Fluorescence Polarization Screening Assays for Small Molecule Allosteric Modulators of ABL Kinase Function

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmacology

Background:

• The ABL protein-tyrosine kinase is crucial in cellular signaling and implicated in various cancers.
• ABL kinase activity is regulated by intramolecular interactions involving its N-terminal cap, SH3, and SH2 domains.
• Allosteric modulators targeting non-catalytic domains offer potential for selective ABL probes.

Purpose of the Study:

• To develop a screening assay for identifying chemical modulators of ABL kinase activity.
• To target the regulatory interaction between the SH3 domain and the SH2-kinase linker.
• To discover novel allosteric regulators of ABL kinase function.

Main Methods:

• Developed a fluorescence polarization (FP) assay using a recombinant ABL protein (N32L) and a fluorescently labeled peptide probe.
• Validated the assay by demonstrating specific binding of the probe peptide to the N32L protein and confirming signal loss with mutant proteins.
• Conducted a pilot screen of 1200 FDA-approved drugs to identify modulators of the SH3-linker interaction.

Main Results:

• The FP assay successfully detected and quantified the interaction between the ABL SH3 domain and its linker.
• A pilot screen identified four compounds that modulated the FP signal, with dipyridamole showing significant ABL activation in vitro.
• Docking studies suggested dipyridamole binds to the SH3-linker interface, disrupting the regulatory interaction and activating ABL kinase.

Conclusions:

• Screening assays targeting ABL's non-catalytic domains can effectively identify allosteric small molecule regulators.
• Dipyridamole emerges as a promising ABL activator, demonstrating the potential of this approach for selective kinase drug discovery.
• This strategy provides a novel pathway for developing targeted therapies for ABL-related cancers.