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George Hoppe, Joseph Potter, Jonathan E. Sears; A Novel Class of Hydrazones Confers Nonhypoxic Activation of HIFα by Potent Inhibition of Proline Hydroxylation. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3989.
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© ARVO (1962-2015); The Authors (2016-present)
Hypoxia inducible factor (HIF) prolyl hydroxylase (PHD) inhibition stabilizes the catabolism of HIF, thereby exerting pleiotropic effects on hematopoiesis and vascular integrity including prevention of oxygen induced retinopathy. We have recently discovered a novel class of hydrazones that are extremely potent activators of HIF. In this study we set out to determine whether these compounds act as hypoxia mimetics, i.e., control HIF hydroxylation and stability.
25 compounds were selected from a Chembridge, LOPAC and NPL chemical libraries using a screening strategy that employed a hypoxia response element fused to the luciferase reporter gene. High, medium, and low inducers of luminescence were chosen to be further tested in hepatocytes and Müller cells for their effects on VEGF and erythropoietin (Epo) secretion, and HIF-1α hydroxylation. Anti-HIF-1α antibody was used to probe Müller (MIO-M1) and Hep3B lysates by Western blot. VEGF and Epo ELISA tested secretion of the proteins into MIO-M1 and Hep3B media. Anti-hydroxy-HIF-1α (Pro564) antibody was used in Western blot to determine whether these compounds inhibited HIF PHD after administration of a proteasome inhibitor MG132.
Submicromolar concentrations of various hydrazone molecules induced robust accumulation of HIF-1α and HIF-2α proteins. These increases correlated to both VEGF and Epo secretion in a hierarchal manner although compound D5 induced both VEGF and Epo to a greater extent in Hep3B cells than in MIO-M1 cells, whereas compound D1 was more active in the latter. Cultured Müller cells did not produce Epo. All compounds efficiently prevented hydroxylation of HIF-1α Pro564, the degree of which correlated to the expression levels of HIF downsteam targets.
This new class of compounds stabilizes HIF-1α and 2α with associated increases in Epo and VEGF, two of which were cell type specific. In addition, these compounds prevent α subunit hydroxylation, and therefore are direct inhibitors of HIF PHD with a potential for neurovascular protection and repair in retinal ischemic disease.
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