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High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy
Published on: February 7, 2019
Optimization of a high-throughput fluorescence polarization assay for STAT5B DNA binding domain-targeting inhibitors
Pimyupa Manaswiyoungkul1, Fettah Erdogan1, Olasunkanmi O Olaoye1
1Department of Chemical and Physical Sciences, University of Toronto Mississauga, 3359 Mississauga Rd N., Mississauga, Ontario L5L 1C6, Canada; Department of Chemistry, University of Toronto, 80 St. George Street, Toronto, Ontario M5S 3H6, Canada.
Abstract:
Signal transducer and activator of transcription 5B (STAT5B) 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 STAT phosphorylation, a prerequisite for STAT5B transcriptional activation. An alternative approach for STAT5B 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 STAT5B DBD fluorescence polarization (FP) assay, which facilitates rapid screening of small molecules targeting the STAT5B 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 STAT5B 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.
Insights
We developed a novel fluorescence polarization assay to screen for drugs targeting the DNA-binding domain of Signal transducer and activator of transcription 5B (STAT5B). This assay enables rapid identification of STAT5B inhibitors for cancer therapy.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Signal transducer and activator of transcription 5B (STAT5B) is frequently activated in cancers.
- Current therapies target the SH2 domain, but STAT5B DNA-binding domain (DBD) inhibition is underexplored.
- A high-throughput assay for STAT5B DBD engagement is lacking.
Purpose of the Study:
- To develop and optimize a fluorescence polarization (FP) assay for STAT5B DNA-binding domain (DBD) engagement.
- To enable high-throughput screening of small molecules targeting the STAT5B DBD.
- To facilitate the discovery of novel STAT5B-targeted cancer therapeutics.
Main Methods:
- Development of a STAT5B DBD fluorescence polarization (FP) assay.
- Assay utilizes displacement of a fluorescently labeled oligonucleotide.
- Optimization for speed, signal stability, and robustness across various conditions.
Main Results:
- The STAT5B DBD FP assay provides a complete DNA-binding profile in 10 minutes.
- Assay exhibits signal stability up to 2 hours and tolerance to glycerol, DMSO, NaCl, BSA, and EDTA.
- Demonstrated suitability for high-throughput screening with a Z' factor of 0.68 ± 0.07 and S/N ratio of 6.7 ± 0.84.
- Compatible with both phosphorylated and unphosphorylated STAT5B.
Conclusions:
- A robust and efficient STAT5B DBD fluorescence polarization assay has been developed.
- This assay facilitates rapid screening for novel STAT5B-targeting small molecules.
- Enables exploration of STAT5B DBD as a therapeutic target for cancer treatment.
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