Fig 1.
Hydroxylation of HIFα and the chemical structures of IOX4 and other PHD inhibitors used in this study.
(a) Prolyl-hydroxylation (as catalyzed by the PHDs) of HIFα. (b) Structures of the dihydropyrazoles (1 and IOX4) in comparison to structures of 2-oxoglutarate (2OG), N-oxalylglycine (NOG) (a catalytically inactive analogue of 2OG), dimethyloxalylglycine (DMOG) (a cell-permeable ester derivative of NOG) and IOX2 [9]. Chemical structures of previously reported PHD inhibitors (compound 2, bicyclic isoquinolinyl inhibitor IOX3 and bicyclic naphthalenylsulfone hydroxythiazole BNS) used in this study are also shown.
Table 1.
Selectivity profiling of the dihydropyrazoles 1 and IOX4 against a panel of human 2OG-dependent dioxygenases.
Fig 2.
Comparison of the binding modes of PHD inhibitors.
Views from crystal structures of PHD2 complexed with 1 (a), IOX3 (b), 2 (e) and NOG (h) Compound 1 coordinates the active site metal in a bidentate manner via the nitrogens of its pyridine (trans to His374 Nε2) and pyrazolone (trans to the Asp315 Oδ1) rings as shown in a. A model of IOX4 binding based on that of 1 (d) and the overlay of a and d (g) are shown for comparison. This coordination mode enables 1 to competitively inhibit PHD2 with respect to 2OG (as observed with the other inhibitors described here); the triazole ring of 1 is located in the 2OG C-5 carboxylate binding site whilst the carboxylate side chain of 1 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) [39]. Compare a, b and c for differences in binding modes between 1 and IOX3; a, e and f for differences between 1 and 2; a, h and i for differences between 1 and NOG. PDB ID: 4BQX (PHD2.IOX3) [9], 4BQW (PHD2.IOX2) [9]; 3HQR (PHD2.NOG.CODD) [39].
Fig 3.
Cellular inhibition of HIF prolyl-hydroxylases by IOX4 leads to HIFα induction.
(a-b) Immunoblots showing selective inhibition of the HIF1α prolyl-, over asparaginyl-hydroxylation in HIFα-stablized RCC4 cells by 1, IOX2 and IOX4. (c) Immunoblots showing the dose-dependent upregulation of HIF1α in HeLa cells by 1 and IOX4. (d-f) Immunoblots highlighting the dose-dependent induction of HIF1α in MCF-7 (d), Hep3B (e) and U2OS (f) cells by IOX2 and IOX4. Note the higher potency of IOX4 compared to IOX2, and the lack of inhibition of FIH-catalyzed HIF1α asparaginyl-hydroxylation at concentrations in which maximal HIF1α induction was observed. (g) The dose-dependent upregulation of HIF1α in MCF-7, Hep3B and U2OS cells by IOX2 and IOX4 as measured using a quantitative HIF1α immunoassay. Each data point represents the average signal ± standard deviation, n = 3. HyPro402: hydroxyproline402; HyPro564: hydroxyproline564; HyAsn803: hydroxyasparagine803; l.e.: long exposure. See Materials and Methods for details (including antibodies used).
Fig 4.
(a) Immunoblots showing HIF1α and HIF2α induction in various mouse tissues (liver, brain, kidney, heart) after 1 h treatment at equimolar concentrations of IOX2 (37.7 mg/kg), IOX4 (35 mg/kg) or dimethyl N-oxalylglycine DMOG (75 mg/kg). (b-c) Immunoblot showing dose-dependent induction of HIF1α and HIF2α in the mouse liver (b) and in the mouse brain (c) after 1 h treatment by various doses of IOX4 (17.5 to 70 mg/kg) in comparison to vehicle control and DMOG (160 mg/kg). n.s.: non-specific; l.e.: long exposure.