Evidence for inhibition of HIF-1α prolyl hydroxylase 3 activity by four biologically active tetraazamacrocycles

Abstract

Hypoxia inducible factor 1 (HIF-1) is central to the hypoxic response in mammals. HIF-1α prolyl hydroxylase 3 (PHD3) degrades HIF through the hydroxylation of HIF-1α. Inhibition of PHD3 activity is crucial for up-regulating HIF-1α levels, thereby acting as HIF-dependent diseases therapy. Macrocyclic polyamines which display high stability on iron-chelating may well inhibit the enzyme activity. Thus inhibition and interaction on catalytic PHD3 by four biologically active tetraazamacrocycles (1–4), which have two types of parent rings to chelate iron(II) dissimilarly, were studied. The apparent IC₅₀ values of 2.56, 1.91, 5.29 and 2.44 μM, respectively, showed good inhibition potency of the four compounds. K_I values were 7.86, 3.69, 1.59 and 2.92 μM for 1–4, respectively. Different inhibition actions of the two groups of compounds were identified. Circular dichroism (CD) and fluorescence spectrometries proved that one type of compound has significant effects on protein conformation while another type does not. Computational methodology was constructed to employ the equilibrium geometry of enzyme active site with the presence of substrate competitive inhibitor. Iron(II) coordination in the active site by inhibitors of this kind induces conformational change of the enzyme and blocks substrate binding.