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Title: Inhibition studies on hypoxia-inducible factor (HIF) hydroxylases
Author: Chan, Mun Chiang
ISNI:       0000 0004 6060 5968
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2014
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The hypoxia-inducible factor (HIF) is a key regulator of transcriptional responses to hypoxia in animals. As part of the cellular response to decreased oxygen concentrations, the transcriptional activity of a heterodimeric complex consisting of HIFα and HIFβ activates the expression of hundreds of target genes, including those involved in cellular growth, apoptosis, energy metabolism and angiogenesis. HIFα prolyl hydroxylation, as catalysed by the HIF prolyl hydroxylases (PHDs), leads to subsequent HIFα polyubiquitination and proteasomal degradation. HIFα asparaginyl hydroxylation as catalysed by factor inhibiting HIF (FIH), blocks the binding of HIFα to the co-activators CBP/p300, leading to reduced HIF transcriptional activity. The activities of the HIF hydroxylases (the PHDs and FIH) can be suppressed under limiting oxygen, resulting in the stabilisation of HIFα and activation of the HIF pathway. The development of PHD inhibitors in order to mimic aspects of the natural hypoxic response has been reported, although the selectivity of the inhibitors has not been investigated. Identification of selective small molecule inhibitors for the PHDs would enable further investigations into the differential role of the HIF hydroxylases in mediating the transcriptional response to hypoxia. Both the PHDs and FIH are part of the Fe(II) and 2-oxoglutarate (2OG)-dependent dioxygenase family, which includes histone and nucleic acid demethylases that are involved in gene regulation. The transcriptional response to hypoxia and/or the effects of non-selective PHD inhibitors could thus be mediated by their effects on these closely related enzymes. In the work described in this thesis, in vitro hydroxylation assays for PHD2 were developed for the identification of PHD inhibitors and determination of their inhibitory potencies. The development of a cellular assay for HIF levels is also described and used to measure the efficacy of PHD inhibitors. The utilisation of these assays led to the identification of potent, selective and cell-permeable PHD inhibitors suitable for use as chemical probes to study the biological roles of the PHDs. To aid in selectivity studies with the PHD isoforms, a cellular model system was developed by the re-expression of individual PHD isoforms in PHD-null mouse embryonic fibroblast cells. PHD inhibitors, including one of the PHD chemical probes identified, were used in a pan-genomic study of the transcriptional response by hypoxia in human breast cancer MCF-7 cells. The results reveal that inhibition of the PHDs, together with FIH, does not fully induce the full set of hypoxia upregulated genes, suggesting that the activation of HIF transcriptional activity alone may not be sufficient to invoke the transcriptional response to hypoxia.
Supervisor: Schofield, Chris ; Flashman, Emily Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available