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Updated: Aug 16, 2025
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Published on: September 9, 2021
A High-Throughput Fluorescence Polarization-Based Assay for the SH2 Domain of STAT4
Angela Berg1, Martin Gräber1,2, Sebastian Schmutzler3
1Institute of Organic Chemistry, Leipzig University, Johannisallee 29, 04103 Leipzig, Germany.
Abstract:
The signal transducer and activation of transcription (STAT) proteins are a family of Src homology 2 (SH2) domain-containing transcription factors. The family member STAT4 is a mediator of IL-12 signalling and has been implicated in the pathogenesis of multiple autoimmune diseases. The activity of STAT4 requires binding of phosphotyrosine-containing motifs to its SH2 domain. Selective inhibitors of the STAT4 SH2 domain have not been published to date. Here, we present a fluorescence polarization-based assay for the identification of inhibitors of the STAT4 SH2 domain. The assay is based on the interaction between the STAT4 SH2 domain and the fluorophore-labelled peptide 5-carboxyfluorescein-GpYLPQNID (Kd = 34 ± 4 nM). The assay is stable with respect to DMSO concentrations of up to 10% and incubation times of at least 8 h. The Z'-value of 0.85 ± 0.01 indicates that the assay is suited for use in high-throughput screening campaigns aimed at identifying new therapeutic modalities for the treatment of autoimmune diseases.
Insights
Researchers developed a new assay to find inhibitors for STAT4, a protein involved in autoimmune diseases. This high-throughput screening method targets the STAT4 SH2 domain, potentially leading to new treatments.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Signal transducer and activation of transcription (STAT) proteins are key transcription factors.
- STAT4 mediates IL-12 signaling and is implicated in autoimmune disease pathogenesis.
- STAT4 activity depends on phosphotyrosine-motif binding to its SH2 domain.
Purpose of the Study:
- To develop a fluorescence polarization-based assay for identifying STAT4 SH2 domain inhibitors.
- To establish a reliable method for high-throughput screening (HTS) of potential autoimmune disease therapeutics.
Main Methods:
- Utilized a fluorophore-labeled peptide (5-carboxyfluorescein-GpYLPQNID) to probe STAT4 SH2 domain interaction.
- Assessed assay stability across varying DMSO concentrations (up to 10%) and incubation times (at least 8 hours).
- Validated assay performance with a Z'-value of 0.85 ± 0.01, indicating suitability for HTS.
Main Results:
- Established a robust fluorescence polarization assay for STAT4 SH2 domain inhibitor screening.
- Demonstrated assay stability and high Z'-value, confirming its suitability for HTS.
- The assay quantifies the STAT4 SH2 domain interaction with a dissociation constant (Kd) of 34 ± 4 nM.
Conclusions:
- The developed assay is a valuable tool for identifying novel STAT4 SH2 domain inhibitors.
- This screening platform holds promise for discovering new therapeutic strategies for autoimmune diseases.
- The assay's robustness supports its application in large-scale drug discovery campaigns.
