Molecular and cellular mechanisms of HIF prolyl hydroxylase inhibitors in clinical trials

What is it about?

Inhibition of the human 2-oxoglutarate (2OG) dependent hypoxia inducible factor (HIF) prolyl hydroxylases (human PHD1-3) causes upregulation of HIF, thus promoting erythropoiesis and is therefore of therapeutic interest. We describe cellular, biophysical, and biochemical studies comparing four PHD inhibitors currently in clinical trials for anaemia treatment, that describe their mechanisms of action, potency against isolated enzymes and in cells, and selectivities versus representatives of other human 2OG oxygenase subfamilies. The ‘clinical’ PHD inhibitors are potent inhibitors of PHD catalyzed hydroxylation of the HIFα oxygen dependent degradation domains (ODDs), and selective against most, but not all, representatives of other human 2OG dependent dioxygenase subfamilies. Crystallographic and NMR studies provide insights into the different active site binding modes of the inhibitors. Cell based results reveal the inhibitors have similar effects on the upregulation of HIF target genes, but differ in the kinetics of their effects and in extent of inhibition of hydroxylation of the N and C terminal ODDs; the latter differences correlate with the biophysical observations.

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Why is it important?

Overall the results reveal that the PHD inhibitors in clinical trials have similar overall mechanisms of action and of potencies, both in terms of assays in cells and against the isolated PHD enzymes. They all work via chelation to the active site iron, though note this mechanism does not necessarily preclude selectivity, as evidenced by assays against other 2OG dependent oxygenases. However, the selectivity results imply that further work on the optimizing selectivity of the inhibitors with respect to the other prolyl hydroxylases may well be beneficial. There are, however, clear differences between the inhibitors, as manifested in the time dependencies of their effects in cells, and, in particular, in diff erences in their relative effects on NODD and CODD binding. The collective results suggest that t he development of PHD inhibitors that manifest, at least a degree, of inhibition selectiv ity for NODD/CODD and maybe HIF1α/HIF2α hydroxylation should be possible. The latter is of particular interest given that it has been reported that HIF1α and HIF2α may have opposing r ole on the progress of cancer. 98 In this regard the development of inhibitors, including tho se not binding to the active site metal, that exploit differences in the binding of different HIF α and nonHIF substrates to the PHDs is also of interest.

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