Alpha screen assay

All reagents were diluted in 50 mM HEPES, 0.1 % BSA, pH 7.5 supplemented with 0.01 % Tween20 and allowed to equilibrate to room temperature prior to addition to plates. Catalytic turnover assays were run in 10 μM volumes in low-volume 384-well plates at RT. The reaction consisted of enzyme (0.5 ~ 25 nM), biotinylated substrate peptide (30 ~ 1000 nM), Fe(II) (1 ~ 10 μM), Ascorbate (100 μM), 2OG (5 ~ 40 μM) and run at RT. EDTA was used to quench the reaction (5 μM) and AlphaScreen donor (Streptavidin-conjugated) and acceptor (ProteinA-conjugated) beads preincubated with peptide product antibodies were added (5 μM). Plates were foil-sealed to protect from light, incubated at room temperature for 60 mins and read on a PHERAstar FS plate reader using an AlphaScreen 680 excitation/570 emission filter set. The final bead concentration in 20 μM reaction was 20 μM/mL. IC50 values were calculated in Prism 5.

Cell lines

RCC4 cells

Preparation method

The solubility of this compound in DMSO is >10 mM. General tips for obtaining a higher concentration: Please warm the tube at 37 ℃ for 10 minutes and/or shake it in the ultrasonic bath for a while. Stock solution can be stored below -20 ℃ for several months.

Reaction Conditions

50 μM

Applications

In RCC4 cells, IOX2 inhibited HIF-1α hydroxylation at 50 μM.

IOX2 is a potent and selective inhibitor of HIF-1α prolyl hydroxylase-2 (PHD2) with an IC50 of 21 nM for PHD2/ELGN-1 in a cell-free assay, IOX2 has shown >100-fold selectivity over JMJD2A, JMJD2C, JMJD2E, JMJD3, or the 2OG oxygenase FIH [1].

PHDs consist three isoforms, named PHD1, PHD2 and PHD3. PHDs are dioxygenases that utilize oxygen as co-substrate providing the molecular basis for the oxygen-sensing function of these enzymes. It has been reported that PHDs are strikingly sensitive to graded levels of hypoxia, mirroring the progressive increases in HIF-1α protein and DNA binding activity that are observed when cells are exposed to gradual hypoxia [2]. In RCC4 cells, IOX2 inhibited HIF-1α hydroxylation at 50 μM [1].

References:
[1] Murray J K, Balan C, Allgeier A M, et al.  Dipeptidyl-quinolone derivatives inhibit hypoxia inducible factor-1α prolyl hydroxylases-1,-2, and-3 with altered selectivity[J]. Journal of combinatorial chemistry, 2010, 12(5): 676-686.
[2] Berra E, Benizri E, Ginouvès A, et al.  HIF prolyl‐hydroxylase 2 is the key oxygen sensor setting low steady‐state levels of HIF‐1α in normoxia[J]. The EMBO journal, 2003, 22(16): 4082-4090.