Cellular Context Influences Kinase Inhibitor Selectivity.

Area of Science:

• Biochemistry
• Pharmacology
• Chemical Biology

Background:

• Kinase inhibitor selectivity profiling is essential for chemical probe and drug development.
• Gauging the number of on- and off-targets for kinase inhibitors is critical.
• Cellular pharmacology necessitates in-cell assessment of target engagement.

Purpose of the Study:

• To systematically compare cell-free kinase inhibitor profiling with in-cell target engagement NanoBRET assays.
• To evaluate how different assay types influence chemical probe prioritization.
• To identify kinase interactions specific to cellular environments.

Main Methods:

• Profiling ten kinase inhibitors using both cell-free and cellular NanoBRET assays across a broad kinase panel.
• Comparing selectivity profiles obtained from biochemical assays versus intact-cell assays.
• Characterizing TPKI-39's in-cell selectivity profile.

Main Results:

• Divergent selectivity profiling results were observed between cell-free and in-cell assays.
• Unanticipated kinase interactions for type II kinase inhibitors were identified in cells but not in cell-free systems.
• TPKI-39 was characterized as a chemical probe for DDR1, DDR2, and FLT1 based on its in-cell selectivity.

Conclusions:

• In-cell target engagement assays provide a more accurate assessment of kinase inhibitor selectivity compared to cell-free methods.
• Cellular context reveals kinase interactions missed by biochemical assays, influencing chemical probe selection.
• TPKI-39 demonstrates specific in-cell activity, validating its use as a chemical probe for DDR1, DDR2, and FLT1.