Optimization of a high-throughput fluorescence polarization assay for STAT5B DNA binding domain-targeting inhibitors
Journal of Pharmaceutical and Biomedical Analysis, cilt.184, 2020 (SCI-Expanded, Scopus)
- Yayın Türü: Makale / Tam Makale
- Cilt numarası: 184
- Basım Tarihi: 2020
- Doi Numarası: 10.1016/j.jpba.2020.113182
- Dergi Adı: Journal of Pharmaceutical and Biomedical Analysis
- Derginin Tarandığı İndeksler: Science Citation Index Expanded (SCI-EXPANDED), Scopus, Academic Search Premier, Analytical Abstracts, Aquatic Science & Fisheries Abstracts (ASFA), BIOSIS, Biotechnology Research Abstracts, CAB Abstracts, Chimica, EMBASE, International Pharmaceutical Abstracts, MEDLINE, Veterinary Science Database
- Anahtar Kelimeler: Fluorescence polarization, Signal transducer and activator of transcription (STAT), Assay development, DNA-binding domain (DBD), Oligonucleotide, Small molecule inhibitor
- Boğaziçi Üniversitesi Adresli: Hayır
Özet
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.