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Arresting the Spliceosome: Investigating the Binding of Fusidic Acid within the Spliceosome

Soltysiak, Robert Joseph

[Thesis]. Manchester, UK: The University of Manchester; 2016.

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Abstract

Splicing is a process that occurs in the nuclei of all eukaryotic cells, removing non-coding sections from pre-mRNA in the final step of transcription. The process consists of two transesterification reactions carried out via interaction with the spliceosome - a large, highly dynamic RNA/protein complex essential in catalysing splicing.Fusidic acid has been shown previously to inhibit splicing, potentially by interaction with snRNA U5 of the spliceosome. This project attempts to elucidate the mechanism of binding, with a view to improving the inhibitory function of the compound. This was achieved by developing photo-crosslinking compounds which could be used to elucidate the protein structure of the binding site, and subsequently the interactions between U5 and fusidic acid. Chapter one discusses the nature of the splicing, as well as examples of photo-crosslinking ligands and their use in biological studies to date. The early sections of chapter three outline the synthesis of the photo-crosslinking compounds and subsequent incorporation into the skeleton of fusidic acid. The latter sections describe the investigations into the structural modifications to fusidic acid, and in particular how these affect the inhibitory function of the molecule.A number of pathways outlined here have been eliminated as unsuccessful in the functionalisation of fusidic acid. Attachment of both diazirinyl and benzophenone compounds was achieved in reasonable yield. While these compounds were insufficiently active to be taken further in the cross-linking study, active intermediates synthesised during the pathway to these compounds have been discovered. These were investigated further, and one compound in particular has shown promise as a potential therapy.Also explored was iodolactonization of fusidic acid, introducing restriction of movement to the side chain via cyclisation. The ambiguous structures of the products were confirmed using X-ray crystallography, and elimination of iodine was investigated to allow for further functionalisation at this position.