Pre-mRNA (precursor messenger RNA) splicing is a fundamental step in eukaryotic gene expression that is carried out by a macromolecular machine known as the spliceosome. The spliceosome catalyses the removal of intervening sequences (introns), and the joining of exons, from >95 % of human transcripts to produce a functional mRNA template for protein synthesis. Splicing must be carried out with nucleotide precision and high fidelity for correct gene expression. The process of splicing is therefore susceptible to mutations which causes disease. As pre-mRNA splicing defects are implicated in a range of diseases, the development of small molecules that are able to modulate splicing are of significant importance and potential therapeutic benefit. This thesis describes the development of small molecule modulators of splicing which will serve as novel âtoolâ compounds for dissecting and uncovering further information on the intricate mechanism of splicing. The starting point of this research was the design and synthesis of a semisynthetic library of fusidic acid (FA) derivatives, towards the development of a structure-activity relationship (SAR) study. Functionalisation of FA at the C21-COOH and C3-OH groups resulted in the synthesis of a library of 40 molecules, including 31 novel derivatives. The biological activities of the library were evaluated using in vitro splicing assays, bacterial growth assays and cell viability assays against a panel of cancer cell lines. Several molecules were shown to inhibit splicing in vitro and display anti-proliferative effects. From this screening, the most potent splicing inhibitor, C21-para-chloro-benzyl-C3-succinate-fusidate (E6) (IC50 = 15 uM), was selected as the lead molecule. The effects of this lead molecule (E6) on the gene expression profiles, and the global-splicing programs within a panel of human cell lines was then assessed using RNA-Seq. Mis-regulation of splicing was found in each cell line (HEK293, HeLa, SK-HEP-1, and RPE-1) and altered exon skipping was identified as the predominant aberrant splicing event. Gene ontology and pathway analysis revealed splicing changes in genes associated with regulation of mRNA splicing via the spliceosome and translation initiation. This analysis supports the idea that the FA-derivatives target the spliceosome machinery and subsequently modulate alternative splicing. Mis-expression of genes associated with the integrated stress response (EIF2 signalling) and endoplasmic reticulum-stress response pathways were observed, which phenocopied effects observed in an EFTUD2 CRISPR-knockdown HEK293 cell line from our laboratory. Photoaffinity labelling was employed to investigate whether the FA-derived molecules target Snu114/EFTUD2 within the spliceosome. Photoaffinity probes of the lead molecule were designed and synthesised and then evaluated with the in vitro splicing assay and proved to be active in splicing inhibition. The probes were utilised in a photoaffinity labelling pull-down assay and indicated a binding interaction with Snu114 via western blot. Proteomics analysis following crosslinking, and pull-down also indicated potential interactions between the probe and other U5 snRNP proteins, including Brr2. Finally, chemical and modelling approaches were employed to explore other potential scaffolds and these approaches led to the discovery of other triterpenoid based small molecules, including lithocholic and oleanolic acid derivatives, as novel inhibitors of splicing. This thesis therefore highlights several new âtoolâ molecules for the investigation of the splicing mechanism and the cellular effects of splicing perturbation, which may be useful for studying the molecular mechanisms of splicing related disease.
Date of Award | 31 Dec 2023 |
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Original language | English |
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Awarding Institution | - The University of Manchester
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Supervisor | Raymond O'Keefe (Supervisor), Richard Bryce (Supervisor) & Roger Whitehead (Supervisor) |
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Expanding the toolbox for studying the splicing mechanism: Development of semi-synthetic triterpene derivatives as novel small molecule inhibitors of pre-mRNA splicing
Eadsforth, M. (Author). 31 Dec 2023
Student thesis: Phd