Screening chelating inhibitors of HIF-prolyl hydroxylase domain 2 (PHD2) and factor inhibiting HIF (FIH).
Two primary O(2)-sensors for humans are the HIF-hydroxylases, enzymes that hydroxylate specific residues of the hypoxia inducible factor-α (HIF). These enzymes are factor inhibiting HIF (FIH) and prolyl hydroxylase-2 (PHD2), each an α-ketoglutarate (αKG) dependent, non-heme Fe(II) dioxygenase. Although the two enzymes have similar active sites, FIH hydroxylates Asn(803) of HIF-1α while PHD2 hydroxylates Pro(402) and/or Pro(564) of HIF-1α. The similar structures but unique functions of FIH and PHD2 make them prime targets for selective inhibition leading to regulatory control of diseases such as cancer and stroke. Three classes of iron chelators were tested as inhibitors for FIH and PHD2: pyridines, hydroxypyrones/hydroxypyridinones and catechols. An initial screen of the ten small molecule inhibitors at varied [αKG] revealed a non-overlapping set of inhibitors for PHD2 and FIH. Dose response curves at moderate [αKG] ([αKG]~K(M)) showed that the hydroxypyrones/hydroxypyridinones were selective inhibitors, with IC(50) in the μM range, and that the catechols were generally strong inhibitors of both FIH and PHD2, with IC(50) in the low μM range. As support for binding at the active site of each enzyme as the mode of inhibition, electron paramagnetic resonance (EPR) spectroscopy were used to demonstrate inhibitor binding to the metal center of each enzyme. This work shows some selective inhibition between FIH and PHD2, primarily through the use of simple aromatic or pseudo-aromatic chelators, and suggests that hydroxypyrones and hydroxypyridones may be promising chelates for FIH or PHD2 inhibition.
Department of Chemistry, University of Massachusetts, Amherst, MA 01003, USA.
Shannon C
SC
Flagg
Cristina B
CB
Martin
Cornelius Y
CY
Taabazuing
Breanne E
BE
Holmes
Michael J
MJ
Knapp
D000595:Amino Acid Sequence; D001665:Binding Sites; D002396:Catechols / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000737:chemistry; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D007501:Iron / Q000737:chemistry*; D007502:Iron Chelating Agents / Q000737:chemistry*; D007656:Ketoglutaric Acids / Q000737:chemistry; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000737:chemistry*; D008969:Molecular Sequence Data; D011393:Procollagen-Proline Dioxygenase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D011485:Protein Binding; D011725:Pyridines / Q000737:chemistry; D011728:Pyridones / Q000737:chemistry; D011753:Pyrones / Q000737:chemistry; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D012097:Repressor Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D013379:Substrate Specificity
D002396:Catechols; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007502:Iron Chelating Agents; D007656:Ketoglutaric Acids; D011725:Pyridines; D011728:Pyridones; D011753:Pyrones; D011994:Recombinant Proteins; D012097:Repressor Proteins; D007501:Iron; D006899:Mixed Function Oxygenases; C439790:HIF1AN protein, human; C490190:EGLN1 protein, human; D011393:Procollagen-Proline Dioxygenase; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases