IOX2(Glycine)

Kinase Assay [1]
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].

Cell Assay [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].

Animal Administration [1]
Zebrafish[1] Phd3:gfpsh144/sh144 fish (Danio rerio) are incrossed to produce phd3:gfpsh144/sh144 embryos, these are raised at 28°C in E3 medium. The phd3:gfpsh144/sh144 line is a hypoxia reporter line created by BAC recombination of the phd3 reporter GFP construct. At 3 days post fertilization, potential inhibitors (e.g., IOX2) are added in fresh medium at 10 μM in 1% DMSO. The embryos are incubated with the compounds for a further 48 h. Embryos are anesthetized at 5 dpf by immersion in tricaine. Lateral view images of the embryos are taken using a fluorescent dissecting stereomicroscope (both bright-field and fluorescent). Fluorescent images are analyzed using Image J.

References:
[1]. Chowdhury R, et al. Selective small molecule probes for the hypoxia inducible factor (HIF) prolyl hydroxylases. ACS Chem Biol. 2013 Jul 19;8(7):1488-96. [2]. Deppe J, et al. Impairment of hypoxia-induced HIF-1α signaling in keratinocytes and fibroblasts by sulfur mustard is counteracted by a selective PHD-2 inhibitor. Arch Toxicol. 2016 May;90(5):1141-50.