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Updated: Jan 20, 2026
High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes
Published on: March 3, 2015
Physiologically relevant orthogonal assays for the discovery of small-molecule modulators of WIP1 phosphatase in
Victor Clausse1, Dingyin Tao2, Subrata Debnath3
1Synthetic Bioactive Molecules Section, Laboratory of Bioorganic Chemistry, NIDDK, National Institutes of Health, Bethesda, Maryland 20892.
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.
Insights
WT P53-Induced Phosphatase 1 (WIP1) is a cancer target. Researchers developed new assays to find small-molecule inhibitors, successfully identifying potential drug candidates for cancer therapy.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- WT P53-Induced Phosphatase 1 (WIP1) is a serine/threonine protein phosphatase overexpressed in many human cancers, correlating with poor prognosis.
- WIP1 is a challenging therapeutic target due to difficulties in developing selective small-molecule modulators with good bioavailability.
- Lack of robust, physiologically relevant phosphatase assays hinders high-throughput screening for WIP1 inhibitors.
Purpose of the Study:
- To develop and validate orthogonal biochemical assays for WT P53-Induced Phosphatase 1 (WIP1) activity.
- To identify potent, selective small-molecule WIP1 inhibitors using high-throughput screening.
- To establish physiologically relevant assays suitable for miniaturized, high-throughput screening of phosphatase modulators.
Main Methods:
- Developed orthogonal biochemical WIP1 activity assays using phosphopeptides from native substrates.
- Optimized a mass spectrometry (MS) assay in a 384-well format and a fluorescence assay in a 1,536-well format for WIP1 activity measurement.
- Validated assays via quantitative high-throughput screening of the NCATS Pharmaceutical Collection and confirmed hits using secondary assays and surface plasmon resonance.
Main Results:
- Successfully optimized miniaturized, physiologically relevant, and orthogonal WIP1 activity assays.
- Identified and validated five small-molecule inhibitors of WIP1 activity through high-throughput screening.
- Demonstrated the utility of these assays for discovering modulators of challenging phosphatase targets.
Conclusions:
- The developed orthogonal WIP1 assays are effective for high-throughput screening and discovery of small-molecule modulators.
- These assays represent a significant advancement for targeting challenging enzymes like WIP1 in cancer therapy.
- The identified inhibitors warrant further investigation as potential therapeutic agents for WIP1-overexpressing cancers.
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