Aspartate/asparagine-β-hydroxylase: a high-throughput mass spectrometric assay for discovery of small molecule inhibitors

Abstract The human 2-oxoglutarate dependent oxygenase aspartate/asparagine-β-hydroxylase (AspH) catalyses the hydroxylation of Asp/Asn-residues in epidermal growth factor-like domains (EGFDs). AspH is upregulated on the surface of malign cancer cells; increased AspH levels correlate with tumour inva...

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Autores principales:	Lennart Brewitz, Anthony Tumber, Inga Pfeffer, Michael A. McDonough, Christopher J. Schofield
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2020
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/86f4a7c44ed5487c8df924d7752ecc2e
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spelling	oai:doaj.org-article:86f4a7c44ed5487c8df924d7752ecc2e2021-12-02T14:47:31ZAspartate/asparagine-β-hydroxylase: a high-throughput mass spectrometric assay for discovery of small molecule inhibitors10.1038/s41598-020-65123-92045-2322https://doaj.org/article/86f4a7c44ed5487c8df924d7752ecc2e2020-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-020-65123-9https://doaj.org/toc/2045-2322Abstract The human 2-oxoglutarate dependent oxygenase aspartate/asparagine-β-hydroxylase (AspH) catalyses the hydroxylation of Asp/Asn-residues in epidermal growth factor-like domains (EGFDs). AspH is upregulated on the surface of malign cancer cells; increased AspH levels correlate with tumour invasiveness. Due to a lack of efficient assays to monitor the activity of isolated AspH, there are few reports of studies aimed at identifying small-molecule AspH inhibitors. Recently, it was reported that AspH substrates have a non-canonical EGFD disulfide pattern. Here we report that a stable synthetic thioether mimic of AspH substrates can be employed in solid phase extraction mass spectrometry based high-throughput AspH inhibition assays which are of excellent robustness, as indicated by high Z’-factors and good signal-to-noise/background ratios. The AspH inhibition assay was applied to screen approximately 1500 bioactive small-molecules, including natural products and active pharmaceutical ingredients of approved human therapeutics. Potent AspH inhibitors were identified from both compound classes. Our AspH inhibition assay should enable the development of potent and selective small-molecule AspH inhibitors and contribute towards the development of safer inhibitors for other 2OG oxygenases, e.g. screens of the hypoxia-inducible factor prolyl-hydroxylase inhibitors revealed that vadadustat inhibits AspH with moderate potency.Lennart BrewitzAnthony TumberInga PfefferMichael A. McDonoughChristopher J. SchofieldNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 10, Iss 1, Pp 1-14 (2020)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Lennart Brewitz Anthony Tumber Inga Pfeffer Michael A. McDonough Christopher J. Schofield Aspartate/asparagine-β-hydroxylase: a high-throughput mass spectrometric assay for discovery of small molecule inhibitors
description	Abstract The human 2-oxoglutarate dependent oxygenase aspartate/asparagine-β-hydroxylase (AspH) catalyses the hydroxylation of Asp/Asn-residues in epidermal growth factor-like domains (EGFDs). AspH is upregulated on the surface of malign cancer cells; increased AspH levels correlate with tumour invasiveness. Due to a lack of efficient assays to monitor the activity of isolated AspH, there are few reports of studies aimed at identifying small-molecule AspH inhibitors. Recently, it was reported that AspH substrates have a non-canonical EGFD disulfide pattern. Here we report that a stable synthetic thioether mimic of AspH substrates can be employed in solid phase extraction mass spectrometry based high-throughput AspH inhibition assays which are of excellent robustness, as indicated by high Z’-factors and good signal-to-noise/background ratios. The AspH inhibition assay was applied to screen approximately 1500 bioactive small-molecules, including natural products and active pharmaceutical ingredients of approved human therapeutics. Potent AspH inhibitors were identified from both compound classes. Our AspH inhibition assay should enable the development of potent and selective small-molecule AspH inhibitors and contribute towards the development of safer inhibitors for other 2OG oxygenases, e.g. screens of the hypoxia-inducible factor prolyl-hydroxylase inhibitors revealed that vadadustat inhibits AspH with moderate potency.
format	article
author	Lennart Brewitz Anthony Tumber Inga Pfeffer Michael A. McDonough Christopher J. Schofield
author_facet	Lennart Brewitz Anthony Tumber Inga Pfeffer Michael A. McDonough Christopher J. Schofield
author_sort	Lennart Brewitz
title	Aspartate/asparagine-β-hydroxylase: a high-throughput mass spectrometric assay for discovery of small molecule inhibitors
title_short	Aspartate/asparagine-β-hydroxylase: a high-throughput mass spectrometric assay for discovery of small molecule inhibitors
title_full	Aspartate/asparagine-β-hydroxylase: a high-throughput mass spectrometric assay for discovery of small molecule inhibitors
title_fullStr	Aspartate/asparagine-β-hydroxylase: a high-throughput mass spectrometric assay for discovery of small molecule inhibitors
title_full_unstemmed	Aspartate/asparagine-β-hydroxylase: a high-throughput mass spectrometric assay for discovery of small molecule inhibitors
title_sort	aspartate/asparagine-β-hydroxylase: a high-throughput mass spectrometric assay for discovery of small molecule inhibitors
publisher	Nature Portfolio
publishDate	2020
url	https://doaj.org/article/86f4a7c44ed5487c8df924d7752ecc2e
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Aspartate/asparagine-β-hydroxylase: a high-throughput mass spectrometric assay for discovery of small molecule inhibitors

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