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Free keywords:
POLO-BOX DOMAIN; MITOCHONDRIA-DERIVED ACTIVATOR; SMALL-MOLECULE
MIMETICS; X-LINKED INHIBITOR; SIGNAL TRANSDUCER; BINDING ASSAY; KINASE
1; TRANSCRIPTION; APOPTOSIS; POTENTBiochemistry & Molecular Biology; assay development; fluorescence polarization; protein-protein
interactions; SH2 domain; STAT4;
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 (K-d = 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.
