A fluorescence lifetime based binding assay to characterize kinase inhibitors

Area of Science:

• Biochemistry
• Chemical Biology
• Drug Discovery

Background:

• Kinase inhibitors are crucial therapeutics, but their development requires robust binding assays.
• Existing assays may not fully capture binding to different kinase conformational states.

Purpose of the Study:

• To develop and validate a fluorescence lifetime-based assay for identifying and characterizing small-molecule kinase inhibitors.
• To assess the assay's utility across a broad range of kinases and different kinase activation states.

Main Methods:

• Utilized a staurosporine-Alexa Fluor 647 conjugate and fluorescence lifetime detection to monitor probe displacement from the kinase ATP-binding site.
• Screened 257 kinases and validated inhibitor binding data against selected serine/threonine and tyrosine kinases.
• Compared binding data with kinase activity inhibition and assessed probe affinity for activated versus nonactivated kinase forms.

Main Results:

• The assay successfully identified kinase binders, with approximately half of the screened kinases amenable to this method.
• Rank-order potencies from binding assays generally correlated with activity assays, with notable differences observed.
• A kinase-dependent difference in probe binding affinity was observed between activated and nonactivated kinase forms.

Conclusions:

• The fluorescence lifetime assay provides a simple and effective method for identifying and characterizing small-molecule kinase inhibitors.
• The assay can be applied to a wide range of kinases and offers potential for identifying binders to both active and inactive kinase conformations.