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Development of a Combined Chemical Biology Tool Using DEVELOPMENT OF A COMBINED CHEMICAL BIOLOGY TOOL USING SYNTHETIC PROBES AND TANDEM MASS SPECTROMETRY TO ELUCIDATE HEAT SHOCK PROTEIN 90 KDA'S C-TERMINAL- DOMAIN BINDING SITES Xuexia Huang Nov 2018 A thesis submitted for the degree of Doctor of Philosophy of the University College London The School of Pharmacy, Faculty of Life Sciences University College London 1 Plagiarism statement This project was written by me and in my own words, except for quotations from published and unpublished sources which are clearly indicated and acknowledged as such. I am conscious that the incorporation of material from other works or a paraphrase of such material without acknowledgement will be treated as plagiarism, subject to the custom and usage of the subject, according to the University Regulations on Conduct of Examinations. The source of any picture, map or other illustration is also indicated, as is the source, published or unpublished, of any material not resulting from my own experimentation, observation or specimen-collecting. Signature: Date: 18/11/2018 2 Acknowledgements As my supervisors, Dr Min Yang and Dr Geoff Wells deserve thanks for too many things. Particularly, for creating the research environment in which I completed my research. They both provided timely guidance and support at some very crucial moments in my PhD, that I will be forever grateful. Professor Ijeoma Uchegbu and Dr Jasmina Jovanovic also offered generous help when very much needed and I feel very blessed to have had them in this journey. I am particularly grateful to Dr Min Yang, for giving me this Hsp90 project, which is a very interesting topic to investigate. More and more evidences are emerging towards the binding site we proposed, making me more and more confident about our work. Thank Matt, Sisi and Peixi, without you people, I may have finished this PhD earlier. I hope our adventure into the real world to be meaningful and full of success. I will be forever indebted to my parents, who supported me whole heartedly through every stage of my life. 3 Abstract Heat shock protein 90 kDa (Hsp90) has long been an appealing drug target for cancer treatment due to its pivotal role in conformational maturation and refolding of many proteins directly related to malignant progression. Previously various Hsp90 N-terminal domain (NTD) inhibitors have progressed to clinical trials in patients, unfortunately these studies were suspended due to high toxicity-related issues. On the other hand, the lack of a high-resolution crystal structure of the C-terminal domain (CTD) of human Hsp90 hampers the discovery of next generations of CTD inhibitors and presents challenges for further structure-based drug design. This study attempts to provide validated evidence for the Novobiocin binding site in yeast Hsp90 CTD by combinatorial use of photochemistry-based affinity protein labelling and analytical tools including NMR and mass spectrometry. This research on completion will shed light on the development of Hsp90 CTD inhibitors, which provide an alternative route to effective development of cancer drug candidates based on inhibition/interruption of Hsp90. The use of ligands conjugated to photoaffinity labelling (PAL) reagents for chemical modification of active site residues can circumvent limitations such as lack of complete crystal structure in CTD region, allowing detailed information on the sites of protein-ligand interactions to be acquired through high resolution MS and MS/MS experiments. Bifunctional PAL compounds incorporated a diazirine group and a novobiocin component, based on a previously reported glucosyl-novobiocin scaffold, which has been reported to demonstrate 200-fold increase in CTD inhibitory effects towards Hsp90 activities compared to that of novobiocin. The synthesis of the diazirine group we developed alleviated the high temperature and high- pressure conditions reported in literature. Also, in the functionalisation of the affinity providing novobiocin was based on the Williamson ether mechanism and resulted in regioisomers. An isomer due to rearrangement from coumarin to flavone in novobiocin core was identified by advanced NMR, and this 4 phenomenon has recently been reported by our group. Further detailed studies of photoactivation revealed the physiochemical properties of these probes, and such information was very critical for the ease of data analysis in the protein labelling stage. These novobiocin derivatives were activated under UV irradiation in the presence of Hsp90 proteins and adducts were detected using detailed MS and MS/MS analysis. The protein-PAL complexes were identified using MALDI- TOF MS and related techniques to confirm the labelling of yeast Hsp90. Complexes were further subjected to enzymatic digestion and result products were analysed by tandem MS (Orbitrap MS). Currently, the peptide mapping of chemically labelled proteins and localisation of chemically labelled amino acid are frequently carried out by manual analysis, which is time-consuming and requires relatively high level of modified peptide in the enzymatic digestion mixture. In this project, by using PeptideShaker with custom modification of the pre-identified mass difference, the modified peptide (at W585) was identified at relatively low protein labelling yield. W585 in yeast Hsp90 has been recently reported to be related to client remodelling coincident with stabilizing yHsp90 in an open conformation. The peptide region in the vicinity of this residue was further analysed and the binding pocket was identified by correlating the experimental results to ICM Pro based molecular docking, providing a direct visualisation of the yeast Hsp90 CTD binding site. Even these probes demonstrated low affinity towards human Hsp90, given the close homology between Hsp90 tertiary structures from yeast and human origins, the combination of both experimental and computational results obtained yeast Hsp90 labelling is expected to cast light on the understanding of Hsp90-novobiocin binding rationale, and contribute to future structure based drug design (SBDD) in the search for more potent Hsp90 C-terminal domain inhibitors. 5 Impact Statement The workflow presented in this thesis demonstrate an indirect but rational approach towards the identification of Hsp90 CTD binding site and to gain deeper understanding of the inhibition mechanism. This workflow can significantly circumvent the great effort and time needed in crystallography experiments in order to obtain high resolution 3-D structures, while providing specific information about protein-inhibitor interaction in the binding site. As a result, this method can be transferred to other similar research projects, revealing more information of substrate-protein binding using covalent binding and MS assisted analysis. More importantly, the binding site we have identified in this research has echoed with many other site directed mutagenesis studies relating to Hsp90 CTD, and with the topology of the binding site now made available, chemists from academia and industrial backgrounds can use the information to initiate rational structure based drug design for next generation of CTD that are more specific and safer for patients who suffer from cancer and also potentially for Alzheimer's disease patients as well. This research has also captured interests from major international pharmaceutical companies both for its application in analysis of covalently labelled protein identification and from contract research organizations for the potential to be accelerating characterising antibody drug conjugates. The research results have also been shared with British mass spectrometry community and evoked discussion over peptide mapping analysis that are not included currently available databases. This research project also led to publication of characterisation of the affinity probe regioisomers using advanced NMR, which underpins the potential complexity in chemically modifying Novobiocin and the approach to distinguish by-products from target Novobiocin derivatives. 6 List of Abbreviations 17AAG 17-(allylamino)-17-demethoxygeldanamycin 17-dimethylaminoethylamino-17- 17-DMAG demethoxygeldanamycin 2D- PAGE two dimensional polyacrylamide gel electrophoresis AA amino acid AC alternating current ACN acetonitrile AHA1 ATPase homologue 1 AIBN azobisisobutyronitrile APS ammonium persulfate ATP adenosine triphosphate BOC British Oxygen Company BRI1 brassinosteroid insensitive 1 BTMAC benzyl trimethyl ammonium chloride BTZ bortezomib CBDD computational based drug design CCl4 carbon tetrachloride CHCA α-cyano-4-hydroxycinnamic acid CID collision induced dissociation COSY homonuclear correlation spectroscopy CTD carboxy-terminal domain DC direct current DCM dichloromethane DEPT distortionless enhancement by polarization transfer DIGE difference gel electrophoresis DMAP 4-(dimethylamino)pyridine DMF dimethylformamide DMSO dimethyl sulfoxide EA ethyl acetate ECD electron-capture dissociation EGCG (−)-epigallocatechin-3-gallate EI electron ionization ESI electrospray ionization Et3N triethylamine ETD electron transfer dissociation FT Fourier transform FT-ICR Fourier transform ion cyclotron resonance GDA geldanamycin GeLCMS gel-enhanced LC-MS 7 GHKL gyrase, hsp90, histidine kinase and mutl Glc-NB glucosyl-novobiocin HCD higher energy collisional dissociation HMBC heteronuclear multiple bond correlation HMQC heteronuclear multiple quantum coherence HOP hsc70/hsp90-organizing protein HPLC high performance liquid chromatography HSOSA hydroxylamine-o-sulfonic acid Hsp
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