Active site profiling reveals coupling between domains in SRC-family kinases

Area of Science:

• Biochemistry
• Chemical Biology
• Molecular Biology

Background:

• Protein kinases are crucial regulators of intracellular signal transduction and significant drug targets.
• Chemical proteomic tools are vital for studying kinase regulation and screening inhibitors.
• Understanding protein kinase active site dynamics is key to developing targeted therapies.

Purpose of the Study:

• To develop a novel crosslinking strategy for rapid and quantitative profiling of protein kinase active sites.
• To identify conformation-specific inhibitors for SRC-family kinases (SFKs).
• To investigate the role of selective ligands in modulating kinase regulatory domain interactions.

Main Methods:

• Development of a new crosslinking strategy for protein kinase active site profiling.
• Application of the methodology to SRC and HCK kinases in cell lysates and live cells.
• Identification and characterization of ATP-competitive inhibitors with specific conformational preferences.

Main Results:

• The new crosslinking method allows for efficient and quantitative analysis of kinase active sites.
• A series of conformation-specific, ATP-competitive inhibitors targeting the autoinhibited forms of SRC and HCK were identified.
• Selective ligands were shown to modulate intermolecular binding interactions of SRC and HCK regulatory domains.

Conclusions:

• The developed crosslinking strategy is a powerful tool for studying protein kinases and their inhibitors.
• Conformation-specific inhibitors targeting autoinhibited SFKs offer new avenues for therapeutic development.
• These findings enhance our understanding of the regulatory mechanisms governing tyrosine kinases.