title: 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.
authors.affiliation: Chair of Proteomics and Bioanalytics, Technische Universität München , Emil Erlenmeyer Forum 5, 85354 Freising, Germany.
authors.forename: Simone
authors.initials: S
authors.lastname: Lemeer
authors.forename: Corina
authors.initials: C
authors.lastname: Zörgiebel
authors.forename: Benjamin
authors.initials: B
authors.lastname: Ruprecht
authors.forename: Kristian
authors.initials: K
authors.lastname: Kohl
authors.forename: Bernhard
authors.initials: B
authors.lastname: Kuster
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D064127:Aurora Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005326:Fetal Proteins / Q000378:metabolism; D016044:Fusion Proteins, bcr-abl / Q000378:metabolism; D006801:Humans; D020014:K562 Cells / Q000187:drug effects; D010879:Piperazines / Q000378:metabolism / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000378:metabolism; D003521:Cyclopropanes; D011720:Pyrazoles; D011743:Pyrimidines
chemical_list: D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D064127:Aurora Kinases; D005326:Fetal Proteins; D016044:Fusion Proteins, bcr-abl; D010879:Piperazines; D047428:Protein Kinase Inhibitors; D011505:Protein-Tyrosine Kinases; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C484810:tozasertib; D003521:Cyclopropanes; D011720:Pyrazoles; D011743:Pyrimidines; C098672:TNK1 protein, human; C069329:RPS6KA1 protein, human; C069330:ribosomal protein S6 kinase, 90kDa, polypeptide 3