Four compounds in clinical trials for anaemia treatment are potent inhibitors of the hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs), but differ in potency and how they interact with HIF at the PHD active site.

In human cells oxygen levels are 'sensed' by a set of ferrous iron and 2-oxoglutarate dependent dioxygenases. These enzymes regulate a broad range of cellular and systemic responses to hypoxia by catalysing the post-translational hydroxylation of specific residues in the alpha subunits of hypoxia inducible factor (HIF) transcriptional complexes. The HIF hydroxylases are now the subject of pharmaceutical targeting by small molecule inhibitors that aim to activate or augment the endogenous HIF transcriptional response for the treatment of anaemia and other hypoxic human diseases. Here we consider the rationale for this therapeutic strategy from the biochemical, biological and medical perspectives. We outline structural and mechanistic considerations that are relevant to the design of HIF hydroxylase inhibitors, including likely determinants of specificity, and review published reports on their activity in pre-clinical models and clinical trials.

Human prolyl‐hydroxylases (PHDs) are hypoxia‐sensing 2‐oxoglutarate (2OG) oxygenases, catalysis by which suppresses the transcription of hypoxia‐inducible factor target genes. PHD inhibition enables the treatment of anaemia/ischaemia‐related disease. The PHD inhibitor Molidustat is approved for the treatment of renal anaemia; it differs from other approved/late‐stage PHD inhibitors in lacking a glycinamide side chain. The first reported crystal structures of Molidustat and IOX4 (a brain‐penetrating derivative) complexed with PHD2 reveal how their contiguous triazole, pyrazolone and pyrimidine/pyridine rings bind at the active site. The inhibitors bind to the active‐site metal in a bidentate manner through their pyrazolone and pyrimidine nitrogens, with the triazole π‐π‐stacking with Tyr303 in the 2OG binding pocket. Comparison of the new structures with other PHD inhibitor complexes reveals differences in the conformations of Tyr303, Tyr310, and a mobile loop linking β2–β3, which are involved in dynamic substrate binding/product release.

As part of the cellular adaptation to limiting oxygen availability in animals, the expression of a large set of genes is activated by the upregulation of the hypoxia-inducible transcription factors (HIFs). Therapeutic activation of the natural human hypoxic response can be achieved by the inhibition of the hypoxia sensors for the HIF system, i.e. the HIF prolyl-hydroxylases (PHDs). Here, we report studies on tricyclic triazole-containing compounds as potent and selective PHD inhibitors which compete with the 2-oxoglutarate co-substrate. One compound (IOX4) induces HIFα in cells and in wildtype mice with marked induction in the brain tissue, revealing that it is useful for studies aimed at validating the upregulation of HIF for treatment of cerebral diseases including stroke.