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Published on: September 28, 2018
Novel multiplexed assay for identifying SH2 domain antagonists of STAT family proteins
Kazuyuki Takakuma1, Naohisa Ogo, Yutaka Uehara
1Center for Drug Discovery, Graduate School of Pharmaceutical Sciences, University of Shizuoka, Suruga-ku, Shizuoka, Japan.
Abstract:
Some of the signal transducer and activator of transcription (STAT) family members are constitutively activated in a wide variety of human tumors. The activity of STAT depends on their Src homology 2 (SH2) domain-mediated binding to sequences containing phosphorylated tyrosine. Thus, antagonizing this binding is a feasible approach to inhibiting STAT activation. We have developed a novel multiplexed assay for STAT3- and STAT5b-SH2 binding, based on amplified luminescent proximity homogeneous assay (Alpha) technology. AlphaLISA and AlphaScreen beads were combined in a single-well assay, which allowed the binding of STAT3- and STAT5b-SH2 to phosphotyrosine peptides to be simultaneously monitored. Biotin-labeled recombinant human STAT proteins were obtained as N- and C-terminal deletion mutants. The spacer length of the DIG-labeled peptide, the reaction time, and the concentration of sodium chloride were optimized to establish a HTS system with Z' values of greater than 0.6 for both STAT3- and STAT5b-SH2 binding. We performed a HTS campaign for chemical libraries using this multiplexed assay and identified hit compounds. A 2-chloro-1,4-naphthalenedione derivative, Compound 1, preferentially inhibited STAT3-SH2 binding in vitro, and the nuclear translocation of STAT3 in HeLa cells. Initial structure activity relationship (SAR) studies using the multiplexed assay showed the 3-substituent effect on both the activity and selectivity of STAT3 and STAT5b inhibition. Therefore, this multiplexed assay is useful for not only searching for potential lead compounds but also obtaining SAR data for developing new STAT3/STAT5b inhibitors.
Insights
Researchers developed a novel multiplexed assay to inhibit signal transducer and activator of transcription (STAT) activation by targeting STAT3 and STAT5b SH2 domain binding. This assay identified potential inhibitors for cancer therapy.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- Constitutive activation of signal transducer and activator of transcription (STAT) proteins, particularly STAT3 and STAT5b, is common in human cancers.
- STAT protein activity relies on SH2 domain binding to phosphotyrosine residues, making this interaction a target for therapeutic intervention.
- Inhibiting STAT SH2 domain binding offers a promising strategy to block aberrant STAT activation in tumors.
Purpose of the Study:
- To develop and validate a novel multiplexed assay for simultaneously monitoring STAT3-SH2 and STAT5b-SH2 domain binding.
- To utilize this assay for high-throughput screening (HTS) of chemical libraries to identify STAT inhibitors.
- To investigate structure-activity relationships (SAR) for STAT3 and STAT5b inhibition.
Main Methods:
- Development of a homogeneous assay using AlphaLISA and AlphaScreen technologies to detect STAT3- and STAT5b-SH2 domain binding to phosphotyrosine peptides.
- Optimization of assay parameters including peptide spacer length, reaction time, and salt concentration to achieve high Z' values (>0.6).
- High-throughput screening of chemical libraries and subsequent structure-activity relationship studies.
Main Results:
- A robust, multiplexed HTS assay for STAT3- and STAT5b-SH2 binding was established with excellent performance metrics.
- Screening identified hit compounds, including a 2-chloro-1,4-naphthalenedione derivative (Compound 1) that selectively inhibited STAT3-SH2 binding.
- Initial SAR studies revealed the impact of 3-substituents on the inhibitory activity and selectivity against STAT3 and STAT5b.
Conclusions:
- The developed multiplexed Alpha assay is effective for simultaneously assessing STAT3 and STAT5b SH2 domain binding.
- This assay serves as a valuable tool for identifying novel STAT inhibitors and generating crucial SAR data.
- The findings support the development of targeted therapies against STAT3/STAT5b pathways in cancer.
