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A fluorescence polarization based Src-SH2 binding assay
B A Lynch1, K A Loiacono, C L Tiong
1ARIAD Pharmaceuticals Inc., Cambridge, Massachusetts 02139, USA.
Abstract:
The tyrosine kinase pp60c.src has been implicated as being a potential therapeutic target in several human diseases including cancer and osteoporosis. An important region within this kinase is the SH2 domain (Src homology 2) which binds to phosphorylated tyrosine residues contained within specific peptide sequences. Homologous domains are found in a variety of cytoplasmic proteins and have been shown to be essential for controlling many important signaling pathways. Developing specific inhibitors of SH2 interactions would therefore be extremely useful for modulating a variety of signaling pathways and potentially be useful for the treatment of human disease. Current methodology for the development of organic molecules as drug leads requires the ability to test thousands of individual compounds or natural product extracts in biochemical assays. Such tests must be reproducible, simple, and versatile. This paper describes an assay based on fluorescence polarization for measuring the binding of compounds to the Src-SH2 domain. The assay is insensitive to changes in fluorescence intensity working even in solutions with moderate optical density and functions in the presence of up to 20% dimethyl sulfoxide. These features make it especially useful for high-throughput screening of both natural and synthetic compound libraries.
Insights
We developed a fluorescence polarization assay to screen for inhibitors of the Src-SH2 domain, crucial for signaling pathways in diseases like cancer. This assay is robust and suitable for high-throughput screening of drug compounds.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Discovery
Background:
- The tyrosine kinase pp60c-src is a therapeutic target for diseases such as cancer and osteoporosis.
- The Src homology 2 (SH2) domain binds to phosphorylated tyrosine residues, regulating critical signaling pathways.
- Inhibiting SH2 domain interactions offers potential therapeutic benefits for various human diseases.
Purpose of the Study:
- To develop a versatile and reproducible assay for measuring compound binding to the Src-SH2 domain.
- To facilitate high-throughput screening (HTS) of potential drug leads targeting SH2 interactions.
Main Methods:
- A fluorescence polarization (FP) assay was designed to quantify the binding of small molecules to the Src-SH2 domain.
- The assay demonstrates robustness, tolerating moderate optical density and up to 20% dimethyl sulfoxide (DMSO).
Main Results:
- The developed FP assay is insensitive to fluorescence intensity variations.
- The assay is suitable for screening large libraries of synthetic and natural compounds.
- This method enables efficient identification of compounds that modulate SH2 domain interactions.
Conclusions:
- The described fluorescence polarization assay is a valuable tool for high-throughput screening of Src-SH2 domain inhibitors.
- This assay can accelerate the discovery of novel therapeutics targeting signaling pathways implicated in cancer and other diseases.