Screening chelating inhibitors of HIF-prolyl hydroxylase domain 2 (PHD2) and factor inhibiting HIF (FIH)

Abstract

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.

Imposing function down a (cupin)-barrel: secondary structure and metal stereochemistry in the αKG-dependent oxygenases

“…In a comparison of inhibitor selectivity toward FIH and PHD2, it was demonstrated that certain functional groups distinguished between the two active sites. 11,14 It was shown that bulky and chiral inhibitors can confer selectivity between FIH and PHD2, as derivatization of the N-oxalylglycine (NOG) framework with chiral amino acid side chains showed that all D-isomers were stronger inhibitors of FIH over PHD2. 11 In related studies demonstrating the potential for further inhibitor selectivity, stereospecific derivatization at the C1 position of NOG did not increase potency when tested with the JMJD2E enzyme; 12 however, the hydrophobic interactions gained from derivatization improved selectivity between αKG dependent hydroxylases.…”

“…11 A related study showed that FIH was more indulgent of sterically bulky inhibitors than PHD2, with EPR data suggesting that inhibitors were more conformationally constrained in the PHD2 active site. 14 …”

Oxygen sensing strategies in mammals and bacteria

“…Although a number of synthetic compounds have been reported to inhibit these enzymes [81-83], challenges include improving both selectivity [81] and the relatively low affinity of the inhibitors (IC 50 > 1μM). Inhibitors for FIH and PHD2 are typically structural mimics of αKG, able to bind to the Fe(II) cofactor via one or two ligation points, opening up the likelihood of cross-reactivity with other αKG oxygenases.…”

Substrate preference of the HIF-prolyl hydroxylase-2 (PHD2) and substrate-induced conformational change

“…Third is the pK A = 7.2 of the Fe 2+ -OH 2 group in (Fe+2OG)PHD2 [23], leading to a significant decrease in activity as pH is increased above the p K a [27]. Based on these divergent Michaelis constants, and the potential for uncoupled oxidative decarboxylation to inflate the observed rates, we felt that the substrate preference and rates could be better defined by directly measuring the formation of hydroxylated product (ODD OH ) using MALDI-TOF [28, 29]. …”