Site-Specific Competitive Kinase Inhibitor Target Profiling Using Phosphonate Affinity Tags.

Protein kinases are prime targets for drug development due to their involvement in various cancers. However, selective inhibition of kinases, while avoiding off-target effects remains a significant challenge for the development of protein kinase inhibitors. Activity-based protein profiling (ABPP), in combination with pan-kinase activity-based probes (ABPs) and mass spectrometry-based proteomics, enables the identification of kinase drug targets. Here, we extend existing ABPP strategies for kinase profiling with a site-specific analysis, allowing for protein kinase inhibitor target engagement profiling with amino acid specificity. The site-specific approach involves highly efficient enrichment of ABP-labeled peptides, resulting in a less complex peptide matrix, straightforward data analysis, and the screening of over ∼100 kinase active sites in a single LC-MS analysis. The complementary use of both trypsin and pepsin in parallel to generate the ABP-labeled peptides considerably expanded the coverage of kinases and pinpoint the exact binding sites. Using the site-specific strategy to examine the on- and off-targets of the Ephrin receptor (Eph) B4 inhibitor NVP-BHG712 showed binding to EphA2 with an IC₅₀ of 17 nM and EphB4 with an IC₅₀ of 20 nM. Next to the known targets, EphA2 and EphB4, NVP-BHG712 bound to the discoidin domain-containing receptor 1 with an IC₅₀ of 2.1 nM, suggesting that a discoidin domain-containing receptor 1-targeting regio-isomer of NVP-BHG712 was used. The promiscuity of XO44 toward ATP-binding pockets on nonkinase proteins facilitated the screening of additional off-target sites, revealing inosine-5'-monophosphate dehydrogenase 2 as a putative off-target. Expanding the search to other amino acids revealed that XO44, in addition to 745 lysines, also covalently linked 715 tyrosines, which significantly expands the competitive ABPP search space and highlights the added value of the site-specific method. Therefore, the presented approach, which can be fully automated with liquid handling platforms, provides a straightforward, valuable new approach for competitive site-specific kinase inhibitor target profiling.