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Combined use of capillary electrophoresis and X-ray crystallography in fragment-based drug discovery

Chen, W; (2014) Combined use of capillary electrophoresis and X-ray crystallography in fragment-based drug discovery. Doctoral thesis , UCL (University College London).

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Abstract

The effectiveness of combined use of capillary electrophoresis (CE) and X-ray crystallography in fragment-based drug discovery has been investigated. Using Heat Shock Protein 90α (Hsp90α) as a test system, CE fragment hits were validated by X-ray crystallography with the added benefit of structural information. The crystal structure of one of the fragment hits in complex with Hsp90α ATPase domain has shown that even weakly binding fragments can cause conformational change of macromolecules upon binding. Another investigation was focused on identifying stabilisers of the tetrameric plasma protein transthyretin (TTR), which can cause amyloidosis when its monomers are misfolded. Two methodologically distinct CE assays were developed to detect TTR binding fragments. 129 fragments were screened using one method and 15 of the 16 initial hits were tested positive by the other (hit rate = 11.6%). Structure activity relationships were built up from analogue screening on two chemically different fragment hits using both CE assays. In parallel, ligand-protein interactions were characterised by X-ray crystallography to assist hit-to-lead development. The CE assays and structural data yielded a detailed perspective on the binding of heterocyclic rings to TTR. Aromaticity and hydrophobicity are favoured in the binding sites but not basicity. Moreover, Ser117 hydrogen bonding with heterocyclic nitrogen atoms was frequently observed. Through the combined use of two biophysical techniques and testing of fewer than 200 compounds, a novel TTR binding scaffold was discovered. The scaffold is likely to be active in vivo as it binds to TTR in neat plasma (IC50 = 34.45μM). It has high ligand efficiency (0.60) with an ITC Kd of 260.42nM and 15 non-hydrogen atoms. Use of CE and X-ray crystallography provides a powerful combination worth considering in early stage drug discovery.

Type: Thesis (Doctoral)
Title: Combined use of capillary electrophoresis and X-ray crystallography in fragment-based drug discovery
Language: English
Additional information: Permission for digitisation not received.
Keywords: Drug discovery, fragment screening, protein crystallography, capillary electrophoresis, transthyretin, Hsp90, amyloidosis
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Population Health Sciences > UCL GOS Institute of Child Health > Infection, Immunity and Inflammation Dept
URI: https://discovery.ucl.ac.uk/id/eprint/1410604
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