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Title: Inhibition and mechanistic studies of FTO
Author: Shishodia, Shifali
ISNI:       0000 0004 7652 3836
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2017
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The Fat Mass and Obesity Associated protein (FTO) is a nucleic acid demethylase that belongs to the 2-oxoglutarate dependent oxygenase superfamily (2OG oxygenases). Like other 2OG oxygenases, FTO uses Fe(II), 2-oxoglutarate (2OG) and molecular oxygen (O2) to catalyse oxidation of its substrates. Mutation of the gene encoding for FTO have been linked to obesity, type II diabetes, cancer, and Alzheimer's disease. One aspect of the work described in this thesis aimed at developing small molecule inhibitors of FTO. An NMR binding assay was developed using 2OG as a reporter ligand. The inhibitors developed in this thesis exhibit high potency and selectivity for FTO (the lead inhibitor has an IC50 value of 1.32 μM). The work also aimed at the synthesis of N6-methyladenosine (m6A) phosphoramidite with the aim of synthesising single stranded RNA (ss-RNA) oligonucleotide containing m6A. Apart from the literature reported substrates, work also focussed on the investigations of other nucleosides as natural and unnatural substrates of FTO. During the study, new synthetic routes to the m6A phosphoramidite, ethanoadenosine, ethylethanoadenosine and methylethanoadenosine were developed. The iodouridine phosphoramidite was also synthesised with the future aim of synthesising a ss-RNA substrate crosslinked to FTO. The reaction of formaldehyde (the FTO co-product) with canonical and non-canonical nucleotides was then investigated using NMR. Various nucleotide/HCHO adducts were identified, including multiple novel species. Among the canonical nucleosides, TMP and UMP reacted the fastest followed by dCMP, dGMP and lastly dAMP. However, dAMP was observed to form the most stable adduct with formaldehyde, while the adducts of TMP and UMP were dynamically favoured. Comparing cytidine, 5-methyldeoxycytidine (5mdC) and 5-hydroxymethyldeoxycytidine (5hmdC) revealed that 5mdC reacts the fastest. Among m6A, N1-methyladenosine (m1A) and adenosine, the initial rate of reaction with formaldehyde was fastest with adenosine. Finally, the FTO and AlkB catalysed oxidative demethylation of substrates was investigated by NMR using [13C] labelled substrates (e.g., N6-[13C]-methyladenosine ([13C]-m6A), N3-[13C]-methythymidine ([13C]-m3T) or N1-methyladenosine ([13C]-m1A)) as probes. The studies reveal the first direct evidence, using NMR, for the formation of N6-hydroxymethyladenosine as an intermediate of FTO- catalysed oxidative demethylation of m6A. The results also confirmed the formation of formaldehyde. The observation of formaldehyde in AlkB-catalysed oxidative demethylation of m1A led to the invalidation of the previously reported hypothesis that a phosphate group attached to a nucleoside is the minimum requirement for AlkB substrates. Overall, the work described in this thesis suggests new approaches to the inhibition of FTO, helps to define the range of substrates for FTO, reassign the requirement for AlkB-catalysed oxidative demethylation, and informs on the reaction of the FTO-catalysed reaction co-product, HCHO, with nucleosides.
Supervisor: Schofield, Christopher J. Sponsor: Felix Scholarship Trust ; Vice Chancellor Scholarship
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available