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Aspartate/asparagine-β-hydroxylase: a high-throughput mass spectrometric assay for discovery of small molecule inhibitors
DOI:
10.1038/s41598-020-65123-9
Authors:
Lennart
Brewitz
(University of Oxford)
,
Anthony
Tumber
(University of Oxford)
,
Inga
Pfeffer
(University of Oxford)
,
Michael A.
Mcdonough
(University of Oxford)
,
Christopher J.
Schofield
(University of Oxford)
Co-authored by industrial partner:
No
Type:
Journal Paper
Journal:
Scientific Reports
, VOL 10
State:
Published (Approved)
Published:
May 2020

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.
Journal Keywords: Biocatalysis; Chemical libraries; Post-translational modifications; Target validation; X-ray crystallography
Subject Areas:
Biology and Bio-materials,
Chemistry
Instruments:
I04-Macromolecular Crystallography
Added On:
11/06/2020 09:41
Documents:
s41598-020-65123-9.pdf
Discipline Tags:
Non-Communicable Diseases
Health & Wellbeing
Cancer
Biochemistry
Catalysis
Chemistry
Structural biology
Organic Chemistry
Life Sciences & Biotech
Technical Tags:
Diffraction
Macromolecular Crystallography (MX)