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Biochemical Characterisation of Human Kifc1 and Eg5, Two Potential Targets for Drug Development in Cancer Chemotherapy

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Biochemical Characterisation of Human Kifc1 and Eg5, Two Potential Targets for Drug Development in Cancer Chemotherapy TableUCL of SCHOOL contents OF PHARMACY BRUNSWICK SQUARE Biochemical characterisation of human KifC1 and Eg5, two potential targets for drug development in cancer chemotherapy Chuin Lean Tham Thesis submitted in accordance with the requirements of UCL School of Pharmacy for the degree of Doctor of Philosophy 6th February 2018 UCL SCHOOL OF PHARMACY 29-39 Brunswick Square London WC1N 1AX 1 | P a g e Plagiarism statement Plagiarism statement This thesis describes research carried out in the University College London School of Pharmacy between January 2015 and February 2018 under the supervision of Prof. Frank Kozielski. I, Tham Chuin Lean, hereby confirm that the work presented in this thesis is my own. Any ideas, quotations, and paraphrasing from other people’s work and publications have been appropriately referenced. I have not violated the UCL School of Pharmacy’s policy on plagiarism. Signature: Date: 2 | P a g e Abstract Abstract Kinesins are ATP-dependent molecular motors that mostly travel unidirectionally along microtubules (MTs) to fulfil their roles in intracellular transport or cell division. Those involved in cell division, known as mitotic kinesins have been implicated in a variety of cancers. As they are crucial for cell division, certain mitotic kinesins such as Eg5 and KifC1 have been considered for their potential as novel drug targets. This is with the hope that these drugs will have fewer toxic side effects than conventional cancer therapies using taxanes and vinca alkaloids. Today, several inhibitors targeting Eg5 have entered Phase I, II and III clinical trials either as monotherapies or in combination with other drugs. As new members are continually being validated, KifC1 is also becoming increasingly important as a potential new target. My PhD thesis focuses on the characterisation of human mitotic kinesins KifC1 and Eg5, two potential targets for drug development in cancer chemotherapy. The first part of this thesis (chapter 3) covers the investigation of KifC1. Firstly, the development and optimisation of the protocols for the expression and purification of several KifC1 constructs covering full-length KifC1 is presented. Then, secondary structure and biophysical characterisation of each KifC1 domain is examined. Furthermore, biochemical characterisation of KifC1 is described and the kinetic properties of each construct in relation to the biophysical data such as oligomeric state is discussed. After this, attempts to crystallise the KifC1 motor domain are described. Finally, inhibitor screening of 15,000 small molecules for KifC1-specific hits are reported. The results provide a basis for future characterisation of KifC1. The second part of this thesis (chapter 4) involves the investigation of K858, a novel inhibitor of Eg5 bearing the 1,3,4-thiadiazole scaffold, an important core moiety in many clinical drug candidates targeting a variety of diseases. Biochemical and biophysical characterisation of K858 binding to Eg5 is reported. 1.8 Å resolution crystal structure of the Eg5-K858 complex (PDB ID: 6G6Y) is presented, providing the first structural evidence of how the thiadiazole containing scaffolds ARRY-520 is so successful in advancing into phase III clinical trials. Finally, structure-activity relationship (SAR) study of 22 K858 analogues is reported. 3 | P a g e Impact statement Impact statement This PhD research project presented very interesting data about the crystal structure of Eg5-K858 complex, delivering the first structural evidence of how ARRY-520 bearing a thiadiazole motif has become the first kinesin inhibitor to enter Phase III clinical trials with an outstanding anti-cancer activity. The crystal structure data is informative for structure-based drug design for developing more potent inhibitors that can potentially be evaluated in the clinical study. The financial impact of my research is potentially vast, in reducing both the time and the cost for lead identification and optimisation in the pre-clinical drug development. Finally, we have published the structural data thereby addressing the importance of focusing on the unique moiety of thiadiazole for drug development in cancer chemotherapy. 4 | P a g e Acknowledgements Acknowledgements First and foremost, I would like to express my appreciation to my supervisor, Prof. Frank Kozielski, for providing me with and co-funding this exciting PhD project. I would like to express my gratitude to “KifC1 team” Eleni Pavlou, Albino Gurung, and Sujan Shrestha who contributed their help throughout my PhD and we challenged the purification of KifC1 together. I would like to thank Aisha Alnami for her invaluable help for the protein crystallisation work particularly protein crystal dehydration, that eventually I have successfully crystallised the Eg5-Inhibitor complexes at high resolution. A special thank you to Shymaa Damfo for her friendship support and I miss the time we had dinner every Friday to chat about our PhD journey and life. Thank Dr. Isa Cruz for her supervision and guidance at the beginning of my PhD study. I would like to thank Dr. Rajshekhar Karpoomath and colleagues from the University of KwaZulu-Natal (South Africa) and Dr. Simone Carradori and colleagues from the University of Chieti-Pescara (Italy) for providing excellent opportunities for collaboration and we have published our exciting data, especially our solved crystal structure of Eg5-K858 complex, that has the key value for structural biologists and clinical scientists to improve the human health. Last but not least, I would like to express my appreciation to my family in Malaysia for love and support, I miss you all so much! To all my friends in UK especially Kar Mun Lau and Samantha Tock for making me feel less homesick. 5 | P a g e Lists of manuscripts Lists of manuscripts Co-first author publication Talapatra, S. K., Tham, C. L., Guglielmi, P., Cirilli, R., Chandrasekaran, B., Karpoormath, R., Carradori, S. & Kozielski, F. (2018). Crystal structure of the Eg5 - K858 complex and implications for structure-based design of thiadiazole-containing inhibitors. Eur J Med Chem, 156, 641-651 (IF = 4.8). Co-author publication Balakumar, C., Ramesh, M., Tham, C. L., Khathi, S. P., Kozielski, F., Srinivasulu, C., Hampannavar, G. A., Sayyad, N., Soliman, M. E. & Karpoormath, R. (2017). Ligand and structure based in silico studies to identify kinesin spindle protein (KSP) inhibitors as potential anticancer agents. J. Biomol. Struct. Dyn., 1-18 (IF = 3.1). Khathi, S. P., Chandrasekaran, B., Karunanidhi, S., Tham, C. L., Kozielski, F., Sayyad, N. & Karpoormath, R. (2018). Design and synthesis of novel thiadiazole-thiazolone hybrids as potential inhibitors of the human mitotic kinesin Eg5. Bioorg Med Chem Lett, 28, 2930-2938 (IF = 2.4). Manuscripts in preparation First author manuscript Chuin Lean Tham, Albino Gurung, Sujan Shrestha, Sandeep K. Talapatra, Frank Kozielski. Biochemical characterisation of human KifC1/HSET, a motor involved in centrosome clustering in tumour cells. Manuscript in preparation. Target journal: J. Biolg. Chem. (IF (2016) = 4.125). 6 | P a g e Table of contents Table of contents Table of Contents Plagiarism statement ........................................................................................................ 2 Abstract ............................................................................................................................. 3 Impact statement .............................................................................................................. 4 Acknowledgements ........................................................................................................... 5 Lists of manuscripts ........................................................................................................... 6 Table of contents .............................................................................................................. 7 Abbreviations .................................................................................................................. 10 1. Introduction ................................................................................................................ 13 1.1 Motor proteins ................................................................................................. 13 1.2 Kinesins ............................................................................................................. 15 1.3 Structural organisation of kinesins ................................................................... 17 1.3.1 Motor domain ........................................................................................... 17 1.3.2 Neck linker and neck coiled-coil ................................................................ 18 1.4 Kinesin mechanochemical mechanisms ........................................................... 19 1.5 Mitotic kinesins and cancer .............................................................................. 22 1.5.1 Tumour suppressor p53 and cancer .............................................................. 26 1.6 Inhibitor screening of mitotic kinesins ............................................................. 26 1.6.1 Mitotic kinesins as an attractive drug target ............................................ 26 1.6.2 Validating human Eg5 as a drug target .......................................................... 27 1.6.3 Human KifC1 as a potential drug target ......................................................... 30 2. Materials and methods ..............................................................................................
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