Target Landscape of Clinical Kinase Inhibitors
Here, we provide a comprehensive overview of the selectivity profiles of 242 kinase inhibitors currently in clinical trials. We screened available inhibitors against human protein kinases by using the Kinobead technology resulting in a drug matrix identifying the druggable kinome and related proteins.
Sample Processing Protocol
Kinobead selectivity profiling was performed as described previously (Medard, G., Pachl, F., Ruprecht, B., Klaeger, S., Heinzlmeir, S., Helm, D., Qiao, H., Ku, X., Wilhelm, M., Kuehne, T., Wu, Z., Dittmann, A., Hopf, C., Kramer, K., and Kuster, B. (2015) Optimized chemical proteomics assay for kinase inhibitor profiling, J Proteome Res 14, 1574-1586.) Briefly, 5 mg of a protein mixture of the four cell lines or a single cell line were incubated with compound dilution series in DMSO (3 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1 µM, 3 µM, 30 µM and DMSO) or single compound dose (5 µM) for 45 min at 4°C. The preincubation step was followed by incubation with kinobeads (35 µl settled beads). Bound proteins were eluted with LDS sample buffer (NuPAGE, Invitrogen) containing 50 mM DTT. For the calculation of a correction factor, the flowthrough of the DMSO control was incubated with fresh beads for a second time. Eluates were alkylated wit CAA and ingel digestion was performed. Peptides were then measured via LC-MS/MS on a nanoLCUltra 1D+ (Eksigent) coupled to a LTQ Orbitrap Elite. Up to 15 precursorer where subjected to fragmentation by HCD in a data dependent setup. A kinase peptide inclusion list was enabled.
Data Processing Protocol
Peptide and protein identification and quantification was performed using MaxQuant (version 220.127.116.11), searching against a human Uniprot reference database (version 22.03.2016, only canonical entries, annotated with PFAM) with Andromeda. Label free quantification and match between runs were enabled. Search results were filtered for 1% peptide and protein FDR, contaminants and reverse identifications.
Klaeger S, Heinzlmeir S, Wilhelm M, Polzer H, Vick B, Koenig PA, Reinecke M, Ruprecht B, Petzoldt S, Meng C, Zecha J, Reiter K, Qiao H, Helm D, Koch H, Schoof M, Canevari G, Casale E, Depaolini SR, Feuchtinger A, Wu Z, Schmidt T, Rueckert L, Becker W, Huenges J, Garz AK, Gohlke BO, Zolg DP, Kayser G, Vooder T, Preissner R, Hahne H, Tõnisson N, Kramer K, Götze K, Bassermann F, Schlegl J, Ehrlich HC, Aiche S, Walch A, Greif PA, Schneider S, Felder ER, Ruland J, Médard G, Jeremias I, Spiekermann K, Kuster B. The target landscape of clinical kinase drugs. Science. 2017 358(6367) PubMed: 29191878