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Updated: May 4, 2026
Identification of Kinase-substrate Pairs Using High Throughput Screening
Published on: August 29, 2015
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.
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.
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.
