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Title: Towards high-throughput fragment screening by mass spectrometry
Author: Maple, Hannah Jane
ISNI:       0000 0004 2722 8101
Awarding Body: University of Bristol
Current Institution: University of Bristol
Date of Award: 2011
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Screening for protein-ligand binding interactions is a key step during early stage drug discovery programmes. The fragment-based screening approach has gained popularity in recent years as a highly promising alternative to traditional high-throughput screening (HTS) , which has not yielded the success rate expected in the 'post-genomic' drug discovery era. The increasing use of fragment-based drug discovery (FBDD), however, places additional demands on biophysical screening techniques, and requires that the techniques used are capable of detecting very weak non-covalent interactions (mM Ko values). Existing screerung techniques that have been applied to FBDD, such as nuelear magnetic resonance (NMR), surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and X-ray crystallography, are all associated with one or more of the following drawbacks: low throughput, high sample consumption and dynamic range limitations. The use of nano- electrospray mass spectrometry (nano-ESI MS) as a means of screening for non-covalent complexes is a relatively recent addition to existing methodologies that shows promise as an orthogonal screening technique. The advantages it offers: high throughput, low sample consumption, generation of stoichiometric information and the ability to determine dissociation constants make this an attractive approach. Presented here is the development and validation of a fully automated screen by nano-ESI MS, capable of detecting fragment binding into the mM KD range. The method was applied for screening against the anti-apoptotic protein target, Bel-XL, and mass spectrometry results were validated using STD-NMR, HSQC-NMR and ITC experiments. Agreement between techniques suggests that mass spectrometry offers a powerful, complementary approach for primary screening. Two alternate, or secondary screerung techniques are also explored. Equilibrium dialysis, combined with nano-ESI MS and STD-NMR, was investigated for the identification of bioactive atropisomers to therapeutic protein targets, Bel-XL and Bcl-2, from mixtures of rotameric compounds. The methodology developed offers an alternative, 'ligand-detect' approach to screening by nano-ESI MS for cases where direct detection of the protein-ligand complex is not possible. Preliminary data are also shown for the application of a microflow capillary NMR probe to automated screening by HSQC NMR experiments. Capillary probes offer excellent mass sensitivity and can be fully automated through interfacing with a liquid handling system. This has the potential to offer a novel fragment screening platform that provides information on the location of compound binding through chemical shift perturbations.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available