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J Biol Chem. 2019 Nov 15;294(46):17654-17668. doi: 10.1074/jbc.RA119.010201. Epub 2019 Sep 3.

Physiologically relevant orthogonal assays for the discovery of small-molecule modulators of WIP1 phosphatase in high-throughput screens.

Author information

1
Synthetic Bioactive Molecules Section, Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
2
National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20850.
3
Laboratory of Cell Biology, Center for Cancer Research, NCI, National Institutes of Health, Bethesda, Maryland 20892.
4
Imaging Probe Development Center, NHLBI, National Institutes of Health, Rockville, Maryland 20850.
5
Reaction Biology Corporation, 1 Great Valley Parkway, Suite 2, Malvern, Pennsylvania 19355.
6
Francis Crick Institute, 1 Midland Road, London NW1 AT, United Kingdom.
7
National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20850 hallma@mail.nih.gov.
8
National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland 20850 coussensn@mail.nih.gov.

Abstract

WT P53-Induced Phosphatase 1 (WIP1) is a member of the magnesium-dependent serine/threonine protein phosphatase (PPM) family and is induced by P53 in response to DNA damage. In several human cancers, the WIP1 protein is overexpressed, which is generally associated with a worse prognosis. Although WIP1 is an attractive therapeutic target, no potent, selective, and bioactive small-molecule modulator with favorable pharmacokinetics has been reported. Phosphatase enzymes are among the most challenging targets for small molecules because of the difficulty of achieving both modulator selectivity and bioavailability. Another major obstacle has been the availability of robust and physiologically relevant phosphatase assays that are suitable for high-throughput screening. Here, we describe orthogonal biochemical WIP1 activity assays that utilize phosphopeptides from native WIP1 substrates. We optimized an MS assay to quantify the enzymatically dephosphorylated peptide reaction product in a 384-well format. Additionally, a red-shifted fluorescence assay was optimized in a 1,536-well format to enable real-time WIP1 activity measurements through the detection of the orthogonal reaction product, Pi We validated these two optimized assays by quantitative high-throughput screening against the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection and used secondary assays to confirm and evaluate inhibitors identified in the primary screen. Five inhibitors were further tested with an orthogonal WIP1 activity assay and surface plasmon resonance binding studies. Our results validate the application of miniaturized physiologically relevant and orthogonal WIP1 activity assays to discover small-molecule modulators from high-throughput screens.

KEYWORDS:

RapidFire; Wip1; assay; cancer; cancer therapy; enzyme kinetics; fluorescence; high-throughput screening (HTS); kinetics; mass spectrometry (MS); oncogene; phosphatase; phosphorylation; therapeutics

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