Abstract
Inhibition of the dioxygen sensing hypoxia-inducible factor prolyl hydroxylases has potential therapeutic benefit for treatment of diseases, including anaemia. We describe the discovery of a small-molecule probe useful for monitoring binding to human prolyl hydroxylase domain 2 (PHD2) via fluorescence polarisation. The assay is suitable for high-throughput screening of PHD inhibitors with both weak and strong affinities, as shown by work with clinically used inhibitors and naturally occurring PHD inhibitors.
Highlights
Zhihong Li,a Shuai Zhen,a Kaijun Su,a Anthony Tumber,b Quanwei Yu,a Ying Dong,a Michael McDonough, b Christopher J
We describe the discovery of a smallmolecule probe useful for monitoring binding to human prolyl hydroxylase domain 2 (PHD2) via fluorescence polarisation
Since hypoxia-inducible transcription factors (HIFs) targets induce erythropoietin and vascular endothelial growth factor, PHD inhibition is of therapeutic interest;[4] PHD inhibitors that are 2OG competitors have recently been approved for the treatment of anaemia in chronic kidney disease and others are in clinical trials.[5]
Summary
Zhihong Li,a Shuai Zhen,a Kaijun Su,a Anthony Tumber,b Quanwei Yu,a Ying Dong,a Michael McDonough, b Christopher J. ChemComm assays are not ideal for monitoring the binding of compounds with weak affinities, e.g., fragments and natural metabolites, including tricarboxylic acid (TCA) cycle related compounds.[13] Here we report the discovery of a fluorescence polarisation (FP) utilising affinity assay for PHD2; importantly, the assay employs the metal Fe(II) cofactor and is suitable for screening metal-binding fragment-like inhibitors.
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