ABSTRACT
Signal transducer and activator of transcription 5b (STAT5b) is constitutively activated in many human tumors. Activity of STAT5b requires binding of its Src homology 2 (SH2) domain to certain phosphotyrosine-containing sequences. We have developed a high-throughput assay based on fluorescence polarization that allows screening of chemical libraries for compounds that inhibit STAT5b by interfering with the function of its SH2 domain. The assay, which is based on binding between a fluorescein-labeled phosphotyrosine peptide derived from the erythropoietin receptor to the STAT5b SH2 domain, is stable with regard to dimethyl sulfoxide concentration and time and has a Z' value of 0.66+/-0.11 in a 384-well format.
Subject(s)
Drug Evaluation, Preclinical/methods , Fluorescence Polarization/methods , STAT5 Transcription Factor/antagonists & inhibitors , STAT5 Transcription Factor/metabolism , Small Molecule Libraries/pharmacology , Amino Acid Sequence , Dimethyl Sulfoxide/pharmacology , Fluorescent Dyes/metabolism , Humans , STAT5 Transcription Factor/chemistry , Sodium Chloride/pharmacology , src Homology DomainsABSTRACT
Signal transducers and activators of transcription (STATs) are a family of latent cytoplasmic transcription factors that transmit signals from the cell membrane to the nucleus. One family member, STAT3, is constitutively activated by aberrant upstream tyrosine kinase activities in a broad spectrum of cancer cell lines and human tumors. Screening of chemical libraries led to the identification of Stattic, a nonpeptidic small molecule shown to selectively inhibit the function of the STAT3 SH2 domain regardless of the STAT3 activation state in vitro. Stattic selectively inhibits activation, dimerization, and nuclear translocation of STAT3 and increases the apoptotic rate of STAT3-dependent breast cancer cell lines. We propose Stattic as a tool for the inhibition of STAT3 in cell lines or animal tumor models displaying constitutive STAT3 activation.
Subject(s)
Cyclic S-Oxides/metabolism , STAT3 Transcription Factor/antagonists & inhibitors , Apoptosis/drug effects , Cell Line, Tumor , Cyclic S-Oxides/pharmacology , Dimerization , Humans , Phosphorylation/drug effects , src Homology DomainsABSTRACT
Signal transducer and activator of transcription 3 (STAT3) is overactive in a wide variety of human tumors. Activity of STAT3 requires its own SH2-domain-mediated binding to phosphotyrosine-containing sequences. We have developed a high-throughput binding assay, based on fluorescence polarization, which allows screening for small molecules that bind to the STAT3 SH2 domain and thereby inhibit its activity. The basis of this assay is the binding of a fluorescein-labeled phosphotyrosine-peptide derived from the interleukin-6 receptor subunit gp130 to unphosphorylated STAT3 with a K(d) of 150 nM. The assay is stable with regard to salt concentration, dimethyl sulfoxide concentration, and time. It has been adapted to a 384-well format, with a Z' value of 0.87, and can be used to screen for small molecules that bind to the STAT3 SH2 domain.
