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The Identification and Development of Small Molecule Inhibitors of Amyloid Β Aggregation

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The Identification and Development of Small Molecule Inhibitors of Amyloid Β Aggregation The Identification and Development of Small Molecule Inhibitors of Amyloid β Aggregation Súil Collins Downing College University of Cambridge September 2017 Supervised by Professor David R. Spring A dissertation submitted to the University of Cambridge for the degree of Doctor of Philosophy Abstract The Identification and Development of Small Molecule Inhibitors of Amyloid β Aggregation Súil Collins Amyloid β (1-42) (Aβ42) is a seminal neuropathic agent in Alzheimer’s disease (AD), a multifaceted neurodegenerative disorder for which no preventative measures or disease modifying therapies currently exist. Aggregation of this peptide plays a key role in the synaptic dysfunction and neuronal death associated with the disease. Perturbing the aggregation process, therefore, represents a key strategy for the development of new AD therapeutics. A variety of issues with current screening methods, including lack of reproducibility, high reagent consumption and spectral interference from the test molecules, can limit efforts to identify new small molecule inhibitors. Furthermore, the lack of robust, time- and cost-efficient methods for screening compounds in cellular or in vivo models limits the throughput with which seemingly active small molecules can be validated and prioritised. Herein, this thesis describes efforts to overcome such limitations through the development of a unified in vitro to in vivo assay system, in which hits identified in the ‘nanoFLIM’ microfluidic-based assay can quickly be tested in cellular and whole organism disease models. The assay platform designed relies on the use of an amyloid aggregation fluorescence lifetime sensor. Aβ42 aggregation is monitored by changes in the fluorescence lifetime of an attached fluorophore, which is significantly quenched upon amyloid formation. To take advantage of the benefits associated with miniaturisation, an in vitro microfluidic platform was employed. A microfluidic chip capable of trapping 110 precisely ordered droplets was designed, allowing for increased sample size and greatly lowering reagent consumption relative to conventional assay formats. Optimisation of the lifetime sensor technique permitted real-time compound screening in SH-SY5Y neuroblastoma cells, as well as in disease model Caenorhabditis elegans (C. elegans). To demonstrate the potential of this assay, a selection of novel chemical libraries developed in the Spring research group was screened, resulting in the identification of a key library of interest. The inhibitory activity of the lead compound from this collection was validated using a variety of biophysical tests, and was also shown to suppress amyloid aggregation in the live cell fluorescence lifetime sensor assay, as well as in whole organism disease model C. elegans. i Whilst assay development was underway, additional screening of structurally diverse chemical libraries was performed using a conventional Thioflavin T spectroscopic assay. Such work identified another molecular scaffold capable of exerting a strong inhibitory effect against Aβ42 aggregation. A selection of analogues was synthesised to improve the in vivo profile of this library, giving rise to a second lead inhibitory compound. The activity of this compound was subsequently validated in biophysical and cellular tests, and was also tested in disease model Drosophila melanogaster. The aggregation of Aβ42 lies at the root of Alzheimer’s disease. In light of the relatively few drug candidates in clinical trials for this disorder, the development of improved translational screening approaches and continued screening of novel chemical libraries is necessary to identify new potential therapeutics. In this study, an in vitro to in vivo fluorescence lifetime imaging assay has been established. Using this assay system and conventional screening approaches, two Aβ42 aggregation inhibitors have been identified and validated. These represent promising candidates for the development of new AD therapeutic agents, or for use as molecular probes to further dissect the mechanisms underlying this devastating disease. ii Declaration This dissertation is submitted in fulfilment of the requirements for the degree of Doctor of Philosophy. It describes work carried out in the Department of Chemistry, the Department of Biochemistry and the Department of Chemical Engineering and Biotechnology, at the University of Cambridge between June 2014 and September 2017, under the supervision of Professor David Spring. The work presented has not been submitted for any other degree. It does not exceed the prescribed word limit for the Physics and Chemistry Degree Committee. Signed, Date: _________________________ _____________________ Súil Collins Downing College, Cambridge iii iv To my family, with all my love and appreciation v vi Acknowledgements First and foremost, I would like to thank Prof David Spring for inviting me to join his research group and for giving me the freedom to develop a project that I feel so strongly about. I truly appreciate it. I would like to express my sincerest gratitude to Dr Gabriele Kaminski Schierle, her constant stream of ideas and unrivalled enthusiasm has been inspiring. Many thanks to Prof Florian Hollfelder, his guidance and constructive critique have kept me, and my project, on track. Prof Clemens Kaminski and Dr Damian Crowther also deserve special thanks, for taking the time to introduce me to microscopy and Drosophila studies, respectively. I greatly enjoyed all this work. I am very grateful to the BBSRC and the Cambridge Trust, for their generous funding and for giving me the opportunity to participate in such an exciting interdisciplinary programme. I have been incredibly lucky to work in three inspiring research groups, each filled with exceptional individuals, who freely gave their time and expertise to help me and my research. Thank you to all the Spring group members, past and present, for sharing your chemistry knowledge and for all the fun and laughter we’ve shared, both in the lab, and out. Sean, Steve, Sarah, Joe, Josie, Tommy, Twigg and Warren deserve special acknowledgement for many helpful discussions and day-to-day moral support. Thanks to the members of the Laser Analytics and Molecular Neuroscience Groups for their kindness, encouragement and invaluable input. I am indebted to Claire, Colin and Amberley for helping me to get to grips with the biological experiments, and to Romain and Weiyue for sharing their boundless FLIM knowledge with me. Thanks to everyone in the Hollfelder group, for their continuous help and advice, and for being such great friends. I would also like to express my deepest appreciation to Liisa, for welcoming me to the group with a smile, and to Fabrice, for his instrumental help in getting my project up and running. I am extremely grateful to the many wonderful friends, both old and new, who have made the last few years so special. Thanks to the basketball club for helping to keep me sane after long hours in the lab. Molly, Michelle and Tati – three-time Varsity champs – thank you for never accepting my attempts to retire. Thanks to Sylwia and Timo, my Cambridge family; Nicole, Sara and Alice for always being there for me with a smile and a hug; Yuteng, for letting me follow him to Cambridge; Eilíse, a breath of fresh Irish air when I needed one; Rita, for making my internship such an enjoyable one; Tom Cotton, my favourite Hot Numbers date; and Kerri, my partner-in-crime from early undergraduate years and hopefully for many years to come. To those who I have neither the space nor word-count to mention, I thank you for your patience, support and encouragement. vii I reserve special thanks for David, who came into my life during a period when my research was being especially uncooperative. Thank you for being so exceptionally tolerant and patient. Compassionate from miles away, never passive in the things you say. You make every day happier. I will finish with thanks to my family, for always setting the bar high and giving me the space and encouragement to rise to the challenge. Much love and thanks to both my grannies, for providing inspirational examples of strong and determined women. Thanks to Aingeal, our surrogate auntie and wonderful friend. Leanbh, Eibh, and Tom, thank you for your support at every step of the journey. Your motivational messages, random jokes and Christmas countdown reminders have kept me smiling. Mariusz, Gar and Doc, thank you for providing family support, when I was otherwise distracted. Mum and dad, you deserve more thanks than I can ever put in words. Thanks to Dad for giving me my first science kit, and to mum for taking away all the dangerous pieces. Your constant love and unfaltering support have filled me with the hope, joy, and the confidence to be the best that I can be. I am eternally grateful to you both. A final thanks to my new baby niece, who has kindly waited to make her debut until after I’ve submitted. We’re ready for you now, Dotty. viii Abbreviations 3D three-dimensional °C degrees Celsius Å Ångström(s) μ micro λ wavelength τ fluorescence lifetime Aβ amyloid β peptide Aβ40 amyloid β peptide 40 residues in length Aβ42 amyloid β peptide 42 residues in length Aβ42-488 synthetic Aβ42 labelled with Hilyte™ Fluor 488 AD Alzheimer’s disease ADME absorption, distribution, metabolism, and excretion AFM atomic force microscopy APP amyloid precursor protein approx. approximately a.u. arbitrary units BACE beta-secretase BBB blood brain barrier (c)LogD (calculated) octanol-water distribution coefficient
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