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Potent and Selective Triazole-Based Inhibitors of the Hypoxia-Inducible Factor Prolyl-Hydroxylases with Activity in the Murine Brain.

Chan MC, Atasoylu O, Hodson E, Tumber A, Leung IK, Chowdhury R, Gómez-Pérez V, Demetriades M, Rydzik AM, Holt-Martyn J, Tian YM, Bishop T, Claridge TD, Kawamura A, Pugh CW, Ratcliffe PJ, Schofield CJ - PLoS ONE (2015)

Bottom Line: Therapeutic activation of the natural human hypoxic response can be achieved by the inhibition of the hypoxia sensors for the HIF system, i.e. the HIF prolyl-hydroxylases (PHDs).Here, we report studies on tricyclic triazole-containing compounds as potent and selective PHD inhibitors which compete with the 2-oxoglutarate co-substrate.One compound (IOX4) induces HIFα in cells and in wildtype mice with marked induction in the brain tissue, revealing that it is useful for studies aimed at validating the upregulation of HIF for treatment of cerebral diseases including stroke.

View Article: PubMed Central - PubMed

Affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, United Kingdom; Centre for Cellular and Molecular Physiology, University of Oxford, Oxford, United Kingdom.

ABSTRACT
As part of the cellular adaptation to limiting oxygen availability in animals, the expression of a large set of genes is activated by the upregulation of the hypoxia-inducible transcription factors (HIFs). Therapeutic activation of the natural human hypoxic response can be achieved by the inhibition of the hypoxia sensors for the HIF system, i.e. the HIF prolyl-hydroxylases (PHDs). Here, we report studies on tricyclic triazole-containing compounds as potent and selective PHD inhibitors which compete with the 2-oxoglutarate co-substrate. One compound (IOX4) induces HIFα in cells and in wildtype mice with marked induction in the brain tissue, revealing that it is useful for studies aimed at validating the upregulation of HIF for treatment of cerebral diseases including stroke.

No MeSH data available.


Related in: MedlinePlus

Cellular inhibition of HIF prolyl-hydroxylases by IOX4 leads to HIFα induction.(a-b) Immunoblots showing selective inhibition of the HIF1α prolyl-, over asparaginyl-hydroxylation in HIFα-stablized RCC4 cells by 1, IOX2 and IOX4. (c) Immunoblots showing the dose-dependent upregulation of HIF1α in HeLa cells by 1 and IOX4. (d-f) Immunoblots highlighting the dose-dependent induction of HIF1α in MCF-7 (d), Hep3B (e) and U2OS (f) cells by IOX2 and IOX4. Note the higher potency of IOX4 compared to IOX2, and the lack of inhibition of FIH-catalyzed HIF1α asparaginyl-hydroxylation at concentrations in which maximal HIF1α induction was observed. (g) The dose-dependent upregulation of HIF1α in MCF-7, Hep3B and U2OS cells by IOX2 and IOX4 as measured using a quantitative HIF1α immunoassay. Each data point represents the average signal ± standard deviation, n = 3. HyPro402: hydroxyproline402; HyPro564: hydroxyproline564; HyAsn803: hydroxyasparagine803; l.e.: long exposure. See Materials and Methods for details (including antibodies used).
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pone.0132004.g003: Cellular inhibition of HIF prolyl-hydroxylases by IOX4 leads to HIFα induction.(a-b) Immunoblots showing selective inhibition of the HIF1α prolyl-, over asparaginyl-hydroxylation in HIFα-stablized RCC4 cells by 1, IOX2 and IOX4. (c) Immunoblots showing the dose-dependent upregulation of HIF1α in HeLa cells by 1 and IOX4. (d-f) Immunoblots highlighting the dose-dependent induction of HIF1α in MCF-7 (d), Hep3B (e) and U2OS (f) cells by IOX2 and IOX4. Note the higher potency of IOX4 compared to IOX2, and the lack of inhibition of FIH-catalyzed HIF1α asparaginyl-hydroxylation at concentrations in which maximal HIF1α induction was observed. (g) The dose-dependent upregulation of HIF1α in MCF-7, Hep3B and U2OS cells by IOX2 and IOX4 as measured using a quantitative HIF1α immunoassay. Each data point represents the average signal ± standard deviation, n = 3. HyPro402: hydroxyproline402; HyPro564: hydroxyproline564; HyAsn803: hydroxyasparagine803; l.e.: long exposure. See Materials and Methods for details (including antibodies used).

Mentions: We then tested the cellular inhibition of 1 and IOX4 using a von Hippel-Lindau (VHL)-defective human renal cell carcinoma (RCC4) cell line, in which HIFα proteins are constitutively stabilized [22]. In this cell line, the extent of the differential HIF1α prolyl hydroxylation at the N-terminal and C-terminal oxygen-dependent degradation domains (HyPro402 and HyPro564, respectively), as well as HIF1α asparaginyl hydroxylation at the C-terminal transactivation domain (HyAsn803) can be interrogated by immunoblotting using hydroxylation specific antibodies [22,23]. Dimethyloxalylglycine, DMOG (a cell-permeable ester of the generic 2OG-dependent dioxygenase inhibitor, NOG) was used as a control. The results show that both 1 and IOX4 block prolyl-hydroxylation of HIF1α at both NODD (HyPro402) and CODD (HyPro564) without markedly affecting the levels of PHD2, consistent with their proposed mode of action via the displacement of 2OG (Fig 3A and 3B). IOX4 was clearly more potent in inhibiting HIF1α prolyl-hydroxylation than IOX2 (Fig 3B); some apparent inhibition of HIF1α asparaginyl-hydroxylation (HyAsn803) was observed at 50 μM of IOX4. In VHL-competent HeLa cells, both 1 and IOX4 induced HIF1α, with IOX4 being substantially more potent (activity being observed at ≥ 1 μM, Fig 3C). Induction of HIF1α levels were observed in MCF-7, Hep3B and U2OS cells treated with IOX2 and IOX4, with IOX4 apparently being markedly more potent than IOX2 (Fig 3D–3F). These cellular results are consistent with the in vitro data indicating that IOX4 is a substantially more potent PHD inhibitor than IOX2.


Potent and Selective Triazole-Based Inhibitors of the Hypoxia-Inducible Factor Prolyl-Hydroxylases with Activity in the Murine Brain.

Chan MC, Atasoylu O, Hodson E, Tumber A, Leung IK, Chowdhury R, Gómez-Pérez V, Demetriades M, Rydzik AM, Holt-Martyn J, Tian YM, Bishop T, Claridge TD, Kawamura A, Pugh CW, Ratcliffe PJ, Schofield CJ - PLoS ONE (2015)

Cellular inhibition of HIF prolyl-hydroxylases by IOX4 leads to HIFα induction.(a-b) Immunoblots showing selective inhibition of the HIF1α prolyl-, over asparaginyl-hydroxylation in HIFα-stablized RCC4 cells by 1, IOX2 and IOX4. (c) Immunoblots showing the dose-dependent upregulation of HIF1α in HeLa cells by 1 and IOX4. (d-f) Immunoblots highlighting the dose-dependent induction of HIF1α in MCF-7 (d), Hep3B (e) and U2OS (f) cells by IOX2 and IOX4. Note the higher potency of IOX4 compared to IOX2, and the lack of inhibition of FIH-catalyzed HIF1α asparaginyl-hydroxylation at concentrations in which maximal HIF1α induction was observed. (g) The dose-dependent upregulation of HIF1α in MCF-7, Hep3B and U2OS cells by IOX2 and IOX4 as measured using a quantitative HIF1α immunoassay. Each data point represents the average signal ± standard deviation, n = 3. HyPro402: hydroxyproline402; HyPro564: hydroxyproline564; HyAsn803: hydroxyasparagine803; l.e.: long exposure. See Materials and Methods for details (including antibodies used).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4492579&req=5

pone.0132004.g003: Cellular inhibition of HIF prolyl-hydroxylases by IOX4 leads to HIFα induction.(a-b) Immunoblots showing selective inhibition of the HIF1α prolyl-, over asparaginyl-hydroxylation in HIFα-stablized RCC4 cells by 1, IOX2 and IOX4. (c) Immunoblots showing the dose-dependent upregulation of HIF1α in HeLa cells by 1 and IOX4. (d-f) Immunoblots highlighting the dose-dependent induction of HIF1α in MCF-7 (d), Hep3B (e) and U2OS (f) cells by IOX2 and IOX4. Note the higher potency of IOX4 compared to IOX2, and the lack of inhibition of FIH-catalyzed HIF1α asparaginyl-hydroxylation at concentrations in which maximal HIF1α induction was observed. (g) The dose-dependent upregulation of HIF1α in MCF-7, Hep3B and U2OS cells by IOX2 and IOX4 as measured using a quantitative HIF1α immunoassay. Each data point represents the average signal ± standard deviation, n = 3. HyPro402: hydroxyproline402; HyPro564: hydroxyproline564; HyAsn803: hydroxyasparagine803; l.e.: long exposure. See Materials and Methods for details (including antibodies used).
Mentions: We then tested the cellular inhibition of 1 and IOX4 using a von Hippel-Lindau (VHL)-defective human renal cell carcinoma (RCC4) cell line, in which HIFα proteins are constitutively stabilized [22]. In this cell line, the extent of the differential HIF1α prolyl hydroxylation at the N-terminal and C-terminal oxygen-dependent degradation domains (HyPro402 and HyPro564, respectively), as well as HIF1α asparaginyl hydroxylation at the C-terminal transactivation domain (HyAsn803) can be interrogated by immunoblotting using hydroxylation specific antibodies [22,23]. Dimethyloxalylglycine, DMOG (a cell-permeable ester of the generic 2OG-dependent dioxygenase inhibitor, NOG) was used as a control. The results show that both 1 and IOX4 block prolyl-hydroxylation of HIF1α at both NODD (HyPro402) and CODD (HyPro564) without markedly affecting the levels of PHD2, consistent with their proposed mode of action via the displacement of 2OG (Fig 3A and 3B). IOX4 was clearly more potent in inhibiting HIF1α prolyl-hydroxylation than IOX2 (Fig 3B); some apparent inhibition of HIF1α asparaginyl-hydroxylation (HyAsn803) was observed at 50 μM of IOX4. In VHL-competent HeLa cells, both 1 and IOX4 induced HIF1α, with IOX4 being substantially more potent (activity being observed at ≥ 1 μM, Fig 3C). Induction of HIF1α levels were observed in MCF-7, Hep3B and U2OS cells treated with IOX2 and IOX4, with IOX4 apparently being markedly more potent than IOX2 (Fig 3D–3F). These cellular results are consistent with the in vitro data indicating that IOX4 is a substantially more potent PHD inhibitor than IOX2.

Bottom Line: Therapeutic activation of the natural human hypoxic response can be achieved by the inhibition of the hypoxia sensors for the HIF system, i.e. the HIF prolyl-hydroxylases (PHDs).Here, we report studies on tricyclic triazole-containing compounds as potent and selective PHD inhibitors which compete with the 2-oxoglutarate co-substrate.One compound (IOX4) induces HIFα in cells and in wildtype mice with marked induction in the brain tissue, revealing that it is useful for studies aimed at validating the upregulation of HIF for treatment of cerebral diseases including stroke.

View Article: PubMed Central - PubMed

Affiliation: Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, United Kingdom; Centre for Cellular and Molecular Physiology, University of Oxford, Oxford, United Kingdom.

ABSTRACT
As part of the cellular adaptation to limiting oxygen availability in animals, the expression of a large set of genes is activated by the upregulation of the hypoxia-inducible transcription factors (HIFs). Therapeutic activation of the natural human hypoxic response can be achieved by the inhibition of the hypoxia sensors for the HIF system, i.e. the HIF prolyl-hydroxylases (PHDs). Here, we report studies on tricyclic triazole-containing compounds as potent and selective PHD inhibitors which compete with the 2-oxoglutarate co-substrate. One compound (IOX4) induces HIFα in cells and in wildtype mice with marked induction in the brain tissue, revealing that it is useful for studies aimed at validating the upregulation of HIF for treatment of cerebral diseases including stroke.

No MeSH data available.


Related in: MedlinePlus