title: Affinity profiling of the cellular kinome for the nucleotide cofactors ATP, ADP, and GTP.
abstract: Most kinase inhibitor drugs target the binding site of the nucleotide cosubstrate ATP. The high intracellular concentration of ATP can strongly affect inhibitor potency and selectivity depending on the affinity of the target kinase for ATP. Here we used a defined chemoproteomics system based on competition-binding assays in cell extracts from Jurkat and SK-MEL-28 cells with immobilized ATP mimetics (kinobeads). This system enabled us to assess the affinities of more than 200 kinases for the cellular nucleotide cofactors ATP, ADP, and GTP and the effects of the divalent metal ions Mg(2+) and Mn(2+). The affinity values determined in this system were largely consistent across the two cell lines, indicating no major dependence on kinase expression levels. Kinase-ATP affinities range from low micromolar to millimolar, which has profound consequences for the prediction of cellular effects from inhibitor selectivity profiles. Only a small number of kinases including CK2, MEK, and BRAF exhibited affinity for GTP. This extensive and consistent data set of kinase-nucleotide affinities, determined for native enzymes under defined experimental conditions, will represent a useful resource for kinase drug discovery.
authors.affiliation: Cellzome GmbH, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Becher
authors.forename: Mikhail M
authors.initials: MM
authors.lastname: Savitski
authors.forename: Maria Fälth
authors.initials: MF
authors.lastname: Savitski
authors.forename: Carsten
authors.initials: C
authors.lastname: Hopf
authors.forename: Marcus
authors.initials: M
authors.lastname: Bantscheff
authors.forename: Gerard
authors.initials: G
authors.lastname: Drewes
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001667:Binding, Competitive; D045744:Cell Line, Tumor; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D008958:Models, Molecular; D010770:Phosphotransferases / Q000378:metabolism*
chemical_list: D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases