Prolyl hydroxylases (PHDs) target hypoxia-inducible factor-1α (HIF-1α) for degradation. Hypoxia inactivates PHDs, causing accumulation of HIF-1α. In turn, HIF-1 further transactivates PHDs.
Asparaginyl hydroxylation also regulates HIFα. Asparaginyl hydroxylation is a modification that blocks the recruitment of the transcriptional co-activators CBP/p300 to HIFα. As a result, this effect causes reduced HIF transcriptional activity. In this study, Mun Chiang Chan et al identify IOX4. IOX4 is a potent and selective inhibitor of PHD2 in vitro. In particular, IOX4 potently inhibits PHD2 with an IC50 value of 1.6 nM using an antibody-based in vitro hydroxylation assay for PHD2 catalysis. In addition, IOX4 induces HIFα in cells and in wildtype mice with marked induction in the brain tissue.

IOX4 blocks prolyl-hydroxylation of HIF1α at both NODD (HyPro402) and CODD (HyPro564). On the contrary, IOX4 does not markedly affect the levels of PHD2. In von Hippel-Lindau (VHL)-competent HeLa cells, IOX4 induces HIF1α, with IOX4 being substantially more potent (activity being observed at ≥ 1 μM). Moreover, IOX4 induces HIF1α levels with EC50 values of 11.7 μM, 11.1 μM and 5.6 μM in MCF-7, Hep3B, and U2OS cells, respectively. IOX4 is a substantially more potent PHD inhibitor than IOX2.

IOX4 as a highly potent and selective inhibitor of human PHD2. Importantly, IOX4 is effective at inducing HIFα in the mouse brain. IOX4 has an improved blood-brain barrier penetration compared to IOX2.

All in all, IOX4 is a potent and selective inhibitor of the hypoxia-inducible factor prolyl-hydroxylases with activity in the murine brain. IOX4 is useful for studies aimed at validating the upregulation of HIF for the treatment of cerebral diseases including stroke.

Chan MC, et al. Potent and Selective Triazole-Based Inhibitors of the Hypoxia-Inducible Factor Prolyl-Hydroxylases with Activity in the Murine Brain. PLoS One. 2015 Jul 6;10(7):e0132004.