Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.553973
Title: Synthesis of bespoke matrices to investigate a novel anti-tumour molecular target using affinity chromatography : the design, synthesis and evaluation of biotinylated biarylheterocycles used as novel affinity probes in the identification of anti-tumour molecular targets
Author: Evans, Hayley Ruth
Awarding Body: University of Bradford
Current Institution: University of Bradford
Date of Award: 2010
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Abstract:
Three novel, synthetic biarylheterocycles bearing imidazole terminal groups had previously been discovered with high cytotoxicity (IC₅₀ 16-640 nM) against a number of human tumour cell lines. Notably, this biological activity was independent of duplex DNA binding affinity. The compounds were tested in the NCI 60-cell line panel and COMPARE analysis suggests they have a novel mechanism of action, targeting the product of a 'gene-like sequence' of unidentified function. The identity of likely protein targets was explored using a chemical proteomic strategy. Bespoke affinity matrices for chromatography were prepared in which test compounds were attached to a solid support through a biotin tag. A synthetic route to hit compounds containing a biotin moiety in place of one of the imidazole sidechains was developed. Chemosensitivity studies confirmed that the biotinylated compounds retained their activity showing IC₅₀ = 6.25 μM in a susceptible cell line, compared with > 100 μM for an insensitive cell line. The biotinylated ligands were complexed to a streptavidin-activated affinity column and exposed to cell lysates from the susceptible cell lines. Bound proteins were eluted from the column and separated using SDS-PAGE. Proteins were characterised by MALDI MS and MS/MS and identified using Mascot database searches. Heterogeneous nuclear ribonuclear protein A2/B1 was found to selectively bind to the affinity probes.
Supervisor: Wheelhouse, Richard T. ; Sutton, Chris W. ; Phillips, Roger M. Sponsor: Yorkshire Cancer Research, BMSS, School of Life Sciences and the Frank Hudson Memorial Fund
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.553973  DOI: Not available
Keywords: Anti-tumour molecular target ; Affinity chromatography ; Biotinylated biarylheterocycles ; Affinity probes ; Cytotoxicity ; Human tumour cells ; Chemosensitivity studies
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