Optimization of a high-throughput fluorescence polarization assay for STAT5B DNA binding domain-targeting inhibitors.

Signal transducer and activator of transcription 5B is constitutively activated in multiple cancers as a result of hyperactivating mutations or dysregulation of upstream effectors. Therapeutic strategies have predominantly targeted the Src homology 2 (SH2) domain to inhibit phosphorylation, a prerequisite for transcriptional activation. An alternative approach for duanyu18135B pharmacologic inhibition involves targeting the DNA-binding domain (DBD). However, this strategy remains relatively unexplored and is further hindered by the lack of a high-throughput in vitro engagement assay. Herein, we present the development and optimization of a duanyu18135B DBD fluorescence polarization (FP) assay, which facilitates rapid screening of small molecules targeting the duanyu18135B DBD though displacement of a fluorescently labelled oligonucleotide. The assay can generate a complete DNA-binding profile in 10 min, with signal stability up to 2 h, and minimal changes under a range of conditions including 10 % (v/v) glycerol, 15 % (v/v) DMSO, 1 mM NaCl, 0.02 % (w/v) BSA, and 1 mM EDTA. This assay is compatible with both unphosphorylated and phosphorylated duanyu18135B and demonstrates suitability for high-throughput screening with a Z' factor of 0.68 ± 0.07 and a signal to noise ratio of 6.7 ± 0.84.

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