Targeting hypoxia signalling for the treatment of ischaemic and inflammatory diseases

Abstract

Hypoxia-inducible factors (HIFs) are stabilized during adverse inflammatory processes associated with disorders such as inflammatory bowel disease, pathogen infection and acute lung injury, as well as during ischaemia-reperfusion injury. HIF stabilization and hypoxia-induced changes in gene expression have a profound impact on the inflamed tissue microenvironment and on disease outcomes. Although the mechanism that initiates HIF stabilization may vary, the final molecular steps that control HIF stabilization converge on a set of oxygen-sensing prolyl hydroxylases (PHDs) that mark HIFs for proteasomal degradation. PHDs are therefore promising therapeutic targets. In this Review, we discuss the emerging potential and associated challenges of targeting the PHD-HIF pathway for the treatment of inflammatory and ischaemic diseases.

Figure 1. Molecular mechanism of oxygen sensing and signalling

Depicted here is the biochemical pathway of hypoxia-inducible factor (HIF) hydroxylation through a combination of α-ketoglutarate (αKG), molecular oxygen (O₂), one or more of the prolyl hydroxylase (PHD) isoenzymes and the asparaginyl hydroxylase factor inhibiting HIF (FIH) in normoxia. In normoxia, the von Hippel–Lindau disease tumour suppressor protein (VHL)-containing E3 ubiquitin ligase recognizes the α-subunit of HIFs and targets the subunit for polyubiquitylation and subsequent degradation. Regulation of the E3 ligase is maintained by the covalent modification of the ubiquitin-like protein NEDD8. The functional E3 ligase requires the COP9 signalosome to bind NEDD8 to CUL2. When oxygen becomes limited (hypoxia), the α-subunit is no longer hydroxylated and becomes stabilized. In the nucleus, the α-subunit binds to the HIF1β subunit and the complex becomes transcriptionally active upon binding to the hypoxia-response element (HRE) consensus sequence on DNA. This binding results in the transcriptional activation of genetically controlled survival pathways, which are central to the regulation of inflammatory outcomes. HIF activation can be elicited pharmacologically using PHD inhibitors, thereby promoting normoxic HIF stabilization and concomitant alterations in gene expression. Similarly, targeting of cullin 2 (CUL2) neddylation (for example, inhibition of NEDD8-activating enzyme) results in CUL2 deneddylation and a loss of E3 ubiquitin ligase activity with concomitant stabilization of HIFα. CBP, CREB-binding protein; RBX, RING-box protein.

Figure 2. PHD inhibitor treatment of inflammatory bowel disease

Inflammatory bowel disease is a result of intestinal inflammation, in which profound changes in metabolic supply and demand lead to an imbalance in oxygen supply. This imbalance causes severe hypoxia of the inflamed mucosa. Mucosal hypoxia during intestinal inflammation can be visualized using nitroimidazole compounds that stain hypoxic tissues^,. Infiltrating polymorphonuclear neutrophils (PMNs) contribute to tissue hypoxia of the inflamed intestinal mucosa by localized oxygen depletion and concomitant stabilization of hypoxia-inducible factor (HIF), thereby contributing to the resolution of inflammation. Inflammatory hypoxia causes the activation of hypoxia-elicited gene programmes. Coordinated gene programmes result in the increased production and signalling of extracellular adenosine. This gene programme is under the control of SP1-dependent induction of CD39, HIF-dependent induction of CD73 and the adenosine A_2A and A_2B receptors. These transcriptional changes lead to an increased turnover rate of the extracellular nucleotides ATP and ADP to AMP (via CD39) and subsequently via CD73 to adenosine. This pathway provides robust protection during intestinal inflammation. Importantly, orally available inhibitors of prolyl hydroxylases (PHDs) are available for the treatment of patients and are effective in increasing erythropoietin responses. Such compounds can promote intestinal protection from inflammation via enhancing extracellular adenosine production and signalling through adenosine receptors.

Figure 3. Mechanism of HIF1α stabilization during acute lung injury

Patients with acute lung injury (ALI) frequently require mechanical ventilation of their lungs to maintain sufficient oxygen levels in their blood to oxygenate critical organs such as the brain, the kidneys or the heart. Cyclic mechanical stretch conditions during mechanical ventilation in vivo or during cyclic mechanical stretch exposure of the alveolar epithelial cells in vitro result in hypoxia-inducible factor 1, α-subunit (HIF1α) stabilization. Stretch-induced HIF1α stabilization is mediated by the inhibition of succinate dehydrogenase (SDH), causing normoxic stabilization of alveolar epithelial HIF1α. Functional studies implicate alveolar epithelial HIF1α in optimizing carbohydrate metabolism of the injured lungs by increasing glycolytic capacity and tricarboxylic acid (TCA) cycle flux, and by optimizing mitochondrial respiration via induction of complex IV. HIF1α-dependent prevention of mitochondrial dysfunction during ALI is associated with increased alveolar epithelial capacity to produce ATP, while concomitantly preventing reactive oxygen species (ROS) accumulation and attenuating lung inflammation.

Figure 4. Structure of the prolyl hydroxylase inhibitor AKB-4924

The AKB-4924 structure contains an iron-binding α-hydroxycarbonyl group similar to that of the iron chelator L-mimosine (outlined).