A Probe-Based Target Engagement Assay for Kinases in Live Cells

Area of Science:

• Biochemistry
• Pharmacology
• Chemical Biology

Background:

• Kinase inhibitor drug efficacy and safety depend on selectivity.
• Current profiling methods use cell extracts, not live cells.
• Developing intracellular assays is crucial for accurate profiling.

Purpose of the Study:

• To develop and validate a robust intracellular chemoproteomics assay for kinase inhibitor profiling.
• To compare intracellular target engagement with traditional lysate-based assays.
• To identify off-target interactions of kinase inhibitors in live cells.

Main Methods:

• Developed a novel covalent probe with a trans-cyclooctene handle for enhanced kinase capture.
• Optimized probe concentration, incubation time, and used isobaric mass tagging for quantification.
• Compared intracellular profiles of dasatinib and dinaciclib with lysate-based kinobeads assays.

Main Results:

• The new protocol demonstrated robust kinase capture and enrichment.
• Intracellular profiling showed excellent agreement with lysate-based assays in rank-order binding.
• Observed shifts in IC50 values for dinaciclib, indicating influence of intracellular factors.
• Identified sepiapterin reductase (SPR) and ABCC1 as off-targets for kinase inhibitor scaffolds.

Conclusions:

• The developed intracellular chemoproteomics assay provides a more accurate assessment of kinase inhibitor target engagement.
• Intracellular conditions significantly influence drug target profiles and binding affinities.
• Identification of off-targets like SPR and ABCC1 has implications for kinase inhibitor efficacy and safety.