Repurposing Drug Scaffolds: A Tool for Developing Novel Therapeutics with Applications in Malaria and Lung Cancer A Thesis Submitted by: Hannah Elizabeth Cook In partial fulfilment of the requirements for the degree of: Doctor of Philosophy September 2018 Supervisors: Professor Matthew J. Fuchter & Professor Anthony G. M. Barrett Department of Chemistry Imperial College London 2 Declaration of Originality I, Hannah Cook, hereby confirm that the work presented within this thesis is entirely my own, conducted under the supervision of Professor Matthew J. Fuchter and Professor Anthony G. M. Barrett, 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. Hannah E. Cook September 2018 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. 3 Abstract The definition of repurposing in the context of drug discovery encompasses a variety of strategies designed to redirect current therapeutic knowledge towards new disease indications. This approach can be successful for the design of new drugs to treat diseases of the developing world such as Malaria, where there are limited resources to fund new drug discovery campaigns. Moreover, it can be used to decrease the drug development time for diseases in which there is high drug attrition rates coupled with high mortality rates, which is the case for some cancers. To this end, two separate medicinal chemistry projects are presented herein which are both based on current drug scaffolds originally used for alternative therapeutic indications. The first is the design of an inhibitor of Plasmodium Falciparum Myosin A (PfMyoA), a motor protein implicated in the blood stage invasion of Malaria. Structural predictions of the myosin structure have been made in the form of a homology model using the crystal structure of a well characterised homologue, myosin II and its inhibitor, (S)-Blebbistatin. Plasmodium specific inhibitors were designed in silico based on the structure of (S)-Blebbistatin and synthesised for validation of the model using biological testing of their effect on myosin activity and importantly, on parasite invasion. Interestingly, compounds were found to reduce parasitaemia in red blood cells, independently of PfMyoA, suggesting interaction with an alternative essential myosin. The second project described is the synthesis of compounds that inhibit 90-kDa ribosomal S6 kinase 4 (RSK4), a promotor of lung cancer metastasis and resistance to chemotherapeutic treatments. Inhibitors were synthesised based on the structures of fluoroquinolone antibiotics Moxifloxacin and Trovafloxacin which were identified as moderately potent inhibitors of RSK4. Derivatives were designed with enhanced solubility and tested in a cell-based assay in which 10 novel compounds were identified that inhibit RSK4 activation by at least 50%. 4 Acknowledgements First and foremost, I would like to thank my two supervisors, Professor Matthew Fuchter and Professor Anthony Barrett for granting me the opportunity to work on two interesting and unique projects, and for their continued support throughout my PhD. I would also like to thank Jochen Brandt and Sandeep Sundriyal for their guidance and reassurance in my early days in the lab. In addition, special thanks must also go to Ainoa Zubiaurre who has always gone the extra mile to help me, especially during my write up period. I’d also like to thank Professor Jake Baum and his group at Imperial College for their work on the biological aspects of the Myosin A project. In particular, I thank Tom Blake and Linda Makhlouf who carried out the biological assays and provided me with their invaluable insights. Thanks also to Dr Olivier Pardo and Professor Michael Seckl for offering their expertise and guidance at the beginning of the RSK4 project. Thanks should also go to the analytical staff at Imperial College; Dr Lisa Haigh, Dick Sheppard and especially Pete Haycock who kindly ran samples for me during the hectic departmental move to White City. During my PhD I have had the pleasure to work with many people from the Fuchter and Barrett group, and I would like to extend my thanks to past and present members for keeping me sane over the years. A special mention must go to Alex, Luiza, Katie and Melis who have been with me since the beginning and have supported (and commiserated with) me the whole way through. Finally, I’d like to thank George for his endless patience and belief in me, and my family for their continued encouragement. It has been a long journey and I am so grateful to have had you all by my side. 5 Contents Declaration of Originality ....................................................................................................... 3 Copyright Declaration............................................................................................................ 3 Abstract................................................................................................................................. 4 Acknowledgements ............................................................................................................... 5 Abbreviations ...................................................................................................................... 10 1. General Introduction .................................................................................................... 14 1.1. Repurposing Strategies in Drug Discovery ............................................................ 14 1.1.1. Target Repurposing ....................................................................................... 15 1.1.2. Drug Repurposing .......................................................................................... 17 Investigating Plasmodium Falciparum Myosin A ................................................................. 19 2. Introduction .................................................................................................................. 20 2.1. Malaria .................................................................................................................. 20 2.1.1. Malaria Cause and Transmission ................................................................... 20 2.1.2. The Invasive Parasite: Merozoites ................................................................. 22 2.1.3. Antimalarials Past and Present ...................................................................... 23 2.1.4. Challenges to Current Treatment ................................................................... 26 2.2. Myosin: The Muscle Motor Protein ........................................................................ 27 2.2.1. Structure and Function ................................................................................... 27 2.2.2. Inhibitors of Myosin ........................................................................................ 31 2.2.3. (S)-Blebbistatin .............................................................................................. 33 2.2.4. Limitations and Derivatives of (S)-Blebbistatin ............................................... 34 6 2.3. Plasmodium Falciparum Myosin A ........................................................................ 38 2.3.1. Parasite Invasion ........................................................................................... 38 2.3.2. The PfMyoA Motor Complex .......................................................................... 40 2.4. (S)-Blebbistatin and Plasmodium Falciparum Invasion ......................................... 42 2.5. Project Aims.......................................................................................................... 44 3. Results and Discussion ................................................................................................ 45 3.1. Part 1: Development of a Homology Model ........................................................... 46 3.1.1. Structure Based Drug Design ........................................................................ 46 3.1.2. Generation of a PfMyoA Homology Model ..................................................... 48 3.1.3. Redesigning (S)-Blebbistatin .......................................................................... 52 3.2. Part 2: Synthesis of 1st Generation (S)-Blebbistatin Analogues ............................ 58 3.2.1. Synthetic Route A – Hydroxylation and Lactam Reduction ............................ 58 3.2.2. Synthetic Route B – Cyanohydrin Formation and N-Arylation ........................ 65 3.2.3. Synthetic Route C – Amide Derivatives.......................................................... 69 3.2.4. Synthetic Route D – Acyl Anion Equivalents for Ketone Synthesis ................. 80 3.2.5. Synthetic Route E - Ketone Derivatives ......................................................... 86 3.3. Part 3: Synthesis of 2nd Generation (S)-Blebbistatin Analogues ........................... 90 3.3.1. A Structural Biologist’s Perspective ................................................................ 90 3.3.2.