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Related Experiment Video

Updated: May 17, 2025

Characterization at the Molecular Level using Robust Biochemical Approaches of a New Kinase Protein
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A Probe-Based Target Engagement Assay for Kinases in Live Cells.

Ursula M Glocker1, Florian Braun2, H Christian Eberl1

  • 1Cellzome, A GSK Company, Heidelberg, Germany.

Molecular & Cellular Proteomics : MCP
|April 5, 2025
PubMed
Summary
This summary is machine-generated.

This study introduces a robust intracellular chemoproteomics assay for profiling kinase inhibitor selectivity. The assay accurately predicts drug target engagement within live cells, revealing off-target effects impacting efficacy and safety.

Keywords:
LC−MS/MSTMT labelingcovalent inhibitorkinasemass spectrometry (MS)proteomicsquantitative target engagement assay

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Last Updated: May 17, 2025

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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.