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

This probe is available from Cayman Chemical, Sigma and Tocris

For any inquiries please contact proberequests@thesgc.org.

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Overview

Biology of the hypoxia inducible factor (HIF) prolyl-hydroxylases

Levels of the Hypoxia Inducible Factor (HIF), 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 proteasome. The requirement of the HIF prolyl-hydroxylases (PHD or EGLN enzymes) for dioxygen as a co-substrate enables them to act as the hypoxia-sensing component of the HIF system. PHDs are members of the 2-oxoglutarate (2OG-dependent) oxygenase superfamily. Inhibition of the PHDs reduces HIFα prolyl-hydroxylation thereby 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.

IOX2: A chemical probe for HIF prolyl-hydroxylases

IOX2, a chemical probe for HIF prolyl-hydroxylases is the result of collaborations with the University of Sevilla, Genome Institute of Singapore, University of Sheffield, University of Oxford, BioNanotechnology Research Center KRIBB and the SGC [1].

Potency Against Target Family

IOX2 displayed an AlphaScreen IC50 of 22nM for inhibition of PHD2

Selectivity Beyond Target Family

IOX2 was found to be inactive against a panel of 55 receptors and ion channels (CEREP panel) at 10µM.

Co-crystal structure

The co-crystal structure of IOX2 with PHD2 has NOT been solved, however, there is a crystal structure with a close analogue (pdb id: 4BQW). This analogue has a methyl rather than benzyl group on the isoquinoline nitrogen. Please click on the 'Co-Crystal structures' tab above for more details.

Cellular Activity

IOX2 showed selective inhibition (by Western blot) of PHD catalysed HIF-1α prolyl-hydroxylation in a variety of human cell lines including renal carcinoma (RCC4) cell line lacking VHL, embryonic kidney (293T), bone osteosarcoma (U2OS) and RCC4 reexpressing VHL (RCC4/VHL) cells.

Properties

(1-benzyl-4-hydroxy-2-oxo-1, 2-dihydroquinoline-3-carbonyl)glycine

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Physical and chemical properties
Molecular weight	352.35
Molecular formula	C₁₉H₁₆N₂O₅
IUPAC name	(1-benzyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carbonyl)glycine
logP	-3.99
PSA
No. of chiral centres	0
No. of rotatable bonds	5
No. of hydrogen bond acceptors	7
No. of hydrogen bond donors	3
Storage	Store at -20°C
Dissolution	Soluble to 100 uM in DMSO

An inactive, negative control for IOX2 has not yet been identified

Selectivity Profile

Potency against Target

IOX2 displayed an AlphaScreen IC50 of 22nM for inhibition of PHD2; values for each data point are averages ± standard deviation (n≥3).

Selectivity against Histone Demethylases

IOX2 was profiled against a panel of human histone Nε-methyl lysine demethylase 2OG-oxygenases in an AlphaScreen assay to determine selectivity.

Selectivity Beyond Target Family

IOX2 was found to be inactive against a panel of 55 receptors and ion channels (CEREP panel) at 10µM.

Click to enlarge

Cell-based Assay Data

Engagement of IOX2 in an in vitro cell assay

Efficacy of IOX2 in a human renal carcinoma (RCC4) cell line lacking VHL (required for the degradation of hydroxylated HIF-α) was established, using antibodies selective for hydroxylated form(s) of a peptide corresponding to the HIF-1α C-terminal oxygen-dependent degradation domain to probe for PHD catalysed inhibition of HIF prolyl-hydroxylation in comparison to FIH catalysed asparaginyl-hydroxylation. IOX2 was further tested in VHL-competent cell lines, including human embryonic kidney (293T), human bone osteosarcoma (U2OS) and RCC4 reexpressing VHL (RCC4/VHL) cells, where increased HIF-1α levels were used as an indicator for PHD inhibition. IOX2 effectively increased HIF-1α levels in all tested VHL-competent cell lines showing that the inhibitory effects are independent of cell type. These results establish that IOX2 is a potent and selective cellular PHD inhibitor.

Selective inhibition of IOX2 in human cell lines. Selectivity of the tested inhibitors for HIF-1α prolyl- over asparaginyl- hydroxylation in RCC4 after 6h of treatment. Upregulation of HIF-1α by inhibitors in various cell types (after 6h of treatment): HEK293T, U2OS and RCC4 stably-transfected with C-HA-tagged wildtype VHL.

Co-crystal structures

A co-crystal structure of PHD2’s catalytic domain with a methyl group on the isoquinoline nitrogen, compound 1, (rather than benzyl group in IOX2) group has been solved (pdb id 4BQW).

Compound 1

The bicyclic heteroaromatic ring of IOX2 is sandwiched between the hydrophobic side chains of Tyr310, Met299 and Trp389. IOX2 coordinates Mn(II) (substituting for Fe(II)) in a bidentate fashion with the side chain amide-carbonyl and phenolic oxygen. The side chain carboxylate of IOX2 is positioned to hydrogen bond with Arg383 and Tyr329. The Tyr303 phenol is positioned to hydrogen bond to the isoquinoline-2-OH. The aromatic rings of IOX2 project through the active site opening and likely make a steric clash with the hydroxylated HIF-1α.

References

Selective Small Molecule Probes for the Hypoxia Inducible Factor (HIF) Prolyl Hydroxylases,
Rasheduzzaman Chowdhury, José Ignacio Candela-Lena, Mun Chiang Chan, David Jeremy Greenald, Kar Kheng Yeoh, Ya-Min Tian, Michael A. McDonough, Anthony Tumber, Nathan R. Rose, Ana Conejo-Garcia, Marina Demetriades, Mathavan Sinnakaruppan, Akane Kawamura, Myung Kyu Lee, Freek van Eeden, Christopher W. Pugh, Peter J. Ratcliffe, Christopher J. Schofield. ACS Chemical Biology 2013; 8: 1488-1496.
Plant Growth Regulator Daminozide Is a Selective Inhibitor of Human KDM2/7 Histone Demethylases,
Rose, N. R., Woon, E. C., Tumber, A., Walport, L. J., Chowdhury, R., Li, X. S., King, O. N., Lejeune, C., Ng, S. S., Krojer, T., Chan, M. C., Rydzik, A. M., Hopkinson, R. J., Che, K. H., Daniel, M., Strain-Damerell, C., Gileadi, C., Kochan, G., Leung, I. K., Dunford, J., Yeoh, K. K., Ratcliffe, P. J., Burgess-Brown, N., von Delft, F., Muller, S., Marsden, B., Brennan, P. E., McDonough, M. A., Oppermann, U., Klose, R. J., Schofield, C. J., and Kawamura, A., J. Med. Chem. 2012, 55, 6639−6643
Monoclonal antibody-based screening assay for factor inhibiting hypoxia-inducible factor inhibitors,
SH Lee, M Jeong Hee, Ah C. Eun, SE Ryu, and L Myung Kyu. Journal of Biomolecular Screening 2008; 13:494−503
Development of homogeneous luminescence assays for histone demethylase catalysis and binding,
Kawamura, A.; Tumber, A.; Rose, N. R.; King, O. N.; Daniel, M.; Oppermann, U.; Heightman, T. D.; Schofield, C. J. Anal Biochem, 2010, 404, 86-93