Reporter Ligand NMR Screening Method for 2-Oxoglutarate Oxygenase Inhibitors

Leung, Ivanhoe K. H.; Demetriades, Marina; Hardy, Adam P.; Lejeune, Clarisse; Smart, Tristan J.; Szöllössi, Andrea; Kawamura, Akane; Schofield, Christopher J.; Claridge, Timothy D. W.

doi:10.1021/jm301583m

Cited by 58 publications

(63 citation statements)

References 89 publications

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“…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. Among them, AS-MS assay and NMR assay can be used for quantitative and site-specific screening of ligand binding to PHD2, which are suitable for the early work.…”

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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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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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“…HIF prolyl-hydroxylation plays a central role in the animal hypoxic response via hydroxylation of HIF, a posttranslational modification that signals for HIF-α degradation in a physiologically relevant oxygen-dependent manner (10,11). Given the tractability of these enzymes as targets for pharmacological modulation by 2OG analogs and related compounds, elucidation of the function of related 2OG oxygenases in biology is an area of current interest (12,13).…”

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confidence: 99%

Sudestada1, a Drosophila ribosomal prolyl-hydroxylase required for mRNA translation, cell homeostasis, and organ growth

Katz

Acevedo

Loenarz

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Genome sequences predict the presence of many 2-oxoglutarate (2OG)-dependent oxygenases of unknown biochemical and biological functions in Drosophila. Ribosomal protein hydroxylation is emerging as an important 2OG oxygenase catalyzed pathway, but its biological functions are unclear. We report investigations on the function of Sudestada1 (Sud1), a Drosophila ribosomal oxygenase. As with its human and yeast homologs, OGFOD1 and Tpa1p, respectively, we identified Sud1 to catalyze prolyl-hydroxylation of the small ribosomal subunit protein RPS23. Like OGFOD1, Sud1 catalyzes a single prolyl-hydroxylation of RPS23 in contrast to yeast Tpa1p, where Pro-64 dihydroxylation is observed. RNAi-mediated Sud1 knockdown hinders normal growth in different Drosophila tissues. Growth impairment originates from both reduction of cell size and diminution of the number of cells and correlates with impaired translation efficiency and activation of the unfolded protein response in the endoplasmic reticulum. This is accompanied by phosphorylation of eIF2α and concomitant formation of stress granules, as well as promotion of autophagy and apoptosis. These observations, together with those on enzyme homologs described in the companion articles, reveal conserved biochemical and biological roles for a widely distributed ribosomal oxygenase.fruit fly | ribosome | dioxygenase | proline | tranlational stress I ron [Fe(II)]-and 2-oxoglutarate (2OG)-dependent oxygenases are a superfamily with diverse biochemical and biological functions. During 2OG oxygenase catalysis, substrate oxidation is coupled to decarboxylation of 2OG, yielding succinate and carbon dioxide (1, 2). Structural studies reveal that the catalytic domain of 2OG oxygenases contains a conserved double-stranded β-helix (DSBH) fold presenting an HXD. . .H facial triad motif that coordinates an Fe(II) cofactor (3, 4). These and other structural features have been used to predict the existence of multiple uncharacterized 2OG oxygenases. In contrast to microorganisms and plants where 2OG oxygenases catalyze a wide variety of oxidative reactions, in animals their biochemical activities appear limited to hydroxylations or demethylations via hydroxylation (1,5,6). Despite progress in making biochemical assignments, the physiological roles of most 2OG oxygenases predicted by bioinformatic analysis of animal genomes are unknown. For instance, we have identified ∼50 putative 2OG oxygenases in the Drosophila genome, but only a few are characterized (7,8).The function of Fatiga, the single Drosophila homolog of human hypoxia inducible transcription factor (HIF) prolyl-4-hydroxylases (PHDs), has been well studied in the context of oxygen sensing (9). HIF prolyl-hydroxylation plays a central role in the animal hypoxic response via hydroxylation of HIF, a posttranslational modification that signals for HIF-α degradation in a physiologically relevant oxygen-dependent manner (10, 11). Given the tractability of these enzymes as targets for pharmacological modulation by 2OG analogs and related co...

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“…70 Human 2OG oxygenases, such as the hypoxia inducible factor (HIF) hydroxylases 71 are validated drug targets for diseases such as cancer, inflammation, ischemia and anemia. 70 Human 2OG oxygenases, such as the hypoxia inducible factor (HIF) hydroxylases 71 are validated drug targets for diseases such as cancer, inflammation, ischemia and anemia.…”

Section: B-o Bond Formation: Boronate Ester Formationmentioning

confidence: 99%

Dynamic combinatorial chemistry: a tool to facilitate the identification of inhibitors for protein targets

2015

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Dynamic combinatorial chemistry (DCC) has emerged as a powerful strategy to identify ligands for biological targets given that it enables the target to direct the synthesis and amplification of its strongest binder(s) from the library of interconverting compounds. Since the first report of DCC applied to the discovery of binders for a protein, this elegant tool has been employed on a range of protein targets at various stages of medicinal-chemistry projects. A series of suitable, reversible reactions that are biocompatible have been established and the portfolio of analytical techniques is growing. Despite progress, in most cases, the libraries employed remain of moderate size. We present here the most recent advances in the field of DCC applied to protein targets, paying particular attention to the experimental conditions and analytical methods chosen.

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Reporter Ligand NMR Screening Method for 2-Oxoglutarate Oxygenase Inhibitors

Cited by 58 publications

References 89 publications

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

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

Sudestada1, a Drosophila ribosomal prolyl-hydroxylase required for mRNA translation, cell homeostasis, and organ growth

Dynamic combinatorial chemistry: a tool to facilitate the identification of inhibitors for protein targets

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