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Hypoxia Signaling Modulation

Conventionally, the study of hypoxic responses has relied on the use of monolayer cell cultures exposed to low oxygen conditions (e.g., 0.02-5% O2) by incubation in gas controlled chambers or incubators with a specific mixture of gases (e.g., 95% N2, 5% CO2- anoxia). However, studying hypoxia signaling can forgo gas exchange requirements by the use of hypoxia-mimetic agents.


  1. Cobalt Chloride (CoCl2) Cobalt inhibits the hydroxylation of HIF-alpha by binding to the iron-binding domain of Prolyl hydroxylases (PHDs). In the absence of Proline 564 hydroxylation, HIF-1 alpha is stabilized and not targeted for ubiquitination and degradation. Cobalt stabilizes HIF-alpha by inhibiting its interaction with the von Hippel-Lindau protein (pVHL). Cobalt binds to the oxygen-dependent degradation domain (ODDD) of hydroxylated HIF-alpha preventing its interaction with pVHL and proteasomal degradation.

  2. Deferoxamine/Desferrioxamine (DFO/DFX) DFO and DFX are iron chelators. By decreasing intracellular iron levels, these agents inhibit the activity of PHDs which require iron ions as cofactors for the hydroxylation of HIF-alpha subunits. The use of DFO or DFX stabilizes HIF-alpha subunits.

  3. Dimethyloxalylglycine (DMOG) DMOG is an ester of N-oxalylglycine and a competitive inhibitor of PHD enzymes that increases the stability of HIF-1 alpha.


HIF hydroxylation by Prolyl hydroxylase

Other biochemical based approaches rely on the use of small molecules to target different steps in HIF signaling. Using inhibitors of key steps or enzymes involved in the regulation of HIF expression helps to uncover how hypoxia-induced signaling operates in specific systems or models under study.

Learn more with Hypoxic and Simulated-Hypoxic Cell Lysate Protocol


Small Molecules: HIF Modulators

hypoxia inducers and inhibitors

Modulators

Mechanism

Potency*

TAT-cyclo-CLLFVY

(5582)

Selective inhibitor of HIF-1α and HIF-1β dimerization

1.3 μM (IC50)

GN 44028

(5655)

Potent inhibitor of HIF-1α transcriptional activity

14 nM (IC50)

TC-S 7009

(5243)

High affinity and selective HIF-2α inhibitor

81 nM (Kd)

Echinomycin

(5520)

Highly potent and selective HIF-1α inhibitor

29.4 pM (IC50)

DMOG

(4408)

Inhibitor of HIF-1α prolylhydroxylase; increases HIF-1α levels

 

VH 298

(6156)

Inhibitor of E3 ubiquitin ligase VHL, Inhibits interaction between VHL and HIF-1α

80-90 nM (Kd)

ML 228

(4565)

HIF pathway activator, Iron chelator

1.23-1.4 μM (EC50)

Available at www.tocris.com *Potency of the various inhibitors and inducers may be system and cell type dependent.


Learn about Methods to Measure the Hypoxic Response