Comparing Immobilized Kinase Inhibitors and Covalent ATP Probes for Proteomic Profiling of Kinase Expression and Drug Selectivity

Abstract

Kinases are involved in the regulation of many cellular processes and aberrant kinase signaling has been implicated in human disease. As a consequence, kinases are attractive drug targets. Assessing kinase function and drug selectivity in a more physiological context is challenging and often hampered by the generally low expression level of kinases and the extensive post-translation modification in vivo. Kinase drug selectivity screens by chemical proteomics have gained attention because they allow the profiling of hundreds of kinases against one drug at the same time. Here, we directly compared two such methods, notably, immobilized broad spectrum kinase inhibitors (kinobeads) and active site labeling using desthiobiotin-ATP and -ADP probes. Affinity purification of ∼ 100 kinases by either kinobeads or ATP/ADP probes was readily achieved using 1 mg of cellular protein. Bioinformatic analysis revealed a high degree of complementarity of the two techniques. Kinobeads covered the Tyrosine Kinase family particularly well and ATP probes enriched higher numbers of STE family kinases. A consecutive combination of both enrichment strategies therefore allowed for the coverage of a larger part of the kinome than any one technique alone. While kinobeads are very selective for kinases, the ATP/ADP probes also enriched a large number of other nucleotide binding proteins. Both methods were applied to the selectivity profiling of the small molecular Aurora kinase inhibitor tozasertib in K562 cells. Our data confirmed Aurora A, B, and BCR-ABL as the main targets of tozasertib and identified TNK1, STK2, RPS6KA1, and RPS6KA3 as submicromolar off targets.

Target Identification in Small Cell Lung Cancer via Integrated Phenotypic Screening and Activity-Based Protein Profiling

“…First, we cannot ensure the entire kinome is being covered by the desthiobiotin-ATP probe used in our studies, which could be limited by the low abundance of certain kinases or the sequences not having a solvent accessible lysine near the active site. Nonetheless, we identified 196 protein kinases in H82 cells and 179 protein kinases in SW210.5 cells, which compares favorably with other reports that employ ABPP (28, 29). However, using these probes results in a certain bias toward serine/threonine kinases over tyrosine kinases compared to approaches with immobilized kinase inhibitors.…”

Global discovery of protein kinases and other nucleotide‐binding proteins by mass spectrometry

“…This notion is further substantiated by the fact that 44% of the identified spectra from the ATP probe experiment belong to proteins with documented nucleotide binding activity, whereas this figure is only 12% in the experiment using kinobeads. In keeping with this finding, the authors also observed that increasing the amount of input material facilitated the detection of more kinases using kinobeads; however, increasing the lysate amount beyond 1 mg in experiments using the ATP/ADP probes led to identification of a large proportion of proteins which were labeled through non‐specific interactions (Lemeer et al, ). Moreover, a significantly larger number of tyrosine kinases were enriched using kinobeads, whereas members of the STE kinase group (mainly the MAP kinases) were better represented with the use of the ATP probes.…”

Measuring Kinase Activity—A Global Challenge

“…Other studies used the KinoBead approach to profile changes in the baseline kinome of different cancer cell lines and other cells [Oppermann et al, 2009;Gholami et al, 2013;Medard et al, 2015]. Interestingly, a high degree of complementarity between these two methods was observed in these studies [Lemeer et al, 2013]. However, no claims were made regarding the ability of these reagents to measure the actual activation state of individual kinases in the kinome.…”