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

Rosen, Mark D.; Venkatesan, Hariharan; Peltier, Hillary M.; Bembenek, Scott D.; Kanelakis, Kimon C.; Zhao, Lucy X.; Leonard, Barry E.; Hocutt, Frances M.; Wu, Xiaodong; Palomino, Heather L.; Brondstetter, Theresa; Haugh, Peter V.; Cagnon, Laurence; Yan, Wen; Liotta, L.; Young, Andrew; Mirzadegan, Taraneh; Shankley, Nigel P.; Barrett, Terrance D.; Rabinowitz, Michael H.

doi:10.1021/ml100198y

Cited by 56 publications

(70 citation statements)

References 25 publications

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“…For example, our research on hypoxia sensing specifically centers on the chemical mechanism of FIH and PHD2, enzymes with very different active site geometry. 3,4 Despite this variety, the Fe cofactor in all αKG oxygenases contains a His 2 (Asp/Glu) facial triad of ligands near the top of the cupin barrel, with one exchangeable ligand site that is frequently occupied by H 2 O. When αKG binds as a bidentate ligand, two stereoisomers of the Fe, Anti and Clock, are observed (Fig.…”

Section: Introductionmentioning

confidence: 99%

Imposing function down a (cupin)-barrel: secondary structure and metal stereochemistry in the αKG-dependent oxygenases

2013

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The Fe(II)/αketoglutarate (αKG) dependent oxygenases catalyze a diverse range of reactions significant in biological processes such as antibiotic biosynthesis, lipid metabolism, oxygen sensing, and DNA and RNA repair. Although functionally diverse, the eight-stranded β-barrel (cupin) and HX(D/E)XnH facial triad motifs are conserved in this super-family of enzymes. Crystal structure analysis of 25 αKG oxygenases reveals two stereoisomers of the Fe cofactor, Anti and Clock, which differ in the relative position of the exchangeable ligand position and the primary substrate. Herein, we discuss the relationship between the chemical mechanism and the secondary coordination sphere of the αKG oxygenases, within the constraints of the stereochemistry of the Fe cofactor. Sequence analysis of the cupin barrel indicates that a small subset of positions constitute the second coordination sphere, which has significant ramifications for the structure of the ferryl intermediate. The competence of both Anti and Clock stereoisomers of Fe points to a ferryl intermediate that is 5 coordinate. The small number of conserved close contacts within the active sites of αKG oxygenases can be extended to chemically related enzymes, such as the αKG-dependent halogenases SyrB2 and CytC3, and the non-αKG dependent dioxygenases isopenicillin N synthase (IPNS) and cysteine dioxygenase (CDO).

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Section: Introductionmentioning

confidence: 99%

Imposing function down a (cupin)-barrel: secondary structure and metal stereochemistry in the αKG-dependent oxygenases

2013

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“…The novel PHD inhibitor described here, 1-(5-chloro-6-(trifluoromethoxy)-1H-benzo[d]imidazol-2-yl)-1H-pyrazole-4-carboxylic acid (JNJ-42041935), is the best characterized compound resulting from a medicinal chemistry program aimed at identifying novel inhibitors of PHD2 (Rosen et al, 2010). JNJ-42041935 is a potent, 2-OG-competitive, reversible, and selective inhibitor of all three PHD isozymes.…”

Section: Introductionmentioning

confidence: 99%

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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“…20 In the assay, excess of Mn II was used to PHD2 to ensure that only the metal-bound holo form was present. Additionally, a stable complex was formed by PHD2 with Mn II , 2-OG, and HIF1α peptide, 19 which indicates that the use of Mn II instead of Fe II has little influence on the binding property of PHD2 protein. Thus, we employ Mn II instead of Fe II as the native metal cofactor to avoid the hydroxylation of the probe FITC-HIF1α (556−574) while maintaining its binding affinity to PHD2 protein.…”

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confidence: 99%

Affinity-Based Fluorescence Polarization Assay for High-Throughput Screening of Prolyl Hydroxylase 2 Inhibitors

Lei

et al. 2015

ACS Med. Chem. Lett.

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Prolyl hydroxylase domain 2 (PHD2) enzyme, a Fe II and 2-oxoglutarate (2-OG) dependent oxygenase, mediates key physiological responses to hypoxia by modulating the levels of hypoxia inducible factor 1-α (HIF1α). PHD2 has been shown to have the therapeutic potentials for conditions including anemia and ischemic disease. Currently, many activity-based assays have been developed for identifying PHD2 inhibitors. Here we report an affinity-based fluorescence polarization method using FITC-labeled HIF1α (556−574) peptide as a probe for quantitative and site-specific screening of small molecule PHD2 inhibitors. KEYWORDS: PHD2, FITC-labeled probe, affinity-based, fluorescence polarization assay H ypoxia is linked to human diseases such as anemia and ischemia. 1,2 In humans, the response to hypoxia is mediated by up-regulation of the hypoxia inducible transcription factor (HIF). 3−5 HIF is a heterodimeric transcription factor composed of an oxygen-dependent α-subunit and constitutively expressed β-subunit. Under normoxic conditions, HIFα, the regulatory subunit of HIF dimer, is constitutively produced with a half-life of approximately 5 min. It is hydroxylated by prolyl hydroxylases1−3 (PHD1−3), recognized by von Hippel Lindau protein (pVHL), and then rapidly ubiquitylated and subsequently degraded by the 26S proteasome. 6 PHDs are members of the dioxygenase family that require O 2 , Fe II , and 2-oxoglutarate (2-OG) for their catalytic activity, which are responsible for the C4 trans hydroxylation of HIFα at Pro402 and Pro564 that initiates the path to protein degradation. 7 It is currently believed that PHD2 plays a dominant role in controlling the cellular HIFα levels. 8 Inhibitor of PHD2 has been pursued as a promising therapy for conditions including anemia and ischemic disease.To discover small molecules that can regulate PHD2 activity, many activity-based assays have been developed. The development of activity-based assay was based on the catalytic activity of PHD2, which utilizes 2-OG and oxygen as cosubstrates to catalyze the prolyl hydroxylations. 9−11 This property has led to the development of several generic activity-based assays, which detected the activity by measuring the ratio of HIFα peptide and its hydroxylated product, such as fluorescence-based assay using o-phenylenediamine, 12 MALDI-TOF MS, 13 AlphaScreen assay, 14 homogeneous time-resolved fluorescence assay, 15 and fluorescence polarization assay based on HIF-von Hippel− Lindau protein-Elongin B−Elongin C (VBC) interaction. 16 However, activity-based assays are not always well-suited to the initial stages of medicinal chemistry, for example, for fragmentbased screening, and are only possible when substrates are available. Recently, affinity-based assays that utilize nondenaturing electrospray ionization mass spectrometry (ESI-MS), 17 affinity selection mass spectroscopy assay (AS-MS), 15 or nuclear magnetic resonance (NMR) 18 technology have been developed for studying the binding of metal ions and small molecules with PHD2 protein. Amo...

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

Cited by 56 publications

References 25 publications

Imposing function down a (cupin)-barrel: secondary structure and metal stereochemistry in the αKG-dependent oxygenases

Imposing function down a (cupin)-barrel: secondary structure and metal stereochemistry in the αKG-dependent oxygenases

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

Affinity-Based Fluorescence Polarization Assay for High-Throughput Screening of Prolyl Hydroxylase 2 Inhibitors

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