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Inhibitors are molecules that reduce enzyme activity by binding to the enzyme. In a normally functioning cell, enzymes are regulated by a variety of inhibitors. Drugs and other toxins can also inhibit enzymes. Some inhibitors bind to the enzyme’s active site, while others inhibit enzymatic activity by binding to other sites on the protein structure.
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Identification of Kinase-substrate Pairs Using High Throughput Screening
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Site-Specific Competitive Kinase Inhibitor Target Profiling Using Phosphonate Affinity Tags.

Wouter van Bergen1, Anneroos E Nederstigt1, Albert J R Heck1

  • 1Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Utrecht, CH, The Netherlands; Netherlands Proteomics Center, Utrecht, CH, The Netherlands.

Molecular & Cellular Proteomics : MCP
|January 18, 2025
PubMed
Summary

This study introduces a site-specific activity-based protein profiling method for precisely identifying protein kinase inhibitor targets. This approach enhances drug discovery by revealing off-target interactions with amino acid specificity.

Keywords:
activity-based protein profilingchemical proteomicsdrug-targetinhibitor selectivitykinase inhibitor

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Area of Science:

  • Biochemistry
  • Proteomics
  • Chemical Biology

Background:

  • Protein kinases are crucial drug targets, particularly in cancer therapy.
  • Selective kinase inhibition is challenging due to off-target effects.
  • Activity-based protein profiling (ABPP) with mass spectrometry aids kinase target identification.

Purpose of the Study:

  • To develop a site-specific ABPP strategy for precise protein kinase inhibitor target engagement profiling.
  • To achieve amino acid-level specificity in identifying kinase and off-kinase targets.
  • To enhance the efficiency and scope of kinase inhibitor profiling.

Main Methods:

  • Developed a site-specific ABPP approach using activity-based probes (ABPs) and mass spectrometry.
  • Employed complementary enzymatic digestion (trypsin and pepsin) for expanded kinase coverage.
  • Utilized highly efficient enrichment of ABP-labeled peptides for complex matrix reduction.

Main Results:

  • Screened over 100 kinase active sites in a single LC-MS analysis.
  • Identified EphA2, EphB4, and discoidin domain-containing receptor 1 as targets for NVP-BHG712.
  • Discovered inosine-5'-monophosphate dehydrogenase 2 as a putative off-target for XO44.
  • Demonstrated XO44 covalently links lysines and tyrosines, expanding the ABPP search space.

Conclusions:

  • The site-specific ABPP method provides a straightforward and valuable approach for kinase inhibitor target profiling.
  • This technique allows for detailed analysis of on- and off-target engagement with amino acid resolution.
  • The approach is automatable and significantly advances the field of drug discovery for kinase inhibitors.