University of East Anglia School of Pharmacy Design and synthesis of novel classes of HDACs and KMTs inhibitors by Remy Thomas Narozny Supervisor: Prof. A. Ganesan Second Supervisor: Prof. Mark Searcey Thesis for the degree of Doctor of Philosophy November 2018 This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived therefrom must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. “Your genetics is not your destiny.” George McDonald Church Abstract For long, scientists thought that our body was driven only by our genetic code that we inherited at birth. However, this determinism was shattered entirely and proven as false in the second half of the 21st century with the discovery of epigenetics. Instead, cells turn genes on and off using reversible chemical marks. With the tremendous progression of epigenetic science, it is now believed that we have a certain power over the expression of our genetic traits. Over the years, these epigenetic modifications were found to be at the core of how diseases alter healthy cells, and environmental factors and lifestyle were identified as top influencers. Epigenetic dysregulation has been observed in every major domain of medicine, with a reported implication in cancer development, neurodegenerative pathologies, diabetes, infectious disease and even obesity. Substantially, an epigenetic component is expected to be involved in every human disease. Hence, the modulation of these epigenetics mechanisms has emerged as a therapeutic strategy. Histone deacetylases (HDAC) are silencing epigenetic markers involved in the mediation of the acetylation system. Aberrant HDAC activities have been associated with the development of a broad range of pathologies, and HDAC inhibition is highly regarded as a potential therapeutic target. This approach became successful with the approval by the FDA of several epidrugs. However, they present undesired side effects. Lack of selectivity was identified as a principal suspect and, therefore, the development of novel compounds that would target more selectively the epigenetics enzymes represent a major axis of research for future pharmacological applications. Accordingly, this work focus on designing new classes of HDAC inhibitors. A variety of structures, build around the use of amino acids, were explored and potential inhibitors were produced with different cores. These series were then evaluated in enzymatic inhibition assays on HDAC and in cell growth inhibition assays on leukaemia cell lines. Additionally, we were also involved in the international consortium A-ParaDDisE that aimed to develop epigenetic modulators as drug candidates against the main parasitic diseases: malaria, schistosomiasis, leishmaniasis and Chagas disease. This collaboration resulted in the identification of critical targets for the different parasites and the development of testing methods to move compounds toward clinical development. The antischistosomal effect of some of our inhibitors was, thus, evaluated. Finally, a side project was carried at the University of Salerno in Italy in a short-term scientific mission that aimed to develop a new class of SETD8 methyltransferase inhibitors. 1 Acknowledgements I must first acknowledge my supervisor Prof. Ganesan who offered me this fantastic opportunity to develop a research project. Thanks, Prof. Ganesan for the stimulating discussions, suggestions and guidance during this PhD work and for the numerous opportunities to attend conferences that highly contributed to my development as a researcher. I especially thank you for the support, over these four years, not only on the project but also on the pursuit of my career. I also thank my second supervisor Prof. Mark Searcey for his support during this project and his help in grant applications. I want to thank Prof. Gianluca Sbardella, from the University of Salerno in Italy, for hosting a research visit for three months. I would also like to thank the member of his group for their warm welcome and their help during this mission and in particular Dr Ciro Milite for his support on this part of my PhD work. Sincere thanks also to Prof. Manfred Jung (University of Freiburg, Germany), Prof. Laurent Bischoff (University of Rouen Normandy, France), Prof. Wolfgang Sippl (Institute of Pharmacy at the Martin-Luther University of Halle-Wittenberg, Germany) and Dr Maria Teresa Borrello (INSERM U1068 Cellular Stress Group, Cancer Research Center of Marseille, France) who contributed to this thesis by operating the biology testing and the molecular docking. I thank the University of East Anglia and the A-ParaDDisE consortium for funding this research project. I would also like to thank the COST action Epichembio for financially supporting my short-term scientific mission at the University of Salerno in Italy. In addition, I express my gratitude to the previous and current members of Prof. Ganesan research group and particularly Teresa Borrello, Carys Thomas, Adam Lee, Mohammed Aldholmi and Philip Schuler for the friendly working environment, the enriching conversation, and the support over these four years. Many thanks as well the undergraduate student who contributed to this project: Alex Chan, Angus Yuen, Faustin Falissard, Karolina Sliwa and Ludmilla Sturm. I am incredibly grateful to my colleagues and friends Mahmoud, Quim, Sara and Hui for their support and all the great deal of time spent together. Thanks, Andrew and Ryan, for your help in the lab, the great football games and the shared drinks. Thank you, Elise, for the cheerful atmosphere and enthusiasm you created in the office, and for being a fantastic geeky friend who always shares my over-excitement for movies, series, comics and more importantly cakes. Thanks to the 2015 survivors group Serena, Valeria, Noe and Liao for our dinners and talks. Thanks, Sarah for all the good moments (although I lost auditory acuity). Thank you Mar and Gerard for the cuckoo times, barbecue, Christmas dinner and all the laughs. Silvia, Foodie, thanks for your 2 support, our discussions, our laughs and your trust. I’m sure you will be there soon. Finally, thank you Lucka for our long runs and endless debate about life, dreams, science, Mars; thanks for the gif talks, the cinematic exchanges, the crazy snaps and your support. With a special mention to my flat 5 family: Bianca, Martina, Bela, Rodrigo, Carter and Pedro. It was fantastic to join your flat when I arrived in Norwich, and I genuinely enjoyed our time there. What a cracking place to live! I hope we will meet again. Enfin, je dédie cette thèse à ma famille, sans qui rien n’aurait été possible. Même si vous ne compreniez pas toujours ce que je voulais faire ni où j’allais, merci de m’avoir toujours soutenu malgré tous les déménagements, les expatriations, et les longues absences. Je n’aurais jamais accompli ce rêve sans vous. 3 Contents Abstract ................................................................................................................................... 1 Acknowledgements .................................................................................................................. 2 List of Figures .......................................................................................................................... 7 List of Scheme ........................................................................................................................10 List of Tables ..........................................................................................................................13 Abbreviations..........................................................................................................................15 Chapter 1. Introduction ...........................................................................................................23 1. Epigenetics .....................................................................................................................23 2. Chromatin .......................................................................................................................24 3. Epigenetic modulators: DNA methylation and histone post-translation modifications ......26 3.1 DNA methylation ......................................................................................................27 3.2 Histone post-translational modifications ....................................................................28 Chapter 2. HDACs and SETD8 inhibitors: overview and perspective. ......................................39 1. SETD8 inhibition ............................................................................................................39 1.1 Introduction ..............................................................................................................39 1.2 Therapeutics applications ..........................................................................................39 1.3 SETD8 inhibitors ......................................................................................................43 1.4 Summary ..................................................................................................................59 2. HDAC inhibition .............................................................................................................59 2.1 Introduction ..............................................................................................................59