STUDIES ON THE INHIBITION OF HUMAN HYPOXIA INDUCIBLE FACTOR (HIF) HYDROXYLASES

Hypoxia inducible factor (HIF) is an α/β-heterodimeric transcription factor that regulates cellular responses to hypoxia in metazoans. The activity and stability of the HIF-α subunits are regulated by prolyl and asparaginyl hydroxylation. Human HIF-1α prolyl hydroxylation occurs at Pro402 and Pro564 while asparaginyl hydroxylation occurs at Asn803. Asparaginyl hydroxylation blocks the HIF-1α interaction with the p300/CBP co-activator, thus inhibiting HIF transcriptional activation. Prolyl hydroxylation promotes the HIF-1α interaction with the von Hippel-Lindau (VHL) ubiquitin E3 ligase complex and targets it for proteasomal degradation. HIF prolyl hydroxylation is catalysed by the PHDs (prolyl hydroxylase domain) 1, 2 and 3, whereas asparaginyl hydroxylation is catalysed by FIH (factor inhibiting HIF). Both the PHDs and FIH are Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases, which utilise oxygen as a co-substrate. In hypoxia, the activity of the PHDs and FIH are suppressed, thus enabling HIF-α subunits to form a productive transcriptional complex. There is widespread interest in developing HIF hydroxylase inhibitors for the treatment of ischemic/hypoxic diseases. The extent to which HIF-1α prolyl and asparaginyl hydroxylation are differentially regulated by chemical reagents is an important question. This thesis describes the development of methods employing immunoblotting and HIF hydroxy-residue specific antibodies to enable the simultaneous measurement of the effects of chemical inhibition at all three HIF-1α hydroxylation sites in cells. The findings reveal that HIF prolyl hydroxylation is substantially more sensitive than asparaginyl hydroxylation to inhibition by iron chelators and transition metal ions, in contrast to predictions from in vitro studies. Studies on a range of 2OG analogue inhibitors resulted in the identification of several cell-permeable PHD specific inhibitors as well as an FIH specific inhibitor that is active in cells. Excessive accumulation of R-2-hydroxyglutarate (R-2HG) in mutated isocitrate dehydrogenase (IDH)-mediated cancers has led R-2HG to be recognised as an 'oncometabolite'. The newly developed antibody assays were used to investigate the effects of cell-permeable 2HG derivatives on the activity of the HIF hydroxylases in cells. This indicated that direct R-2HG inhibition of PHDs does not play a role in mutated IDH-mediated tumourgenesis. The PHDs have been proposed to play a general role as metabolic sensors besides their function as intracellular oxygen sensors. The Krebs cycle metabolite 2OG (a co-substrate of HIF hydroxylases) was therefore investigated as a potential regulator of the PHDs. The cell-based results demonstrate that 2OG elevation results in HIF-α induction, a mechanism suggested to be, at least in part, through PHD inhibition as supported by in vitro and cell-based results. This thesis also describes the first attempt to apply a chemical-genetic approach to functional studies of the PHD isoforms. The in vitro results demonstrate the feasibility of selective inhibition of PHD2 by employing small-molecule-sensitive PHD2 variants. However, attempts to test this approach in mammalian cells have not been successful to date due to the lack of a suitable cell-system. Work on PHD inhibition then describes the development of a new class of diacylhydrazine-based PHD inhibitors. Findings show that some of these compounds are capable of binding to the PHD2 active site and simultaneously inducing the binding of a second iron to PHD2. The reported PHD inhibitor, aspirin metabolite 2,3-dihydroxybenzoylglycine (DHBG), was unexpectedly found to exhibit a concentration-dependent dichotomous effect on HIF stabilisation and HIF suppression in VHL-competent cells. DHBG and other aspirin metabolites including gentisuric acid (GUA) were subsequently found to suppress HIF-1α and HIF-2α levels in renal carcinoma RCC4 VHL-defective cells, suggesting that therapeutic effects of aspirin in cancers may involve potential regulation of HIF-α activity.