without whom I could not have done this. 4 Contents Declaration of Originality ................................................................................................... 2 Copyright Declaration ........................................................................................................ 2 Abstract ............................................................................................................................. 3 Acknowledgements ........................................................................................................... 4 Abbreviations ..................................................................................................................... 9 1. Introduction .................................................................................................................. 11 1.1. Malaria .................................................................................................................. 11 Malaria Lifecycle ............................................................................................ 11 Current Treatments and Challenges .............................................................. 13 Future of Malaria Treatment ........................................................................... 15 1.2. Epigenetics and Malaria ........................................................................................ 16 Chromatin and Transcriptional Control ........................................................... 16 Transcriptional Control in Plasmodium ........................................................... 17 Post-Translational Modifications .................................................................... 19 Epigenetic Markers and Transcriptional Control in P. falciparum.................... 20 1.2.4.1. Antigenic Variation .................................................................................. 22 Epigenetic Machinery: Readers, Writers and Erasers .................................... 23 1.2.5.1. Acetylation .............................................................................................. 25 1.2.5.2. Methylation ............................................................................................. 27 Epigenetics and Drug Discovery .................................................................... 30 1.2.6.1. Epigenetic Therapeutics in Cancer ......................................................... 30 1.2.6.2. Targeting Epigenetics in Malaria ............................................................. 32 5 1.3. The BIX-01294 Compound Series ........................................................................ 34 Antimalarial activity of BIX-01294................................................................... 35 1.4. Proteomics ............................................................................................................ 37 Activity-based protein profiling ....................................................................... 38 Photo-crosslinking probes .............................................................................. 41 Mass Spectrometry Proteomics ..................................................................... 44 Examples of Probes for Malaria ..................................................................... 46 1.5. Project Aims.......................................................................................................... 48 2. Results and Discussion Part I: Development of the SAR .............................................. 49 2.1. Background........................................................................................................... 49 2.2. Expanding the SAR: Improving Pharmacodynamic Properties .............................. 52 Rationale ....................................................................................................... 52 General Synthesis of Diaminoquinazolines .................................................... 53 Synthesis ....................................................................................................... 54 Results........................................................................................................... 55 2.3. Expanding the SAR: Investigating Aromatic Substituents...................................... 58 Rationale ....................................................................................................... 58 Synthesis ....................................................................................................... 59 Results........................................................................................................... 60 2.4. Conclusions .......................................................................................................... 62 3. Results and Discussion Part II: Photo-crosslinkable Probes & Proteomics ................... 63 3.1. Probe Design and Synthesis ................................................................................. 63 Probe Design ................................................................................................. 63 6 Probe Synthesis ............................................................................................. 65 3.1.2.1. Probe 1 ................................................................................................... 65 3.1.2.2. Probe 2 ................................................................................................... 67 3.1.2.3. Probe 3 ................................................................................................... 68 3.1.2.4. Probe 4 ................................................................................................... 69 Probe Antimalarial Activity ............................................................................. 70 3.2. Lysate Labelling and In-Gel Fluorescence ............................................................ 71 Protocol Development ...................................................................................
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