Sensitivity of VHL-1–Regulated Genes to Defects in Extracellular Matrix-Associated Proteins

<div><p>RNase protection assays showing altered expression of VHL-1–regulated genes that are HIF-1 independent (upper six panels) and HIF-1 dependent (F22B5.4) in worms bearing mutations affecting (A) procollagen prolyl and lysyl hydroxylases and (B) other extracellular matrix-associated proteins. A common pattern of upregulation is observed in <i>hif-1; vhl-1, vhl-1, dpy-18, let-268, gon-1, mig-17,</i> and <i>unc-6</i> worms but not other mutants. This contrasts with the HIF-1–dependent gene F22B5.4, which is upregulated in <i>vhl-1</i> worms but none of the other mutants.</p> <p>(C) RNase protection assay for C01B4.9 illustrating DPY-18–mediated changes in expression that are independent of HIF-1.</p></div

Responses of VHL-1–Dependent, HIF-1–Independent Genes to <i>egl-9</i> Inactivation, Hypoxia, and 2-Oxoglutarate Dioxygenase Inhibitors

<p>RNase protection assays showing regulation of VHL-1–dependent, HIF-1–independent genes by (A) EGL-9 and hypoxia and (B) pharmacological inhibitors of 2-oxoglutarate dioxygenases: DIP and DMOG. None of the genes is regulated by EGL-9, but two genes (C01B4.7 and C01B4.8) show modest induction by hypoxia, DIP, and DMOG.</p

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