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Title: 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
Author: Huang, Xuexia
ISNI:       0000 0004 7660 4863
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2019
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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 highpressure 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 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 MALDITOF 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.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available