Targeting hypoxia-inducible factors: therapeutic opportunities and challenges

Abstract

Hypoxia-inducible factors (HIFs) are highly conserved transcription factors that are crucial for adaptation of metazoans to limited oxygen availability. Recently, HIF activation and inhibition have emerged as therapeutic targets in various human diseases. Pharmacologically desirable effects of HIF activation include erythropoiesis stimulation, cellular metabolism optimization during hypoxia and adaptive responses during ischaemia and inflammation. By contrast, HIF inhibition has been explored as a therapy for various cancers, retinal neovascularization and pulmonary hypertension. This Review discusses the biochemical mechanisms that control HIF stabilization and the molecular strategies that can be exploited pharmacologically to activate or inhibit HIFs. In addition, we examine medical conditions that benefit from targeting HIFs, the potential side effects of HIF activation or inhibition and future challenges in this field.

Fig. 1|. Molecular structure of hypoxia-inducible factors

Hypoxia-inducible factors (HIFs) consist of α (HIF1α, HIF2α and HIF3α) and β (HIF1β) subunits, with the α-subunit functioning as the principal regulator of HIF transcriptional activity. HIF1α and HIF2α feature an N-terminal basic helix–loop–helix (bHLH) domain and two Per–Arnt–Sim (PAS-A and PAS-B) domains crucial for dimerization and DNA binding^,. The C-terminal region contains a transcription activation domain (C-TAD) and a second N-terminal activation domain (N-TAD) and N- and C-terminal oxygen-dependent degradation domains (NODD and CODD). Unlike HIF1α and HIF2α, HIF3α lacks the C-TAD and is therefore considered an inhibitory HIF factor^,. Under normoxic conditions, HIF prolyl hydroxylase domain-containing proteins (HIF-PHDs) hydroxylate specific proline residues on HIF1α, HIF2α and HIF3α, which depends on the availability of oxygen, α-ketoglutarate and iron. Following hydroxylation, HIFα is targeted for proteasomal degradation^,. This process involves the binding of the von Hippel–Lindau protein (pVHL) and the initiation of ubiquitylation, facilitated by the complex that includes elongin B, elongin C (EloBC), cullin 2 (CUL2) and RING box protein 1 (RBX1)^,. An additional E3 ubiquitin ligase also contributes to this process, ultimately leading to the proteasomal degradation of HIFα. In addition, factor inhibiting HIF (FIH) hydroxylates Asn803 within the C-TAD of HIF1α and Asn847 within the C-TAD of HIF2α^,, obstructing the binding of coactivators cyclic adenosine monophosphate response element binding protein (CREB) binding protein and histone acetyltransferase p300 (CBP–p300) and inhibiting HIF function. In hypoxic conditions, the activities of PHDs and FIH are decreased, enabling the stabilization and accumulation of active HIFα–HIF1β complexes, leading to the subsequent binding to hypoxia-response elements in target genes and the concomitant induction of HIF target genes. aa, amino acids; Ub, ubiquitin.

Fig. 2|. Crystal structures of PHD2 and HIF-PHDi complexes and HIF2α–PT2385–PT2977 complexes

a, Left: overall view of 2-oxoglutarate (2-OG) C5-carboxyl-binding pockets for prolyl hydroxylase inhibitors (PHDis) within the PHD2 crystal structure. Two enlarged views display electron density maps of the PHD2–vadadustat (Protein Data Bank (PDB): 5OX6; resolution: 1.99 Å) and PHD2–molidustat (PDB: 6ZBO; resolution: 1.79 Å) complexes with key residues shown. The 2-OG dioxygenase domain is depicted in magenta, the β2–β3 finger loop in light yellow and key residues in red. Right: chemical structures of selected PHDis approved for clinical use or late-stage development are ordered by ascending half-maximal inhibitory concentration (IC₅₀) values for PHD2 (ref. 87). b, Binding position for the ‘PT’ series compounds within the entire hypoxia-inducible factor 2α (HIF2α)–HIF1β crystal structure (PDB: 6E3S; resolution: 3.00 Å) on the left (HIF2α, pale cyan; HIF1β, pink), with a close-up view showing the location of PT2385 (PDB: 5TBM; resolution: 1.85 Å) and PT2977 (PDB: 7W80; resolution: 2.75 Å) inside the HIF2α Per–Arnt–Sim (PAS)-B domain (light cyan), along with the surrounding residues (cyan) in the pocket. bHLH, basic helix–loop–helix.

Fig. 3|. Potential therapeutic applications of HIF-PHDis in various diseases

In normoxia (central panel) hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHDis) enhance the stabilization of HIFα. This is achieved by inhibiting proline hydroxylation of HIFα by HIF-PHDs, thereby preventing the recognition of von Hippel–Lindau protein (pVHL) and subsequent proteasomal degradation of HIFα. Once stabilized, HIFα forms a complex with HIF1β, histone acetyltransferase p300 and cyclic adenosine monophosphate response element binding protein (CREB) binding protein (CBP), translocates to the nucleus, and binds to hypoxia-responsive elements (HREs) in the promoter regions of HIF targets to activate their transcription. Pharmacologically enhanced HIF stabilization by HIF-PHDis has shown benefits in diseases such as ischaemia–reperfusion injury (heart and liver), acute kidney injury, inflammatory bowel disease, central nervous system injury, renal anaemia and acute respiratory distress syndrome (ARDS). In both the kidneys and liver, HIF-PHDis stabilize HIF2α, thereby enhancing the production of endogenous erythropoietin (EPO)^,. This process is particularly beneficial in conditions such as renal anaemia, in which EPO production is typically insufficient. EPO promotes the survival and differentiation of bone marrow erythroid progenitors, specifically colony-forming unit erythroid (CFU-e) cells and proerythroblasts, resulting in an increase in red blood cell production. In the liver, the stabilization of HIF2α leads to increased production of transferrin (TF), the plasma protein that carries iron, and a decrease in hepcidin production, which serves as a negative regulator of ferroportin (FPN)^,. FPN is responsible for the export of iron from duodenal enterocytes. Additionally, HIF-PHDis can improve iron absorption and metabolism by inducing divalent metal transporter 1 (DMT1), ferrireductase duodenal cytochrome B (DCYTB) and FPN1 in the enterocytes, further promoting iron homeostasis. With increased iron availability and delivery by TF to marrow erythroblasts, there is an increase in both the size of red blood cells and their haemoglobin content. HIF-PHDis may alleviate ARDS by stabilizing HIFs. HIF1α stabilization in alveolar type II epithelial cells (ATII cells) during ARDS optimizes carbohydrate metabolism. HIF1α also activates extracellular adenosine signalling — particularly through increasing extracellular adenosine levels via enhancement of CD73 and repression of equilibrative nucleoside transporter ENT1 or ENT2 and adenosine kinase^–. Subsequently, extracellular adenosine binds to HIF1α-dependent ADORA2B to reduce lung inflammation. Furthermore, HIF1α attenuates severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication through inhibition of angiotensin-converting enzyme 2 and transmembrane protease, serine 2, two receptors that are important to viral entry, and repression of viral RNA replication. On the other hand, HIF2α maintains endothelial cell barrier integrity by inducing vascular endothelial protein tyrosine phosphatase (VE-PTP), and the resultant VE-cadherin dephosphorylation-mediated assembly of adherens junctions (AJs), thereby preventing vascular permeability and lung oedema. HIFs also regulate immune cell functions, with HIF-mediated glycolysis being essential for macrophage-mediated pathogen clearance^,. HIF-dependent netrin-1 induction in myeloid cells inhibits natural killer cell (NK cell) invasion during endotoxin-induced lung injury.

Fig. 4|. Potential therapeutic applications of HIF inhibition in various diseases

PT2385 and PT2977 (also known as MK-6482 or belzutifan) are small-molecule inhibitors that selectively target hypoxia-inducible factor 2α (HIF2α), blocking its transcriptional activity by preventing heterodimerization with HIF1β^,. These inhibitors offer potential therapeutic options for HIF-driven diseases. For instance, clear cell renal cell carcinoma (ccRCC) is a common form of RCC associated with VHL gene mutations. Such mutations can impair the ability of the von Hippel–Lindau protein (pVHL) to recognize and bind to hydroxylated HIF2α, leading to HIF stabilization and target gene transcription, promoting tumour growth. PT2399, a HIF2α inhibitor, has shown promising results in reducing tumour growth in preclinical studies. PT2385 and PT2977 have undergone clinical trials for patients with ccRCC, with PT2977 (belzutifan) obtaining FDA approval for VHL disease-related tumours owing to its encouraging outcomes. Pacak–Zhuang syndrome is a rare form of multiple paragangliomas associated with polycythaemia and caused by a gain-of-function mutation in the endothelial Per–Arnt–Sim (PAS) domain protein 1 (EPAS1) gene^–. Under normoxic conditions, HIF2α is hydroxylated by prolyl hydroxylase domain-containing proteins (PHD) and undergoes rapid degradation. In Pacak–Zhuang syndrome, the gain-of-function mutations in the EPAS1 gene can lead to a version of HIF2α that is resistant to hydroxylation by PHDs, preventing its degradation^–. As a result, HIF2α accumulates and activates its target genes even under normoxic conditions, which can contribute to the pathophysiological manifestations of the syndrome, such as paragangliomas and polycythaemia^–. In a single-patient trial, belzutifan demonstrated promising results in treating the syndrome, with sustained improvements observed. Other conditions that may benefit from targeting HIF2α stabilization include pulmonary hypertension (PH) and retinal neovascularization. ARG1, arginase 1; bHLH, basic helix–loop–helix; CDH5, cadherin 5; CXCL12, C-X-C motif chemokine ligand 12; EGFR, epidermal growth factor receptor; ET1, endothelin-1; GLUT1, glucose transporter type 1; HRE, hypoxia-responsive element; ICAM1, intercellular adhesion molecule 1; OIR, oxygen-induced retinopathy; PAI-1, plasminogen activator inhibitor 1; PDGFB, platelet-derived growth factor subunit B; RBX1, RING box protein 1; RNAi, RNA interference; siRNA, small interfering RNA; SNAI1, snail family transcriptional repressor 1; SNAI2, snail family transcriptional repressor 2; TGFα, transforming growth factor-α; VEGF, vascular endothelial growth factor.