Related Experiment Video

Updated: Feb 8, 2026

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays
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Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

Published on: October 23, 2019

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Cellular Context Influences Kinase Inhibitor Selectivity

Matthew J Binder1, Frances M Bashore1, Kaitlin K Dunn Hoffman2

  • 1Structural Genomics Consortium, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.

Journal of Medicinal Chemistry
|February 6, 2026
PubMed

Insights

Assessing kinase inhibitor selectivity in cells using NanoBRET assays reveals crucial on- and off-target interactions missed by cell-free methods, impacting chemical probe development.

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.

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