IOX2: A selective Inhibitor of the Hypoxia Inducible Factor (HIF) Prolyl-Hydroxylases

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Overview

2-(1-benzyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetic acid

Levels of the Hypoxia Inducible Factor (HIF), which is a master regulator of the cellular-response to hypoxia, are regulated by the post-translational modification of prolyl-residues in oxygen-dependent degradation domains in the HIFα subunit. HIFα prolyl-hydroxylation signals for its degradation by the proteosome. The requirement of the HIF prolyl-hydroxylases (PHD or EGLN enzymes) for dioxygen as a cosubstrate enables them to act as the hypoxia-sensing component of the HIF system. The PHDs are members of the 2-oxoglutarate (2OG) oxygenase superfamily. Inhibition of the PHDs reduces HIFα prolyl-hydroxylation so elevating HIF levels and artificially inducing the hypoxic response. Because HIF-target genes include those encoding for biomedically important proteins such as erythropoietin (EPO) and vascular endothelial growth factor (VEGF). PHD inhibition is of considerable medical interest. However, many research studies on HIF have employed non-selective 2OG oxygenase inhibitors. IOX2 is a selective PHD inhibitor which is active in cells.

Properties

Physical and chemical properties
Molecular weight	352.11
Molecular formula	C19H16N2O5
IUPAC name	2-(1-benzyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetic acid
logP	0.73
PSA	106.94
Storage	-20°C as powder
Dissolution	Soluble in DMSO at least up to 10mM

2-(1-benzyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamido)acetic acid
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SMILES:

C(C(O)=O)NC(C1=C(c2ccccc2N(Cc2ccccc2)C1=O)O)=O

InChI:

InChI=1S/C19H16N2O5/c22-15(23)10-20-18(25)16-17(24)13-8-4-5-9-14(13)21(19(16)26)11-12-6-2-1-3-7-12/h1-9,24H,10-11H2,(H,20,25)(H,22,23)

InChIKey:

CAOSCCRYLYQBES-UHFFFAOYSA-N

Selectivity Profile

Selectivity
	IC50 µm
PHD2/ELGN-1	0.021
FIH	No inhibition (20µM)
KDM4A/JMJD2A	<100µM (65% inhibition at 100µM)
KDM4C/JMJD2C	<100µM (49% inhibition at 100µM)
KDM4E/JMJD2E	<100µM (47% inhibition at 100µM)
KDM6B/JMJD3	<100µM (14% inhibition at 100µM)

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 10µL volumes in low-volume 384-well plates (ProxiPlateTM-384 Plus, PerkinElmer, USA) at RT (Kawamura et al. 2010). The reaction consisted of enzyme (0.5-25nM), biotinylated substrate peptide (30-1000nM), Fe(II) (1-10µM), Ascorbate (100µM), 2OG (5-40µM) and run at RT. EDTA was used to quench the reaction (5µL) and AlphaScreen donor (Streptavidin-conjugated) and acceptor (ProteinA-conjugated) beads preincubated with peptide product antibodies were added (5µL). Plates were foil-sealed to protect from light, incubated at room temperature for 60 minutes and read on a PHERAstar FS plate reader (BMG Labtech, Germany) using an AlphaScreen 680 excitation/570 emission filter set. The final bead concentration in 20µL reaction was 20µg/mL. IC₅₀ values were calculated in Prism 5 after normalization against corresponding DMSO controls

Cell-based Assay Data

Potency in Cells
PHD (RCC4 cells, western blot)	Inhibition of HIF1α hydroxylation levels (hydroxylated-Pro564) at 50µM
FIH (RCC4 cells, western blot)	No inhibition of HIF1α hydroxylation levels (hydroxylated-Asn803) at 250µM

References

King, O. N. F., Li, X. S., Sakurai, M., Kawamura, A., Rose, N. R., Ng, S. S., Quinn, A. M., Rai, G., Mott, B. T., Beswick, P., Klose, R. J., Oppermann, U., Jadhav, A., Heightman, T. D., Maloney, D. J., Schofield, C. J. & Simeonov, A. (2010). PLoS ONE 5, e15535.
Kawamura, A., Tumber, A., Rose, N. R., King, O. N., Daniel, M., Oppermann, U., Heightman, T. D. & Schofield, C. (2010). Anal Biochem 404, 86-93.
Rose, N.R., Ng, S.S., Mecinovic, J., Lienard, B.M., Bello, S.H., Sun, Z., McDonough, M.A., Oppermann, U., and Schofield, C.J. (2008). J Med Chem 51, 7053-7056.
Hewitson, K.S., Holmes, S.L., Ehrismann, D., Hardy, A.P., Chowdhury, R., Schofield, C.J., and McDonough, M.A. (2008). J Biol Chem 283, 25971-25978.
Flashman, E., Bagg, E.A., Chowdhury, R., Mecinovic, J., Loenarz, C., McDonough, M.A., Hewitson, K.S., and Schofield, C.J. (2007). Journal of Biological Chemistry 283, 3808-3815.
Tian YM, Yeoh KK, Lee MK, Eriksson T, Kessler BM, Kramer HB, Edelmann MJ, Willam C, Pugh CW, Schofield CJ, Ratcliffe PJ.(2011). Differential sensitivity of hypoxia inducible factor hydroxylation sites to hypoxia and hydroxylase inhibitors. Journal of Biological Chemistry 286(15), 13041-51.

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IOX2: A selective Inhibitor of the Hypoxia Inducible Factor (HIF) Prolyl-Hydroxylases

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