Two primary O2-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, nonheme Fe(II) dioxygenase. Although the two enzymes have similar active sites, FIH hydroxylates Asn803 of HIF-1α while PHD2 hydroxylates Pro402 and/or Pro564 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] ~ KM) showed that the hydroxypyrones/hydroxypyridinones were selective inhibitors, with IC50 in the µM range, and that the catechols were generally strong inhibitors of both FIH and PHD2, with IC50 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.

The factor inhibiting hypoxia inducible factor-1α (FIH) is a nonheme Fe(II)/αKG oxygenase using a 2-His-1-Asp facial triad. FIH activates O2 via oxidative decarboxylation of α-ketoglutarate (αKG) to generate an enzyme-based oxidant which hydroxylates the Asn803 residue within the C-terminal transactivation domain (CTAD) of HIF-1α. Tight coupling of these two sequential reactions requires a structural linkage between the Fe(II) and the substrate binding site to ensure that O2 activation occurs after substrate binds. We tested the hypothesis that the facial triad carboxylate (Asp201) of FIH linked substrate binding and O2 binding sites. Asp201 variants of FIH were constructed and thoroughly characterized in vitro using steady-state kinetics, crystallography, autohydroxylation, and coupling measurements. Our studies revealed each variant activated O2 with a catalytic efficiency similar to that of wild-type (WT) FIH (kcat/KM(O2) = 0.17 μM−1 min−1), but led to defects in the coupling of O2 activation to substrate hydroxylation. Steady-state kinetics showed similar catalytic efficiencies for hydroxylation by WT-FIH (kcat/KM(CTAD) = 0.42 μM−1 min−1) and D201G (kcat/KM(CTAD) = 0.34 μM−1 min−1); hydroxylation by D201E was greatly impaired, while hydroxylation by D201A was undetectable. Analysis of the crystal structure of the D201E variant revealed steric crowding near the diffusible ligand site supporting a role for sterics from the facial triad carboxylate in the O2 binding order. Our data support a model in which the facial triad carboxylate Asp201 provides both steric and polar contacts to favor O2 access to the Fe(II) only after substrate binds, leading to coupled turnover in FIH and other αKG oxygenases.

Protein dynamics are crucial for the mechanistically ordered enzymes to bind to their substrate in the correct sequence and perform catalysis. Factor-inhibiting HIF-1 (FIH) is a nonheme Fe(II) αketoglutarate-dependent oxygenase that is a key hypoxia (low p O 2) sensor in humans. As these hypoxia-sensing enzymes follow a multistep chemical mechanism consuming α-ketoglutarate, a protein substrate that is hydroxylated, and O 2 , understanding protein flexibility and the order of substrate binding may aid in the development of strategies for selective targeting. The primary substrate of FIH is the C-terminal transactivation domain (CTAD) of hypoxia-inducible factor 1α (HIF) that is hydroxylated on the side chain of Asn803. We assessed changes in protein flexibility connected to metal and αKG binding, finding that (M+αKG) binding significantly stabilized the cupin barrel core of FIH as evidenced by enhanced thermal stability and decreased protein dynamics as assessed by global amide hydrogen/deuterium exchange mass spectrometry and limited proteolysis. Confirming predictions of the consensus mechanism, (M+αKG) increased the affinity of FIH for CTAD as measured by titrations monitoring intrinsic tryptophan fluorescence.The decreased protein dynamics caused by (M+αKG) enforces a sequentially ordered substrate binding sequence in which αKG binds before CTAD, suggesting that selective inhibition may require inhibitors that target the binding sites of both αKG and the prime substrate. A consequence of the correlation between dynamics and αKG binding is that all relevant ligands must be included in binding-based inhibitor screens, as shown by testing permutations of M, αKG, and inhibitor.

The interplay between active-site chemistry and functionally relevant enzyme motions can provide useful insights into selective enzyme modulation. Modulation of the hypoxia-sensing function of factor-inhibiting-HIF-1 (FIH) enzyme is a potential therapeutic strategy in disease states such as ischemia and cancer. The hypoxia-sensing function of FIH relies in major part on the tight coupling of the first half of the catalytic mechanism which involves O 2 activation and eventual succinate production to the second half which involves HIF-1α/ CTAD substrate hydroxylation. In this study, we demonstrate the role of a loop hinge domain in FIH (FIH102−118) called the 100s loop in maintaining this particular tight coupling. Molecular dynamics patterns from Gaussian Network Model (iGNM) database analysis of FIH identified the 100s loop as one dynamic domain containing a hinge residue (Tyr102) with a potential substrate positioning role. Enzymological and biophysical studies of the 100s loop point mutants revealed altered enzyme kinetics with the exception of the conservative FIH mutant Y102F, which suggests a sterics-related role for this residue. Removal of the bulk of Tyr102 (Y102A) resulted in succinate production, autohydroxylation, and an O 2 binding environment comparable to wild-type FIH. However, the HIF-1α/CTAD substrate hydroxylation of this mutant was significantly reduced which implies that (1) the FIH loop hinge residue Tyr102 does not affect O 2 activation, (2) the stacking steric interaction of Tyr102 is important in substrate positioning for productive hydroxylation, and (3) Tyr102 is important for the synchronization of O 2 activation and substrate hydroxylation.

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