Characterization of a Robust Enzymatic Assay for Inhibitors of 2-Oxoglutarate-Dependent Hydroxylases

Kanelakis, Kimon C.; Palomino, Heather L.; Li, Lina; Wu, Jiejun; Yan, Wen; Rosen, Mark D.; Rizzolio, Michèle; Trivedi, Meghana V.; Morton, Magda F.; Yang, Young Chul; Venkatesan, Hariharan; Rabinowitz, Michael H.; Shankley, Nigel P.; Barrett, Terrance D.

doi:10.1177/1087057109333976

Cited by 14 publications

(21 citation statements)

References 19 publications

(27 reference statements)

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“…The expression, purification, and enzymatic reaction of a human PHD2 construct containing amino acids 181 to 417 (PHD2 ) and a construct containing the full-length sequence for factor-inhibiting HIF (FIH) were described previously (Kanelakis et al, 2009). Full-length sequences of human PHD1, PHD2, and PHD3 were also cloned, expressed, and purified by Emerald Biostructures (Bainbridge Island, WA) using similar methods.…”

Section: Methodsmentioning

confidence: 99%

“…The optimization of the reaction conditions for PHD2 181-417 have been described previously and were used for all PHD enzymatic assays (Kanelakis et al, 2009). The protein concentration was selected to give a good signal-to-noise ratio (ϳ10-fold increase in signal above background), and the K M for 2-OG was determined by examining enzymatic activity over a range of 2-OG concentrations (0.1-20 M).…”

Section: Methodsmentioning

confidence: 99%

“…HIF-1␣ peptide residues 547 to 581 [KNPFSTGDTDLDLEM-LAPYIPMDDDFQLRSFDQLS] (10 M; California Peptide Research Inc., Napa, CA), and [5-14 C]2-oxoglutarate (50 mCi/mmol; Moravek Chemicals, Brea, CA) were incubated in a final volume of 0.5 ml of reaction buffer (40 mM Tris-HCl, pH 7.5, 0.4 mg/ml catalase, 0.5 mM dithiothreitol, and 1 mM ascorbate). The [1-14 C]succinate formed from the enzymatic reaction was separated from [5-14 C]2-oxoglutarate by incubating the reaction mixture with 100 l of 0.16 M 2,4-dinitrophenylhydrazine prepared in 30% perchloric acid and separated by centrifugation (Kanelakis et al, 2009).…”

Section: Methodsmentioning

confidence: 99%

“…The pK I was also estimated using the Cheng-Prussoff correction. A correction factor of 0.35 was calculated from the K M ϭ 0.8 M relative to the 1 M concentration of 2-OG used to determine the pIC 50 (Kanelakis et al, 2009).…”

Section: Methodsmentioning

confidence: 99%

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Pharmacological Characterization of 1-(5-Chloro-6-(trifluoromethoxy)-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic Acid (JNJ-42041935), a Potent and Selective Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor

Barrett¹,

Palomino²,

Brondstetter³

et al. 2011

Mol Pharmacol

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The hypoxia-inducible factor (HIF) prolyl hydroxylase (PHD) enzymes represent novel targets for the treatment of anemia, ulcerative colitis, and ischemic and metabolic disease inter alia. We have identified a novel small-molecule inhibitor of PHD, 1-(5-chloro-6-(trifluoromethoxy)-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic acid (JNJ-42041935), through structurebased drug design methods. The pharmacology of JNJ-42041935 was investigated in enzyme, cellular, and wholeanimal systems and was compared with other compounds described in the literature as PHD inhibitors. JNJ-42041935, was a potent (pK I ϭ 7.3-7.9), 2-oxoglutarate competitive, reversible, and selective inhibitor of PHD enzymes. In addition, JNJ-42041935 was used to compare the effect of selective inhibition of PHD to intermittent, high doses (50 g/kg i.p.) of an exogenous erythropoietin receptor agonist in an inflammationinduced anemia model in rats. JNJ-42041935 (100 mol/kg, once a day for 14 days) was effective in reversing inflammationinduced anemia, whereas erythropoietin had no effect. The results demonstrate that JNJ-42041935 is a new pharmacological tool, which can be used to investigate PHD inhibition and demonstrate that PHD inhibitors offer great promise for the treatment of inflammation-induced anemia.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Pharmacological Characterization of 1-(5-Chloro-6-(trifluoromethoxy)-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic Acid (JNJ-42041935), a Potent and Selective Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor

Barrett¹,

Palomino²,

Brondstetter³

et al. 2011

Mol Pharmacol

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“…A number of benzimidazole-2-glycinamides emerged as targets for chemical synthesis and subsequent evaluation as inhibitors, using a PHD2 enzymatic assay previously reported from this laboratory. 23 The synthesis of this initial set of potential inhibitors began with known or available benzimidazole-2-carboxylic acids 1a-h (Scheme 1). 24 These acids were coupled to amino esters 2a-h, which were then saponified to provide the corresponding benzimidazole acids 3a-h.…”

mentioning

confidence: 99%

Benzimidazole-2-pyrazole HIF Prolyl 4-Hydroxylase Inhibitors as Oral Erythropoietin Secretagogues

Rosen

Venkatesan

Peltier

et al. 2010

ACS Med. Chem. Lett.

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Development of a colorimetric α-ketoglutarate detection assay for prolyl hydroxylase domain (PHD) proteins

Wong

Ringel

Yuan

et al. 2021

Journal of Biological Chemistry

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Since the discovery of the prolyl hydroxylases domain (PHD) proteins and their canonical hypoxia-inducible factor (HIF) substrate two decades ago, a number of in vitro hydroxylation (IVH) assays for PHD activity have been developed to measure the PHD–HIF interaction. However, most of these assays either require complex proteomics mass spectrometry methods that rely on the specific PHD–HIF interaction or require the handling of radioactive material, as seen in the most commonly used assay measuring [ 14 C]O 2 release from labeled [ 14 C]α-ketoglutarate. Here, we report an alternative rapid, cost-effective assay in which the consumption of α-ketoglutarate is monitored by its derivatization with 2,4-dinitrophenylhydrazine (2,4-DNPH) followed by treatment with concentrated base. We extensively optimized this 2,4-DNPH α-ketoglutarate assay to maximize the signal-to-noise ratio and demonstrated that it is robust enough to obtain kinetic parameters of the well-characterized PHD2 isoform comparable with those in published literature. We further showed that it is also sensitive enough to detect and measure the IC 50 values of pan-PHD inhibitors and several PHD2 inhibitors in clinical trials for chronic kidney disease (CKD)-induced anemia. Given the efficiency of this assay coupled with its multiwell format, the 2,4-DNPH α-KG assay may be adaptable to explore non-HIF substrates of PHDs and potentially to high-throughput assays.

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Characterization of a Robust Enzymatic Assay for Inhibitors of 2-Oxoglutarate-Dependent Hydroxylases

Cited by 14 publications

References 19 publications

Pharmacological Characterization of 1-(5-Chloro-6-(trifluoromethoxy)-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic Acid (JNJ-42041935), a Potent and Selective Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor

Pharmacological Characterization of 1-(5-Chloro-6-(trifluoromethoxy)-1H-benzoimidazol-2-yl)-1H-pyrazole-4-carboxylic Acid (JNJ-42041935), a Potent and Selective Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor

Benzimidazole-2-pyrazole HIF Prolyl 4-Hydroxylase Inhibitors as Oral Erythropoietin Secretagogues

Development of a colorimetric α-ketoglutarate detection assay for prolyl hydroxylase domain (PHD) proteins

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