IOX2 (IOX 2) is a selective inhibitor of the Hypoxia Inducible Factor (HIF) Prolyl-Hydroxylases (PHD); active in cells with the IC50 value of 21 nM for PHD2/ELGN-1 and no inhibition at FIH (20uM).

ELGN1:21 nM, PHD2:21 nM

为了探究IOX2作为体内功能探针的实用性，通过在全生物体（即转基因斑马鱼[Danio rerio]）中测试IOX2以增强HIF信号传导，考察其效果。鉴于人类和斑马鱼中PHD3编码基因的表达受HIF调控，因此PHD3水平可作为HIF活性的指示。生成了一个斑马鱼低氧报告系列，该系列通过phd3启动子元素表达GFP。处理含有化合物（10 μM）的转基因野生型胚胎，在受精后3天进行2天的处理，相对于对照组，在肝脏中明显观察到phd3:EGFP表达的增加。使用IOX2[3]观察到GFP水平的显著增加。

Inhibition assays are carried out in 384-well white ProxiPlates in 10 μL of reaction volume. Standard reaction mixtures consisted of the compound (in 2% DMSO final concentration), enzyme mix (0.001 μM of PHD2, 10 μM of Fe(II), 100 μM of ascorbate) and peptide mix (0.06 μM of biotinylated C-terminal oxygen dependent degradation domain (CODD) peptide, 2 μM of 2OG) in 50 mM HEPES pH 7.5, 0.01% Tween-20 and 0.1% BSA buffer. Compounds (e.g., IOX2) are preincubated with the enzyme mix for 15 min before being incubated with peptide mix for 10 min at 22°C. Each reaction is quenched with 5 μL of 30 mM EDTA. The bead mix containing AlphaScreen beads is preincubated for 1h with a rabbit monoclonal antibody selective for hydroxy-HIF1α (Pro564) and are added to the wells for a further 1 h at 22°C. The plates are then analyzed with an Envision plate reader. The IC50 values are calculated using nonlinear regression with normalized dose-response fit using Prism GraphPad (n≥3)[1].

IOX2 is prepared in DMSO and stored, and then diluted with appropriate medium before use[1]. Both VHL-defective (renal carcinoma cells with an empty vector, RCC4) and VHL-competent cells human embryonic kidney HEK293T, osteosarcoma U2OS and RCC4/VHLHA (RCC4 stably transfected with C-terminal HA-tagged wt VHL) are used. Cells are treated with DMSO (control) and tested compounds (e.g., IOX2) (dissolved in DMSO except for DMOG which is dissolved in PBS and added directly to culture medium) for 4-5 h. Cell extracts are probed with antibodies to hydroxy-Pro564 (CODD-OH) and hydroxy-Asn803 (CAD-OH). HIF-1α band intensities are used to normalize hydroxylation signals[1].