Hydroxylation of HIFα and the chemical structures of IOX4 and other PHD inhibitors used in this study.

<p><b>(a)</b> Prolyl-hydroxylation (as catalyzed by the PHDs) of HIFα. <b>(b)</b> Structures of the dihydropyrazoles (<b>1</b> and <b>IOX4</b>) in comparison to structures of 2-oxoglutarate (<b>2OG</b>), <i>N</i>-oxalylglycine (<b>NOG</b>) (a catalytically inactive analogue of 2OG), dimethyloxalylglycine (<b>DMOG</b>) (a cell-permeable ester derivative of NOG) and <b>IOX2</b> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132004#pone.0132004.ref009" target="_blank">9</a>]. Chemical structures of previously reported PHD inhibitors (compound <b>2</b>, bicyclic isoquinolinyl inhibitor <b>IOX3</b> and bicyclic naphthalenylsulfone hydroxythiazole <b>BNS</b>) used in this study are also shown.</p

Comparison of the binding modes of PHD inhibitors.

<p>Views from crystal structures of PHD2 complexed with <b>1</b> (<b>a</b>), <b>IOX3</b> (<b>b</b>), <b>2</b> (<b>e</b>) and <b>NOG</b> (<b>h</b>) Compound <b>1</b> coordinates the active site metal in a bidentate manner via the nitrogens of its pyridine (<i>trans</i> to His374 N<i>ε</i>2) and pyrazolone (<i>trans</i> to the Asp315 O<i>δ</i>1) rings as shown in <b>a</b>. A model of <b>IOX4</b> binding based on that of <b>1</b> (<b>d</b>) and the overlay of <b>a</b> and <b>d</b> (<b>g</b>) are shown for comparison. This coordination mode enables <b>1</b> to competitively inhibit PHD2 with respect to 2OG (as observed with the other inhibitors described here); the triazole ring of <b>1</b> is located in the 2OG C-5 carboxylate binding site whilst the carboxylate side chain of <b>1</b> makes electrostatic interaction with another arginine, R322 (1 carboxylate O–NH1 R322, 2.9 Å) that is located at the entrance of the active site; R322 is directly involved in substrate binding (P564/HIF1α CODD O–NH1 R322/PHD2, 2.6 Å; P564/CODD O–NH1 R322/PHD2, 2.8 Å) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132004#pone.0132004.ref039" target="_blank">39</a>]. Compare <b>a</b>, <b>b</b> and <b>c</b> for differences in binding modes between <b>1</b> and <b>IOX3</b>; <b>a</b>, <b>e</b> and <b>f</b> for differences between <b>1</b> and <b>2</b>; <b>a</b>, <b>h</b> and <b>i</b> for differences between <b>1</b> and <b>NOG</b>. PDB ID: 4BQX (PHD2.IOX3) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132004#pone.0132004.ref009" target="_blank">9</a>], 4BQW (PHD2.IOX2) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132004#pone.0132004.ref009" target="_blank">9</a>]; 3HQR (PHD2.NOG.CODD) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132004#pone.0132004.ref039" target="_blank">39</a>].</p

Selectivity profiling of the dihydropyrazoles 1 and IOX4 against a panel of human 2OG-dependent dioxygenases.

<p>The IC₅₀ values obtained reveal the selectivity of dihydropyrazoles <b>1</b> and <b>IOX4</b> for PHD2 in comparison with <b>IOX2</b> and <b>NOG</b>. Assays were carried out as previously reported [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132004#pone.0132004.ref009" target="_blank">9</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132004#pone.0132004.ref018" target="_blank">18</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132004#pone.0132004.ref019" target="_blank">19</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132004#pone.0132004.ref038" target="_blank">38</a>].</p><p>PHD2: HIF-prolyl hydroxylase-2, JMJD1A (KDM3A): Lysine-specific demethylase 3A, JMJD2A (KDM4A): Lysine-specific demethylase 4A, JMJD2C (KDM4C): Lysine-specific demethylase 4C, JMJD2E (KDM4E): Lysine-specific demethylase 4E, JMJD3 (KDM6B): Lysine-specific demethylase 6B, FBXL11 (KDM2A): Lysine-specific demethylase 2A, JARID1C (KDM5C): Lysine-specific demethylase 5C, BBOX: γ-butyrobetaine hydroxylase, FIH: factor inhibiting HIF, FTO: fat mass and obesity associated protein.</p><p>Selectivity profiling of the dihydropyrazoles 1 and IOX4 against a panel of human 2OG-dependent dioxygenases.</p

IOX4 induces HIFα in mice.

<p>(<b>a</b>) Immunoblots showing HIF1α and HIF2α induction in various mouse tissues (liver, brain, kidney, heart) after 1 h treatment at equimolar concentrations of <b>IOX2</b> (37.7 mg/kg), <b>IOX4</b> (35 mg/kg) or dimethyl <i>N</i>-oxalylglycine <b>DMOG</b> (75 mg/kg). (<b>b-c</b>) Immunoblot showing dose-dependent induction of HIF1α and HIF2α in the mouse liver (<b>b</b>) and in the mouse brain (<b>c</b>) after 1 h treatment by various doses of <b>IOX4</b> (17.5 to 70 mg/kg) in comparison to vehicle control and <b>DMOG</b> (160 mg/kg). n.s.: non-specific; l.e.: long exposure.</p

Selective Small Molecule Probes for the Hypoxia Inducible Factor (HIF) Prolyl Hydroxylases

The hypoxia inducible factor (HIF) system is central to the signaling of low oxygen (hypoxia) in animals. The levels of HIF-α isoforms are regulated in an oxygen-dependent manner by the activity of the HIF prolyl-hydroxylases (PHD or EGLN enzymes), which are Fe­(II) and 2-oxoglutarate (2OG) dependent oxygenases. Here, we describe biochemical, crystallographic, cellular profiling, and animal studies on PHD inhibitors including selectivity studies using a representative set of human 2OG oxygenases. We identify suitable probe compounds for use in studies on the functional effects of PHD inhibition in cells and in animals

Plant Growth Regulator Daminozide Is a Selective Inhibitor of Human KDM2/7 Histone Demethylases

The JmjC oxygenases catalyze the <i>N</i>-demethylation of <i>N</i>^ε-methyl lysine residues in histones and are current therapeutic targets. A set of human 2-oxoglutarate analogues were screened using a unified assay platform for JmjC demethylases and related oxygenases. Results led to the finding that daminozide (<i>N-</i>(dimethylamino)­succinamic acid, 160 Da), a plant growth regulator, selectively inhibits the KDM2/7 JmjC subfamily. Kinetic and crystallographic studies reveal that daminozide chelates the active site metal via its hydrazide carbonyl and dimethylamino groups