Quantitative chemical proteomics identifies novel targets of the anti-cancer multi-kinase inhibitor E-3810.
Novel drugs are designed against specific molecular targets, but almost unavoidably they bind non-targets, which can cause additional biological effects that may result in increased activity or, more frequently, undesired toxicity. Chemical proteomics is an ideal approach for the systematic identification of drug targets and off-targets, allowing unbiased screening of candidate interactors in their natural context (tissue or cell extracts). E-3810 is a novel multi-kinase inhibitor currently in clinical trials for its anti-angiogenic and anti-tumor activity. In biochemical assays, E-3810 targets primarily vascular endothelial growth factor and fibroblast growth factor receptors. Interestingly, E-3810 appears to inhibit the growth of tumor cells with low to undetectable levels of these proteins in vitro, suggesting that additional relevant targets exist. We applied chemical proteomics to screen for E-3810 targets by immobilizing the drug on a resin and exploiting stable isotope labeling by amino acids in cell culture to design experiments that allowed the detection of novel interactors and the quantification of their dissociation constant (Kd imm) for the immobilized drug. In addition to the known target FGFR2 and PDGFRα, which has been described as a secondary E-3810 target based on in vitro assays, we identified six novel candidate kinase targets (DDR2, YES, LYN, CARDIAK, EPHA2, and CSBP). These kinases were validated in a biochemical assay and-in the case of the cell-surface receptor DDR2, for which activating mutations have been recently discovered in lung cancer-cellular assays. Taken together, the success of our strategy-which integrates large-scale target identification and quality-controlled target affinity measurements using quantitative mass spectrometry-in identifying novel E-3810 targets further supports the use of chemical proteomics to dissect the mechanism of action of novel drugs.
From the ‡Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, 20139 Milano, Italy; §Center of Genomic Science, Istituto Italiano di Tecnologia, via Adamello 16, 20139 Milano, Italy;
Mara
M
Colzani
From the ‡Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, 20139 Milano, Italy; §Center of Genomic Science, Istituto Italiano di Tecnologia, via Adamello 16, 20139 Milano, Italy;
Roberta
R
Noberini
From the ‡Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, 20139 Milano, Italy;
Mauro
M
Romanenghi
¶EOS S.p.A., Via Monte di Pietà 1/A, 20121 Milano, Italy; ‖Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Via Amadeo 42, 20133 Milano, Italy;
Gennaro
G
Colella
**Drug Discovery Program, European Institute of Oncology, Via Adamello 16, 20139 Milano, Italy;
Maurizio
M
Pasi
**Drug Discovery Program, European Institute of Oncology, Via Adamello 16, 20139 Milano, Italy;
Daniele
D
Fancelli
**Drug Discovery Program, European Institute of Oncology, Via Adamello 16, 20139 Milano, Italy;
Mario
M
Varasi
From the ‡Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, 20139 Milano, Italy; **Drug Discovery Program, European Institute of Oncology, Via Adamello 16, 20139 Milano, Italy; ‡‡Department of Bioscience, University of Milan, 20133 Milano, Italy.
Saverio
S
Minucci
From the ‡Department of Experimental Oncology, European Institute of Oncology, Via Adamello 16, 20139 Milano, Italy; tiziana.bonaldi@ieo.eu.
Tiziana
T
Bonaldi
D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry; D058990:Molecular Targeted Therapy; D009281:Naphthalenes / Q000008:administration & dosage*; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D047428:Protein Kinase Inhibitors / Q000008:administration & dosage*; D040901:Proteomics; D011804:Quinolines / Q000008:administration & dosage*; D053475:Receptor-Interacting Protein Serine-Threonine Kinase 2 / Q000037:antagonists & inhibitors; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors* / Q000378:metabolism
C000595232:E-3810; D009281:Naphthalenes; D009363:Neoplasm Proteins; D047428:Protein Kinase Inhibitors; D011804:Quinolines; D042461:Vascular Endothelial Growth Factor A; D053475:Receptor-Interacting Protein Serine-Threonine Kinase 2