IN-SILICO INVESTIGATION OF COENZYME A SELECTIVITY FOR AURORA A KINASE AND DEVELOPMENT OF AN AURORA A KINASE- SELECTIVE INHIBITOR AS A POTENTIAL ANTICANCER AGENT. Trang M. Tran Ph.D Supervisor: Prof. Alethea B. Tabor Submitted in partial fulfilment of the requirements of the Doctor of Philosophy in Chemistry of University College London. NOVEMBER 1, 2016 UCL Declaration I, Trang M. Tran, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. London, 1st May, 2016 Trang M Tran 1 Abstract Centrosome amplification has been observed in most cancer cells, and is considered to be a “hallmark” of cancer cells. This process is commonly associated with chromosome segregation process in the mitosis phase in the cell cycle, which is tightly controlled by mitotic kinases. Among these kinases, the Aurora kinase family, Aurora A (AURKA), Aurora B (AURKB) and Aurora C (AURKC), ensures the accurate progression of mitosis, including the formation of a bipolar mitotic spindle, accurate segregation of chromosomes and the completion of cytokinesis. AURKA has been seen to have the largest role in mitotic progression and checkpoint control pathways and overexpression of AURKA is most associated with cancer. Hence, interfering with AURKA activity has been considered to be a promising approach to anticancer agents. Professor Gout’s group has recently shown that Coenzyme A (CoA) selectively inhibits AURKA activity (IC50 of 4.4 µM). However, its large molecular weight (>500) and negatively charged phosphate group make it unsuitable as a drug candidate. This project was set to investigate the possible binding modes of CoA in the catalytic domain of AURKA. The corresponding interactions between CoA and protein residues would provide some insights in the selectivity of CoA towards AURKA. Furthermore, based on the understanding of the interactions between CoA and the catalytic domain of AURKA and the possible reasons behind the selectivity of CoA towards AURKA, in silico design and synthesis of a new highly selective and potent AURKA inhibitor based on the structure of CoA and current lead compounds which are in clinical trials for Aurora kinase inhibitors could be carried out. 2 Acknowledgement I would like to thank my supervisor, Prof. Alethea B Tabor and Prof. Ivan Gout for the opportunity to work on this project. I would like to thank Dr. Edith Chan for her guidance and support in computational study. I would like to thank Dr. Tsuchiya for his support in biological images and data. I would like to thank Ms. Fiona Bellany for her collaboration in synthetic chemistry throughout my research and Ms. Yueyang Huang for her assistance in pharmacological testings. I would like to thank Dr. Richard Bayliss and Dr. Patrick Eyers and Mr. Patrick McIntyre – for their collaborative work in crystallography. I would like to thank Dr. Lalitha Guruprasad for the preliminary docking studies. I would also like to gratefully acknowledge Dr. Kersti Karu and Dr. Lisa D Haigh’s help with running mass spectroscopy samples, Dr. Abil E Aliev for his help with NMR analysis and members of Tabor’s and Hailes’ research groups for their help and advice. My gratitude also goes to all the past and present members who have been working on the projects. Funding. I would like to express my gratitude to UCL for the generous fundings of UCL Graduate Research Scholarship and UCL Overseas Research Scholarship. 3 TABLE OF CONTENTS Declaration ................................................................................................................................ 1 Abstract ..................................................................................................................................... 2 Acknowledgement .................................................................................................................... 3 Abbreviations ............................................................................................................................ 6 1. Introduction of biochemistry background and thesis proposal ...................................... 8 1.1. An introduction on cancer ............................................................................................ 8 1.2. Protein kinases for cancer therapy .............................................................................. 15 1.3. Aurora A kinase (AURKA) for cancer therapy ......................................................... 25 1.4. Biochemical findings ................................................................................................. 38 1.5. Thesis proposal ........................................................................................................... 43 1.5.1. Approach A – The first generation of compounds .............................................. 44 1.5.2. Approach B – The second generation of compounds .......................................... 46 2. Computational study and design ..................................................................................... 47 2.1. Backgrounds on proteins and methods of modelling .................................................. 47 2.1.1. Backgrounds ....................................................................................................... 47 2.1.2. Materials and methods of modelling ................................................................... 54 2.2. Backgrounds on computational tools .......................................................................... 62 2.2.1. PROCHECK for Ramachandran plot ................................................................ 62 2.2.2. MOE for molecular visualisation and amino acid alignment and modification 62 2.2.3. Other bioinformatics tools .................................................................................. 62 2.3. Results and Discussion on the selectivity of CoA towards AURKA .......................... 63 2.3.1. Structural alignment of selected protein kinases for structural comparison ...... 63 2.3.2. Docking results and discussion of the conformation of CoA in AURKA ............ 72 2.4. Ab initio design of a new AURKA selective inhibitor ................................................ 90 2.4.1. Re-examination of preliminary docking results in the 1st generation of compounds .......................................................................................................................... 90 2.4.2. Methods of new design for the 2nd generation of compounds ............................. 93 4 2.5. Conclusion ................................................................................................................ 105 3. Approach A syntheses ..................................................................................................... 107 3.1. Synthesis of the first generation of compounds ........................................................ 107 3.1.1. Literature research and synthetic plan of the 1st generation of compounds ..... 107 3.1.2. Synthetic results of pantothenamide tail ........................................................... 110 3.1.3. Synthesis of linkers and esterification ............................................................... 111 3.2. Heteroaromatic core .................................................................................................. 115 3.2.1. Literature and preliminary research on synthesis of the heteroaromatic head group, the first generation of compounds ......................................................................... 115 3.2.2. Synthetic results ................................................................................................ 119 3.3. Conclusion ................................................................................................................ 123 4. Approach B syntheses ..................................................................................................... 124 4.1. Literature research and proposed approach............................................................... 125 4.2. Synthetic results ........................................................................................................ 127 4.2.1. The feasibility of forming prerequisite alkynes and azides ............................... 127 4.2.2. The feasibility of Click reaction between alkyne 72 and azide 64 .................... 130 4.3. Biological screening results of the second generation of compounds and Discussion ... ………………………………………………………………………………………………137 5. Future work ..................................................................................................................... 138 6. Synthetic experimental ................................................................................................... 139 6.1. Approach A syntheses ............................................................................................... 140 6.1.1. Synthesis of the first generation of compounds ................................................. 140 6.1.2. Heteroaromatic core ......................................................................................... 150 6.2. Approach B syntheses ............................................................................................... 159 Failed synthetic attempts .................................................................................................. 178 7. Appendix .........................................................................................................................